High-Density
Lipoprotein
G. Luc, J.M. Bard, S. Lussier-Cacan,
Particles
D. Bouthillier,
in Octogenarians
H.J. Parra, J.C. Fruchart, and J. Davignon
High-density lipoprotein (HDL) particles exhibit considerable heterogeneity, specifically in apolipoprotein (apo) composition. Thus, apo A-l, the major pcotein of HDL, is present in two types of particles: one species contains both apo A-l and apo A-II (Lp A-l/A-II) while in the other (Lp A-l), apo A-II is absent. We used the hypothesis that octogenarians, who survived periods in life when the incidence of coronary heart disease (CHD) is very high, have several protective factors. We compared HDL-cholesterol (HDL-C), HDL,-cholesterol (HDL,-C), HDL,-cholesterol (HDL,-C), apo A-l, and apo A-II in octogenarians and younger control subjects smoking less than,10 cigarettes/d and not taking drugs known to affect lipid metabolism. Using a new procedure, we also compared the levels of Lp A-l and Lp A-I/A-II. The cholesterol content of total HDL was similar in octogenarian and control (38 + 8 years) men while HDL,-C was higher and HDL,-C, apo A-l, and A-II were lower in octogenarian than in control men. In women, the level of HDL-C and apo A-l was similar in premenopausal and octogenarian subjects but higher in postmenopausal women than in octogenarians, while HDL,-C and apo A-II were similar in the three groups. In contrast, HDL,-C was higher in the two groups of control women (premenopausal and postmenopausal) than in octogenarians. However, Lp A-l was significantly elevated in octogenarian men and women (men: 81 2 14 mg/dL; women: 70 ? 14 mg/dL) by comparison with younger control subjects (men: 48 2 12 mg/dL; premenopausal women: 53 ? 11 mg/dL; postmenopausal women: 83 2 19 mg/dL). On the other hand, Lp A-I/A-II was clearly lower in octogenarians. The distribution of Lp A-l and Lp A-I/A-II values also appeared to be modified by age, a shift of the distribution being observed toward higher values of Lp A-l and lower values of Lp A-I/A-II in octogenarians as compared with younger subjects. This observation could signify that a part of the population with a low level of Lp A-l could have a high frequency of CHD during the sixth or seventh decades. Thus, these results are consistent with the protective function of Lp A-l, which has already been perceived through fundamental and angiographic studies. Copyright
0 1991 by W.B. Saunders Company
E
PIDEMIOLOGIC and clinical studies have determined a number of risk factors for atherosclerosis. Among these, in addition to elevated low-density lipoprotein-cholesterol (LDL-C),’ other alterations have been described, most prominently a reduction of high-density lipoprotein-cholesterol (HDL-C) levels. Reduced HDLcholesterolemia has been confirmed in numerous clinical reports in coronary patients as well as in other noncardiac atherosclerotic syndromes.Z7 Moreover, apolipoprotein (apo) A-I, the major protein of HDL, has been shown in a number of clinical and angiographic studies to be an independent risk factor for coronary heart disease (CHD), especially in patients whose plasma lipoprotein levels are largely norma1.8~‘5 However, it is now established that HDL particles exhibit considerable heterogeneity of size, density, flotation rate, and lipid and apo composition. Also, the cholesterol content of the entire HDL family is a complex function of the composition and particle number of several subclasses.‘6 Specifically, HDL can be separated into several subclasses by different procedures. Evidence of heterogeneity has been provided by the determination of hydrated density, molecular weight, particle size, chemical composition, and hydrodynamic properties.‘7-‘9 Two main fractions, HDL, (density 1.063 to 1.125 g/mL) and HDL, (density 1.125 to 1.21 g/mL) have been isolated by ultracentrifugation and/or
From SERLIA, Institut Pasteur, Lille, France; and the Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. Suppotied in part by the Medical Research Council of Canada (Grant No. MA-5427), the succession JA DeSeve and INSERM. Address reprint requests to G. Luc, MD, SERLIA, Institut Pasteur, rue du Professeur Calmette, 59019 Lille Cedet; France. Copyright 0 1991 by U?B. Saunders Company 0026-0495/91/4012-0002$03.00/O 1238
selective precipitation. Reduction of both HDL subclasses has been associated with CHD but HDL, had the strongest association.zo~z2With regard to the apo composition, it is now recognized that HDL contains two main types of apo A-I-containing particles that may have different metabolic functions and clinical significance.23-26One species contains both apo A-I and A-II (Lp A-I/A-II) while in the other (Lp A-I) apo A-II is absent. We hypothesized that octogenarians, who survived periods in life when CHD mortality is very high, have very few or no risk factors for atherosclerosis and/or several protective factors. Thus, Davignon et al,” postulating an atherogenie role for apo E4, showed a decreased frequency of the ~4 allele (coding for the isoform E4 of the polymorphic apolipoprotein E) in octogenarians. Similarly, one might expect to find a protective profile of lipids or lipoproteins, particularly for HDL particles among octogenarians. MATERIALS
AND METHODS
Subjects The octogenarian subjects in this study were ambulatory and autonomous, had completed their 80th year, were capable of answering a questionnaire, and agreed to submit to a short physical examination and to the drawing of blood. They were recruited from six homes for the aged and one Veterans’ hospital in metropolitan Montreal, Canada. Excluded were individuals with chronic debilitating disease (eg, cancer, Parkinson’s disease), mental illness, alcoholism, hypothyroidism, and diabetes; those taking drugs known to affect lipoprotein metabolism such as hypolipidemic and hypoglycemic drugs, hormones (thyroid hormones, estrogens, progesterone, corticosteroids, or androgenic steroids), betablockers, or thiazides; as well as subjects who had sustained major surgery, weight changes (210%). or a myocardial infarction within the previous 3 months. Smokers defined as subjects smoking more than 10 cigarettes/d were also excluded. Eighty-nine unrelated octogenarians (33 men and 56 women) were included in this report. The mean age was 84 ? 3 (mean + SD) for women and 85 ? 5 years for men. Metabolism,
Vol40, No 12 (December), 1991: pp 1238-1243
HIGH-DENSITY
1239
LIPOPROTEIN IN OCTOGENARIANS
The reference sample consisted of subjects smoking less than 10 cigarettes/d, 48 men and 127 women, who had a desirable blood cholesterol ( ~200 mg/dL) or a borderline-high (200 to 239 mg/dL) blood cholesterol, and a triglycerides concentration less than 150 mg/dL. The mean age of control men was 39 ? 8 years. The control women were divided into two subgroups, one (n = 76) including postmenopausal women not taking estrogenic supplementation with a mean age of 50 -c 3 years, the other (n = 51) including prernenopausal women not taking birth control pills with a mean age of 35 ? 5 years. The postmenopausal status was assessed by typical clinical signs and a hormonal test (follicle-stimulating hormone [FSH] > X0 mUl/mL). Methods Venous blood obtained from subjects was collected into EDTAcontaining tubes (1.5 mg/dL) after an overnight fast. Plasma was immediately separated by centrifugation at 4°C. Sodium azide at a final concentration 0.2% (wtivol) was added to plasma samples. C‘ho’lesterol and triglycerides were determined by automated en2;rimatic procedures.‘X,” HDL-C was determined on fractions separated by combined ultracentrifugation and heparin-manganest precipitation.2” LDL-C was measured by subtracting HDL-C and the cholesterol content in very low-density lipoproteins (VLDL), these last lipoproteins being isolated by sequential uitr,lcentrifugation. from total plasma cholesterol. The cholesterol conlent of HDLz and HDL, was measured according to the method oi Gidez et al.” Apo A-I was determined by electroimmunodiffuslon using the kit Hydragel from Sebia (Issy les Moulineaux, France). Apo A-II was measured by noncompetitive enzymelinked immunoassay.” Lp A-I was determined by electroimmunoassay using a polyclonal antiserum? Briefly, agarose gel plates ((1.9%) containing polyclonal antisera to apo A-I and apo A-II with a high excess of anti-apo A-II. were prepared and mounted onto plastic films. Wells were punched into the gel. The plasma samples were diluted 1:30 in saline and 5 FL of the solution was applied to each well. Electrophoresis was then performed at 150 \ /cm for 3 hours in a buffer containing 0.06 mol/L Tris, 0.07 mol/L glycme, and 0.01 mol/L barbital, pH 8.6. The excess of anti-apo A-11 in the gel, relative to anti-apo A-I, causes a blockage of the Lp A-I A-II particles near the origin, while allowing Lp A-I particles tcl migrate and react with anti-apo A-I. After washing, staining (Coomassie brilliant blue). and destaining, rockets corresponding tc+L.p A-I particles were then visible and measured. The Lp A-I concentration was calculated from a calibration cume using a secondary standard serum. The primary standard was a pure fraction of Lp A-I particles purified by immunoaffinity. The concentration of particles containing both apo A-I and A-II was determined by the difference between total plasma apo A-I and Lp A-I concentrations. Comparisons were made between the octogenarians and the comrol subjects by gender. The statistical significance of the d&.rence in the mean levels of lipids, apo, and lipoprotein particles between groups was evaluated using a nonparametric test ( Mann-Whitnev). RESULTS Octogenarian men and women were clearly different with regard to several parameters. Among plasma lipid levels, total cholesterol, triglycerides, and LDL-C were more elevated in women than in men (Table 1). Among octogenarian women, 19 (34%) were hyperlipidemic inciuding 13 (23%) with hypercholesterolemia alone (cholesterol > 240 mg/dL), 1 (2%) with hypertriglyceridemia (triglycerides > 200 mg/dL), and 5 (9%) with both abnor-
Table 1. Cholesterol, Triglycerides, and LDL-C (mg/dL) in Octogenarian n
Octogenarian men
33
(Mean + SD)
and Control Subjects Cholesterol Triglyceride 186+42t
122%79*
LDL-C
118r36t
Control men
48
189k29
89 2 29
122 -t 28
Octogenarian women
56
226 + 42
130 -t 57
149 t 34
Postmenopausal women
76
203 + 32$
84?33$
124?30$
Premenopausal women
51
173 f 24:
53 rt 19*
107 -t 22*
*P < .05; tP < .Ol compared with octogenarian women. tP
< .Ol compared with octogenarians of corresponding gender
malities. Conversely, only five (15%) among the men were hyperlipidemic, including two with hypercholesterolemia, two with hypertriglyceridemia alone, and one with mixed hyperlipidemia. The average cholesterol, triglyceride, and LDL-C levels were similar in octogenarian men and in controls. In contrast, cholesterol, triglycerides, and LDL-C were significantly higher in octogenarian women when compared with postmenopausal or premenopausal women. Differences also appeared between octogenarian men and women when HDL particle parameters were analyzed (Table 2). Indeed, HDL,-C, apo A-I, apo A-II, and Lp A-I were lower in men than in women and HDL-C, HDL& and Lp A-I/A-II were similar in the two groups. The measurement of cholesterol in HDL and its subfractions HDL-C and HDL,-C gave different results for women and men of different ages (Table 2). Indeed, while HDL-C was not significantly different in octogenarian and in control men, it was higher in postmenopausal women than in octogenarian women. The level of HDL-C was also noted to be higher in premenopausal women than in octogenarian women but this difference was not significant. In men, the partition of HDL-C in HDL,-C and HDL,-C was clearly different in octogenarians than in controls, HDLZ-C being lower and HDL,-C higher in the control group. The relative content of apo A-II was higher in HDL, than in HDL,, and this difference was reflected in the level of apo A-II, which is significantly higher in controls than in octogenarians. Apo A-I was also higher in controls than in octogenarians. In women, HDL,-C was similar in groups of different ages and the higher level of HDL-C in postmenopausal women was due to a higher level of HDL,-C. Moreover, while HDL-C and HDL?-C were not different in octogenarian and premenopausal women, HDL,-C was however higher in this latter group than in octogenarians. The apo A-I levels of the postmenopausal women were more elevated than in octogenarians, but those of premenopausal and octogenarian women were similar. Conversely, apo A-II levels were the same in the octogenarian and in the younger women (Table 2). The concentrations of Lp A-I and Lp A-I/A-II determined from the apo A-I content in each class were clearly different in the octogenarian and control groups (Table 2). The levels of Lp A-I were dramatically higher in octogenarians than in controls for both sexes (P < .01). The levels of Lp A-I/A-II were also different between groups but in the opposite direction, being higher in controls than in octogenarians.
1240
LUC ET AL
Table 2. HDL-C, HDL,, and HDL,-C, Apo A-l, Apo A-II, Lp A-l, and Lp A-I/A-II HDL-C
HDL,-C
HDL,.C
Octogenarian men
42 + 10
20 5 9
21 +4t
Control men
43k
IO
15 I? 78
28 + 55
Octogenarian women
472
11
22 + a
25 2 4
Postmenopausal women
56 2 119
21 k 6
35 2 65
Premenopausal women
52 + 12
23 + 9
29 + 45
(mg/dL)
(Mean k SD) in Octogenarian
ApoA-I
and Control Subjects
ApoA-Ii
Lp A-l
Lp A-I/A-II
26 f 4’
61 f 14t
55 + 18
127 rr 219
29 2 71
48 + 121
79 A 199
129 t- 23
29 -t 5
70 t 14
59 * 20
139 2 ia*
29 f 5
63 r 199
76 -+ 205
137 t 21
27 i 6
53 + 119
a3 2 201
116 t- 19*
*P < .05; tP < .Ol compared with octogenarian women. SP < .05; §P < .Ol compared with octogenarians of corresponding gender.
The distribution of individual values was studied using percentiles in each octogenarian and control group. Figure 1A and B compare distributions of percentiles of apo A-I, apo A-II, Lp A-I, and Lp A-I/A-II for men and women, respectively. Each point corresponds to a percentile (5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 9.5, or 99th) for the control group (on the abscissa) and for the octogenarian group (on the ordinate). The percentile distributions of apo A-I in octogenarians were slightly shifted toward lower values as compared with controls with the exception of the 99th percentile of octogenarian women, which was higher than that of premenopausal women. Furthermore, the percentiles of apo A-II were similar in octogenarian and control women while the distribution was shifted toward lower values in octogenarian men as compared with control men. The percentile distributions of Lp A-I and Lp A-I/A-II in
6
the octogenarian and control men and in octogenarian and control women were, respectively, clearly shifted toward higher and smaller values relative to the means, with the exception of the 95th percentile of Lp A-I in postmenopausal women, which was higher than in octogenarian women. However, this shift was more important for premenopausal than postmenopausal women. The 99th percentile of Lp A-I was always higher in octogenarians than in controls and a fraction of the octogenarians (men and women) presented high values of Lp A-I or low values of Lp A-I/A-II not found in the control groups. DISCUSSION
In the present study, we have looked at different HDL parameters in octogenarians and in control groups of younger subjects. The cholesterol content in HDL and in
PRE MEKOPAUSAI
POST MENOPAUSAL
coNlR0Ls
Fig 1. Comparisons of distributions of percentiles of apo A-l, apo A-II, Lp A-l, and LP A-l /A-II for men and women.
HIGH-DENSITY
LIPOPROTEIN
IN OCTOGENARIANS
each of its major subfractions, HDLz and HDL,, was measured as well as the major HDL apo, apo A-I and apo A-II. Using a new method, we have also determined the concentration of particles containing apo A-I but not apo A-II (Lp A-I) and by calculation, that of particles containing both apo A-I and A-II (Lp A-I/A-II). A number of studies comparing myocardial infarction survivors with control subjects, using CHD incidence as well as angiographic studies, have demonstrated that high levels of HDL-C, HDL2-C, and apo A-I, and possibly HDL,-C and apo A-II are protective against CHD.*“’ We postulated that these values would be elevated in a population with a great longevity (octogenarians) in comparison with a younger control group. However, our results in women did not support this hypothesis. HDL-C, HDL,-C, and apo A-I were lower in octogenarians than in postmenopausal women and HDL,-C in octogenarian than in premenopausal women while the levels of HDL-C, HDL,-C, apo A-I, and apo A-II wcrc not different in the two latter groups. Moreover, potential CHD risk factors, such as high plasma cholesterol and triglycerides, were more pronounced in octogenarians, particularly in women. In men, results were clearly different showing a high level of HDL2-C in octogenarians while other values were lower (HDL,-C, apo A-I, apo A-II) than those in controls or similar (HDL-C) in both groups. Thus, the high level of HDL2-C in octogenarian men would appear to be a strong protective factor. Relatively little data about HDL in the elderly are available. HDL-C has been reported in octogenarians and nonagenarians by a few authors. While Alvarez et al’” noted lower HDL-C values in octogenarian men in comparison with younger (65 to 80 years) subjects, octogenarians and younger women had similar levels. Heckers et ap5 showed the inverse situation in nonagenarian men in comparison with a group of 70-year-old men, while HDL-C was higher in the 70-year-old women than in the nonagenarians. In contrast, Nicholson et alZh and Rifkind et a1j7 showed a tendency for a decrease of HDL-C after the age of 65 years in men and a stability of these levels in women. The most striking observation of the present investigation was the significant elevation of Lp A-I and the decrease of Lp A-I/A-II levels in male and female octogenarians. However, the modification of HDL particle composition according to age, already suggested by the different levels of HDL,-C and HDL,-C in octogenarian and younger subjects, was confirmed by the important difference in levels of Lp 4-I and Lp A-I/A-II and by the clearly different distribution of Lp A-I and Lp A-I/A-II in octogenarian and younger groups. The decrease of total apo A-I in octogenarians as compared with controls was the reflection of a marked reduction in Lp A-I/A-II not compensated by the increase of Lp A-I. A possible explanation of the levels of apo .4-I-containing particles in the different age groups may be that a fraction of the population with high levels of Lp A-I or low levels of Lp A-I/A-II is protected and survives beyond the age of 80 years. This hypothesis is compatible with the observations made in angiographic studies and a case control study, which have shown a lower level of Lp A-I in subjects with CHD.lJ,“’ Thus, the elevated level of
1241
HDL-C in families described by Glueck et al”’ in which subjects survive much longer than the average population could be the consequence of a very high level of Lp A-I. In addition to the putative role of Lp A-I as an antiatherogenic particle, Davignon et al” showed that a decreased frequency of the ~4 allele may prevent an individual from reaching the ninth decade, and a high level of Lp A-I may help attain this ripe old age, especially in men. A few differences appeared when octogenarian women were compared with postmenopausal or premenopausal women. Indeed, the HDL-C level was more elevated in postmenopausal women and this result was the consequence of a higher level of HDL,-C, the HDL2-C level being similar in both control groups. Further, the Lp A-I and Lp A-I/A-II levels of postmenopausal women were intermediary between premenopausal and octogenarian women. However, if the individual distribution of Lp A-I values was clearly different between premenopausal and octogenarian women, the distribution for octogenarians was close to that of postmenopausal women (Fig 1B). So, this variation of mean levels and distributions of Lp A-I in women as a function of age could signify that a part of the population with a low level of Lp A-I could have a higher frequency of CHD from the fifth until the seventh decade and only that part of the female population having a high level of Lp A-I could survive until the ninth decade. The lower frequency of hyperhpidemia in men could mean that atherogenic factors, such as an elevation of cholesterol and LDL-C, were not counterbalanced by protective factors as is evident in women. Thus, a fraction of the male population is more likely to suffer a heart attack before reaching the ninth decade while the female population is protected longer and develops problems later in life. Indeed, the level of apo A-I was clearly higher in octogenarian women than in octogenarian men, this differcncc being the consequence of a difference in Lp A-I levels, Lp A-I/A-II levels being similar in both groups. It is possible that a part of the difference in the incidence of CHD between sexes could be related to the higher level of Lp A-I in women as compared with men of the same age. while the level of Lp A-I/A-II was similar in both sexes (Table 2). However, it should be noted that the Lp A-I levels of premenopausal women, which tended to be higher than those of control men, were not significantly different. With regard to the interaction of lipoproteins with cells, it is possible that the antiatherogenic potential of Lp A-I particles suggested by this study may reside in their putative role in reverse cholesterol transport. Indeed, it was shown by Fielding and Fieldinga” and more recently by Barbaras et al” that the efflux of sterol from cultured cells was mediated by Lp A-I particles while Lp A-I/A-II had little effect on this efflux. In conclusion, the results obtained in this study of octogenarians are consistent with the protective role of Lp A-I. which had already been perceived through fundamcntal and angiographic studies. Furthermore, the availability of a simple technique for the measurement of Lp A-I particles allows for its routine use in cardiovascular risk factor profile assessment.
1242
LUC ET AL
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in human plasma catalyzing sterol efflux from cultured libroblasts and its relationship to lecithin:cholesterol acyltransferase. Proc Nat] Acad Sci USA 6:3911-3914, 1981 41. Barbaras R. Puchois P, Fruchart JC, et al: Cholesterol efflux from cultured adipose cells is mediated by Lp A-I particles but not by Lp A-I/A-II particles. Biochem Biophys Res Commun 142:6369.1987
Lipoprotein
G. Luc, J.M. Bard, S. Lussier-Cacan,
Particles
D. Bouthillier,
in Octogenarians
H.J. Parra, J.C. Fruchart, and J. Davignon
High-density lipoprotein (HDL) particles exhibit considerable heterogeneity, specifically in apolipoprotein (apo) composition. Thus, apo A-l, the major pcotein of HDL, is present in two types of particles: one species contains both apo A-l and apo A-II (Lp A-l/A-II) while in the other (Lp A-l), apo A-II is absent. We used the hypothesis that octogenarians, who survived periods in life when the incidence of coronary heart disease (CHD) is very high, have several protective factors. We compared HDL-cholesterol (HDL-C), HDL,-cholesterol (HDL,-C), HDL,-cholesterol (HDL,-C), apo A-l, and apo A-II in octogenarians and younger control subjects smoking less than,10 cigarettes/d and not taking drugs known to affect lipid metabolism. Using a new procedure, we also compared the levels of Lp A-l and Lp A-I/A-II. The cholesterol content of total HDL was similar in octogenarian and control (38 + 8 years) men while HDL,-C was higher and HDL,-C, apo A-l, and A-II were lower in octogenarian than in control men. In women, the level of HDL-C and apo A-l was similar in premenopausal and octogenarian subjects but higher in postmenopausal women than in octogenarians, while HDL,-C and apo A-II were similar in the three groups. In contrast, HDL,-C was higher in the two groups of control women (premenopausal and postmenopausal) than in octogenarians. However, Lp A-l was significantly elevated in octogenarian men and women (men: 81 2 14 mg/dL; women: 70 ? 14 mg/dL) by comparison with younger control subjects (men: 48 2 12 mg/dL; premenopausal women: 53 ? 11 mg/dL; postmenopausal women: 83 2 19 mg/dL). On the other hand, Lp A-I/A-II was clearly lower in octogenarians. The distribution of Lp A-l and Lp A-I/A-II values also appeared to be modified by age, a shift of the distribution being observed toward higher values of Lp A-l and lower values of Lp A-I/A-II in octogenarians as compared with younger subjects. This observation could signify that a part of the population with a low level of Lp A-l could have a high frequency of CHD during the sixth or seventh decades. Thus, these results are consistent with the protective function of Lp A-l, which has already been perceived through fundamental and angiographic studies. Copyright
0 1991 by W.B. Saunders Company
E
PIDEMIOLOGIC and clinical studies have determined a number of risk factors for atherosclerosis. Among these, in addition to elevated low-density lipoprotein-cholesterol (LDL-C),’ other alterations have been described, most prominently a reduction of high-density lipoprotein-cholesterol (HDL-C) levels. Reduced HDLcholesterolemia has been confirmed in numerous clinical reports in coronary patients as well as in other noncardiac atherosclerotic syndromes.Z7 Moreover, apolipoprotein (apo) A-I, the major protein of HDL, has been shown in a number of clinical and angiographic studies to be an independent risk factor for coronary heart disease (CHD), especially in patients whose plasma lipoprotein levels are largely norma1.8~‘5 However, it is now established that HDL particles exhibit considerable heterogeneity of size, density, flotation rate, and lipid and apo composition. Also, the cholesterol content of the entire HDL family is a complex function of the composition and particle number of several subclasses.‘6 Specifically, HDL can be separated into several subclasses by different procedures. Evidence of heterogeneity has been provided by the determination of hydrated density, molecular weight, particle size, chemical composition, and hydrodynamic properties.‘7-‘9 Two main fractions, HDL, (density 1.063 to 1.125 g/mL) and HDL, (density 1.125 to 1.21 g/mL) have been isolated by ultracentrifugation and/or
From SERLIA, Institut Pasteur, Lille, France; and the Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. Suppotied in part by the Medical Research Council of Canada (Grant No. MA-5427), the succession JA DeSeve and INSERM. Address reprint requests to G. Luc, MD, SERLIA, Institut Pasteur, rue du Professeur Calmette, 59019 Lille Cedet; France. Copyright 0 1991 by U?B. Saunders Company 0026-0495/91/4012-0002$03.00/O 1238
selective precipitation. Reduction of both HDL subclasses has been associated with CHD but HDL, had the strongest association.zo~z2With regard to the apo composition, it is now recognized that HDL contains two main types of apo A-I-containing particles that may have different metabolic functions and clinical significance.23-26One species contains both apo A-I and A-II (Lp A-I/A-II) while in the other (Lp A-I) apo A-II is absent. We hypothesized that octogenarians, who survived periods in life when CHD mortality is very high, have very few or no risk factors for atherosclerosis and/or several protective factors. Thus, Davignon et al,” postulating an atherogenie role for apo E4, showed a decreased frequency of the ~4 allele (coding for the isoform E4 of the polymorphic apolipoprotein E) in octogenarians. Similarly, one might expect to find a protective profile of lipids or lipoproteins, particularly for HDL particles among octogenarians. MATERIALS
AND METHODS
Subjects The octogenarian subjects in this study were ambulatory and autonomous, had completed their 80th year, were capable of answering a questionnaire, and agreed to submit to a short physical examination and to the drawing of blood. They were recruited from six homes for the aged and one Veterans’ hospital in metropolitan Montreal, Canada. Excluded were individuals with chronic debilitating disease (eg, cancer, Parkinson’s disease), mental illness, alcoholism, hypothyroidism, and diabetes; those taking drugs known to affect lipoprotein metabolism such as hypolipidemic and hypoglycemic drugs, hormones (thyroid hormones, estrogens, progesterone, corticosteroids, or androgenic steroids), betablockers, or thiazides; as well as subjects who had sustained major surgery, weight changes (210%). or a myocardial infarction within the previous 3 months. Smokers defined as subjects smoking more than 10 cigarettes/d were also excluded. Eighty-nine unrelated octogenarians (33 men and 56 women) were included in this report. The mean age was 84 ? 3 (mean + SD) for women and 85 ? 5 years for men. Metabolism,
Vol40, No 12 (December), 1991: pp 1238-1243
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The reference sample consisted of subjects smoking less than 10 cigarettes/d, 48 men and 127 women, who had a desirable blood cholesterol ( ~200 mg/dL) or a borderline-high (200 to 239 mg/dL) blood cholesterol, and a triglycerides concentration less than 150 mg/dL. The mean age of control men was 39 ? 8 years. The control women were divided into two subgroups, one (n = 76) including postmenopausal women not taking estrogenic supplementation with a mean age of 50 -c 3 years, the other (n = 51) including prernenopausal women not taking birth control pills with a mean age of 35 ? 5 years. The postmenopausal status was assessed by typical clinical signs and a hormonal test (follicle-stimulating hormone [FSH] > X0 mUl/mL). Methods Venous blood obtained from subjects was collected into EDTAcontaining tubes (1.5 mg/dL) after an overnight fast. Plasma was immediately separated by centrifugation at 4°C. Sodium azide at a final concentration 0.2% (wtivol) was added to plasma samples. C‘ho’lesterol and triglycerides were determined by automated en2;rimatic procedures.‘X,” HDL-C was determined on fractions separated by combined ultracentrifugation and heparin-manganest precipitation.2” LDL-C was measured by subtracting HDL-C and the cholesterol content in very low-density lipoproteins (VLDL), these last lipoproteins being isolated by sequential uitr,lcentrifugation. from total plasma cholesterol. The cholesterol conlent of HDLz and HDL, was measured according to the method oi Gidez et al.” Apo A-I was determined by electroimmunodiffuslon using the kit Hydragel from Sebia (Issy les Moulineaux, France). Apo A-II was measured by noncompetitive enzymelinked immunoassay.” Lp A-I was determined by electroimmunoassay using a polyclonal antiserum? Briefly, agarose gel plates ((1.9%) containing polyclonal antisera to apo A-I and apo A-II with a high excess of anti-apo A-II. were prepared and mounted onto plastic films. Wells were punched into the gel. The plasma samples were diluted 1:30 in saline and 5 FL of the solution was applied to each well. Electrophoresis was then performed at 150 \ /cm for 3 hours in a buffer containing 0.06 mol/L Tris, 0.07 mol/L glycme, and 0.01 mol/L barbital, pH 8.6. The excess of anti-apo A-11 in the gel, relative to anti-apo A-I, causes a blockage of the Lp A-I A-II particles near the origin, while allowing Lp A-I particles tcl migrate and react with anti-apo A-I. After washing, staining (Coomassie brilliant blue). and destaining, rockets corresponding tc+L.p A-I particles were then visible and measured. The Lp A-I concentration was calculated from a calibration cume using a secondary standard serum. The primary standard was a pure fraction of Lp A-I particles purified by immunoaffinity. The concentration of particles containing both apo A-I and A-II was determined by the difference between total plasma apo A-I and Lp A-I concentrations. Comparisons were made between the octogenarians and the comrol subjects by gender. The statistical significance of the d&.rence in the mean levels of lipids, apo, and lipoprotein particles between groups was evaluated using a nonparametric test ( Mann-Whitnev). RESULTS Octogenarian men and women were clearly different with regard to several parameters. Among plasma lipid levels, total cholesterol, triglycerides, and LDL-C were more elevated in women than in men (Table 1). Among octogenarian women, 19 (34%) were hyperlipidemic inciuding 13 (23%) with hypercholesterolemia alone (cholesterol > 240 mg/dL), 1 (2%) with hypertriglyceridemia (triglycerides > 200 mg/dL), and 5 (9%) with both abnor-
Table 1. Cholesterol, Triglycerides, and LDL-C (mg/dL) in Octogenarian n
Octogenarian men
33
(Mean + SD)
and Control Subjects Cholesterol Triglyceride 186+42t
122%79*
LDL-C
118r36t
Control men
48
189k29
89 2 29
122 -t 28
Octogenarian women
56
226 + 42
130 -t 57
149 t 34
Postmenopausal women
76
203 + 32$
84?33$
124?30$
Premenopausal women
51
173 f 24:
53 rt 19*
107 -t 22*
*P < .05; tP < .Ol compared with octogenarian women. tP
< .Ol compared with octogenarians of corresponding gender
malities. Conversely, only five (15%) among the men were hyperlipidemic, including two with hypercholesterolemia, two with hypertriglyceridemia alone, and one with mixed hyperlipidemia. The average cholesterol, triglyceride, and LDL-C levels were similar in octogenarian men and in controls. In contrast, cholesterol, triglycerides, and LDL-C were significantly higher in octogenarian women when compared with postmenopausal or premenopausal women. Differences also appeared between octogenarian men and women when HDL particle parameters were analyzed (Table 2). Indeed, HDL,-C, apo A-I, apo A-II, and Lp A-I were lower in men than in women and HDL-C, HDL& and Lp A-I/A-II were similar in the two groups. The measurement of cholesterol in HDL and its subfractions HDL-C and HDL,-C gave different results for women and men of different ages (Table 2). Indeed, while HDL-C was not significantly different in octogenarian and in control men, it was higher in postmenopausal women than in octogenarian women. The level of HDL-C was also noted to be higher in premenopausal women than in octogenarian women but this difference was not significant. In men, the partition of HDL-C in HDL,-C and HDL,-C was clearly different in octogenarians than in controls, HDLZ-C being lower and HDL,-C higher in the control group. The relative content of apo A-II was higher in HDL, than in HDL,, and this difference was reflected in the level of apo A-II, which is significantly higher in controls than in octogenarians. Apo A-I was also higher in controls than in octogenarians. In women, HDL,-C was similar in groups of different ages and the higher level of HDL-C in postmenopausal women was due to a higher level of HDL,-C. Moreover, while HDL-C and HDL?-C were not different in octogenarian and premenopausal women, HDL,-C was however higher in this latter group than in octogenarians. The apo A-I levels of the postmenopausal women were more elevated than in octogenarians, but those of premenopausal and octogenarian women were similar. Conversely, apo A-II levels were the same in the octogenarian and in the younger women (Table 2). The concentrations of Lp A-I and Lp A-I/A-II determined from the apo A-I content in each class were clearly different in the octogenarian and control groups (Table 2). The levels of Lp A-I were dramatically higher in octogenarians than in controls for both sexes (P < .01). The levels of Lp A-I/A-II were also different between groups but in the opposite direction, being higher in controls than in octogenarians.
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LUC ET AL
Table 2. HDL-C, HDL,, and HDL,-C, Apo A-l, Apo A-II, Lp A-l, and Lp A-I/A-II HDL-C
HDL,-C
HDL,.C
Octogenarian men
42 + 10
20 5 9
21 +4t
Control men
43k
IO
15 I? 78
28 + 55
Octogenarian women
472
11
22 + a
25 2 4
Postmenopausal women
56 2 119
21 k 6
35 2 65
Premenopausal women
52 + 12
23 + 9
29 + 45
(mg/dL)
(Mean k SD) in Octogenarian
ApoA-I
and Control Subjects
ApoA-Ii
Lp A-l
Lp A-I/A-II
26 f 4’
61 f 14t
55 + 18
127 rr 219
29 2 71
48 + 121
79 A 199
129 t- 23
29 -t 5
70 t 14
59 * 20
139 2 ia*
29 f 5
63 r 199
76 -+ 205
137 t 21
27 i 6
53 + 119
a3 2 201
116 t- 19*
*P < .05; tP < .Ol compared with octogenarian women. SP < .05; §P < .Ol compared with octogenarians of corresponding gender.
The distribution of individual values was studied using percentiles in each octogenarian and control group. Figure 1A and B compare distributions of percentiles of apo A-I, apo A-II, Lp A-I, and Lp A-I/A-II for men and women, respectively. Each point corresponds to a percentile (5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 9.5, or 99th) for the control group (on the abscissa) and for the octogenarian group (on the ordinate). The percentile distributions of apo A-I in octogenarians were slightly shifted toward lower values as compared with controls with the exception of the 99th percentile of octogenarian women, which was higher than that of premenopausal women. Furthermore, the percentiles of apo A-II were similar in octogenarian and control women while the distribution was shifted toward lower values in octogenarian men as compared with control men. The percentile distributions of Lp A-I and Lp A-I/A-II in
6
the octogenarian and control men and in octogenarian and control women were, respectively, clearly shifted toward higher and smaller values relative to the means, with the exception of the 95th percentile of Lp A-I in postmenopausal women, which was higher than in octogenarian women. However, this shift was more important for premenopausal than postmenopausal women. The 99th percentile of Lp A-I was always higher in octogenarians than in controls and a fraction of the octogenarians (men and women) presented high values of Lp A-I or low values of Lp A-I/A-II not found in the control groups. DISCUSSION
In the present study, we have looked at different HDL parameters in octogenarians and in control groups of younger subjects. The cholesterol content in HDL and in
PRE MEKOPAUSAI
POST MENOPAUSAL
coNlR0Ls
Fig 1. Comparisons of distributions of percentiles of apo A-l, apo A-II, Lp A-l, and LP A-l /A-II for men and women.
HIGH-DENSITY
LIPOPROTEIN
IN OCTOGENARIANS
each of its major subfractions, HDLz and HDL,, was measured as well as the major HDL apo, apo A-I and apo A-II. Using a new method, we have also determined the concentration of particles containing apo A-I but not apo A-II (Lp A-I) and by calculation, that of particles containing both apo A-I and A-II (Lp A-I/A-II). A number of studies comparing myocardial infarction survivors with control subjects, using CHD incidence as well as angiographic studies, have demonstrated that high levels of HDL-C, HDL2-C, and apo A-I, and possibly HDL,-C and apo A-II are protective against CHD.*“’ We postulated that these values would be elevated in a population with a great longevity (octogenarians) in comparison with a younger control group. However, our results in women did not support this hypothesis. HDL-C, HDL,-C, and apo A-I were lower in octogenarians than in postmenopausal women and HDL,-C in octogenarian than in premenopausal women while the levels of HDL-C, HDL,-C, apo A-I, and apo A-II wcrc not different in the two latter groups. Moreover, potential CHD risk factors, such as high plasma cholesterol and triglycerides, were more pronounced in octogenarians, particularly in women. In men, results were clearly different showing a high level of HDL2-C in octogenarians while other values were lower (HDL,-C, apo A-I, apo A-II) than those in controls or similar (HDL-C) in both groups. Thus, the high level of HDL2-C in octogenarian men would appear to be a strong protective factor. Relatively little data about HDL in the elderly are available. HDL-C has been reported in octogenarians and nonagenarians by a few authors. While Alvarez et al’” noted lower HDL-C values in octogenarian men in comparison with younger (65 to 80 years) subjects, octogenarians and younger women had similar levels. Heckers et ap5 showed the inverse situation in nonagenarian men in comparison with a group of 70-year-old men, while HDL-C was higher in the 70-year-old women than in the nonagenarians. In contrast, Nicholson et alZh and Rifkind et a1j7 showed a tendency for a decrease of HDL-C after the age of 65 years in men and a stability of these levels in women. The most striking observation of the present investigation was the significant elevation of Lp A-I and the decrease of Lp A-I/A-II levels in male and female octogenarians. However, the modification of HDL particle composition according to age, already suggested by the different levels of HDL,-C and HDL,-C in octogenarian and younger subjects, was confirmed by the important difference in levels of Lp 4-I and Lp A-I/A-II and by the clearly different distribution of Lp A-I and Lp A-I/A-II in octogenarian and younger groups. The decrease of total apo A-I in octogenarians as compared with controls was the reflection of a marked reduction in Lp A-I/A-II not compensated by the increase of Lp A-I. A possible explanation of the levels of apo .4-I-containing particles in the different age groups may be that a fraction of the population with high levels of Lp A-I or low levels of Lp A-I/A-II is protected and survives beyond the age of 80 years. This hypothesis is compatible with the observations made in angiographic studies and a case control study, which have shown a lower level of Lp A-I in subjects with CHD.lJ,“’ Thus, the elevated level of
1241
HDL-C in families described by Glueck et al”’ in which subjects survive much longer than the average population could be the consequence of a very high level of Lp A-I. In addition to the putative role of Lp A-I as an antiatherogenic particle, Davignon et al” showed that a decreased frequency of the ~4 allele may prevent an individual from reaching the ninth decade, and a high level of Lp A-I may help attain this ripe old age, especially in men. A few differences appeared when octogenarian women were compared with postmenopausal or premenopausal women. Indeed, the HDL-C level was more elevated in postmenopausal women and this result was the consequence of a higher level of HDL,-C, the HDL2-C level being similar in both control groups. Further, the Lp A-I and Lp A-I/A-II levels of postmenopausal women were intermediary between premenopausal and octogenarian women. However, if the individual distribution of Lp A-I values was clearly different between premenopausal and octogenarian women, the distribution for octogenarians was close to that of postmenopausal women (Fig 1B). So, this variation of mean levels and distributions of Lp A-I in women as a function of age could signify that a part of the population with a low level of Lp A-I could have a higher frequency of CHD from the fifth until the seventh decade and only that part of the female population having a high level of Lp A-I could survive until the ninth decade. The lower frequency of hyperhpidemia in men could mean that atherogenic factors, such as an elevation of cholesterol and LDL-C, were not counterbalanced by protective factors as is evident in women. Thus, a fraction of the male population is more likely to suffer a heart attack before reaching the ninth decade while the female population is protected longer and develops problems later in life. Indeed, the level of apo A-I was clearly higher in octogenarian women than in octogenarian men, this differcncc being the consequence of a difference in Lp A-I levels, Lp A-I/A-II levels being similar in both groups. It is possible that a part of the difference in the incidence of CHD between sexes could be related to the higher level of Lp A-I in women as compared with men of the same age. while the level of Lp A-I/A-II was similar in both sexes (Table 2). However, it should be noted that the Lp A-I levels of premenopausal women, which tended to be higher than those of control men, were not significantly different. With regard to the interaction of lipoproteins with cells, it is possible that the antiatherogenic potential of Lp A-I particles suggested by this study may reside in their putative role in reverse cholesterol transport. Indeed, it was shown by Fielding and Fieldinga” and more recently by Barbaras et al” that the efflux of sterol from cultured cells was mediated by Lp A-I particles while Lp A-I/A-II had little effect on this efflux. In conclusion, the results obtained in this study of octogenarians are consistent with the protective role of Lp A-I. which had already been perceived through fundamcntal and angiographic studies. Furthermore, the availability of a simple technique for the measurement of Lp A-I particles allows for its routine use in cardiovascular risk factor profile assessment.
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