Atherosclerosis, 25 (1976) 131-136 0 Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands


SOAIRA MENDOZA, ROBERT F. LUTMER, CHARLES J. GLUECK, CHENG-YU CHEN, PAULA M. STEINER, RONALD W. FALLAT and MOTI L. KASHYAP Department of Medicine, Geneml Clinical Research Center and Lipid Research Center, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267 (U.S.A.) (Received 2nd March, 1976) (Accepted 1st April, 1976)

Summary The content and percent composition of cholesterol, triglyceride, phospholipids, and total proteins in HDL-2 and HDL-3 were quantitated in 5 women with familial hyperalphalipoproteinemia to determine if there are any distinctive characteristics of the high density lipoproteins in this heritable disorder. The 5 women with familial hyperalphalipoproteinemia (FHA) were compared to 4 normal women, with the groups being comparable in regards to age (40 + 3 and 37 * 5 years), total plasma cholesterol (202 f 9 and 188 + 16 mg/lOO ml), triglyceride (75 f 12 and 95 f 19), and differing in levels of high density lipoprotein cholesterol (C-HDL, 84 + 6 and 61 + 3 mg/lOO ml) respectively. Total cholesterol in the HDL-2 and HDL-3 fractions obtained by ultracentrifugation were 43.2 f 3.3 and 33.8 + 4.1 in FHA subjects, higher than total cholesterol in HDL-2 and HDL-3 in normals, 25.8 f 6.2 and 21.5 f 1.3 mg/lOO ml, P < 0.025. Total concentration of HDL-3 was higher in FHA than in normal subjects, respectively 222.4 f 22.6 and 149 + 7.2 mg/lOO ml, P< 0.025. Lipid-protein percent composition of HDL-2 and HDL-3 in FHA and normals was nearly identical, and polyacrylamide gel electrophoresis revealed no qualitative differences in band migration and appearance of the HDL-2 and HDL-3 fractions in normal and FHA subjects. In these women with FHA, there appears to be an increased concentration of normal HDL-2 and HDL-3. Keywords:







Supported in part by the General Clinical Research Center, RR 00068-13. A portion of this work was done during Dr. Glueck’s tenure as an Established Investigator of the Amencan Heart Association, 1971-1976. Address for reprints: Dr. Charles J. Glueck, General Clinical Research Center. Cincinnati General Hospital, 234 Goodman Street, C2-3 Cincinnati, Ohio 45267, U.S.A.


Introduction Familial hyperalphalipoproteinemia (FHA) is a newly described heritable lipoprotein disorder which is characterized by elevated alphalipoprotein cholesterol (C-HDL) [ 1,2]. In kindreds with familial hyperalphalipoproteinemia (FHA), longevity analysis revealed a significant prolongation of life expectancy, and an apparent rarity of premature cardiac events [ 21. Hyperalphalipoproteinemia may also be secondary to varied hepatic disorders [3], alcoholism [4,5], exogenous estrogen supplementation [6], diabetes mellitus [7] and environmental exposure to chlorinated hydrocarbon pesticides [ 81. Two investigators have examined the lipid composition of high density lipoproteins (HDL) in subjects having familial hyperalphalipoproteinemia [9,10]. Naito et al. [9] separated the HDL fraction in the ultracentrifuge (d 1.063-1.21) in 2 members of kindred with FHA, and reported that the lipid percent composition was similar to normal subjects. Avogaro et al. [lo] found a distinctively elevated percent composition of cholesterol in the lipid fraction only of ultracentrifugally recovered HDL, but did not carry out quantitative studies on the protein moiety. Studies on the subfractions of HDL were not performed by these investigators. High density lipoproteins are heterogeneous; various physiologic and pathologic states are characterized by a selective elevation of HDL-2 (d 1.063-1.125) and/or HDL-3 (d 1.125-1.210) [11,12]. Other studies indicate that HDL-2 is physiologically more important than HDL-3 in that it contains the peptide (apolipoprotein CII) which activates the lipoprotein lipase triglyceride interaction leading to hydrolysis and disposal of circulating triglyceride [ 131. The present study was conducted to quantitate the content and percent composition of cholesterol, triglyceride, phospholipids, and total proteins in HDL-2 and HDL-3 in 5 women with FHA [2] to determine if there are any distinctive characteristics of HDL in this heritable disorder. These women had been initially identified by elevated C-HDL [2] as determined by the heparinMnClz precipitation technique of Burstein [ 141. Materials and Methods Blood samples were obtained after a 12-h fast from 5 women with well documented FHA [2] and 4 healthy normal women. Cholesterol, triglyceride, and C-HDL levels, as well as age are summarized for these 9 subjects in Table 1. All of the FHA and normal women were pre-menopausal, and were not receiving contraceptive steroids or estrogen supplementation. Four FHA women (Nos. l-4) were siblings from a single kindred [ 11, (Kindred No. 6) [2], and one came from a separate family [2]. All of the women were on an ad libitum diet, without recent weight loss. None had diabetes mellitus, excessive alcohol intake, hepatic disorders, or work exposure to chlorinated hydrocarbons. The blood was collected into tubes containing EDTA (1 mg/ml) which were immediately centrifuged at 4”C. Ultracentrifugal separation of HDL fractions was instituted within 2-3 h of obtaining the fasting samples. Total plasma cholesterol [ 151 and triglyceride [ 161 were measured by modification of Auto Analyzer methodology [ 171. Alphalipoprotein cholesterol, C-HDL w,as initially


Total choIestero1


Familial hyperalphalipoproteinemia 1 35 190 2 41 236 3 43 201 4 48 iai 5 33 202

ST? SE NOt7Ud.S 1 2 3 4



40 + 3

202 f 9

28 30 41 50

150 172 210 219



188 f 16


19 102 101 51 44 75+


73 93 71


104 12

51 74 119 134 95 f 19


54 69 64 51 61 f 3

a C-HDL quantitated by the heparin-MnC12 precipitation method 114.171. b P < 0.01, comparison of FHA and normal women.

quantitated by the heparin-MnClz precipitation method [14,17]. Plasma lipoprotein electrophoresis of whole plasma and the supemate of the heparin MnC& precipitation was carried out using agarose-ar [18]. HDL-2 and HDL-3 were isolated from plasma by preparative ultracentrifugation according to the method of Have1 et al. [ 191. A L5-50 Beckman Ultracentrifuge and a 40.3 Beckman fixed angle rotor were used. All densities during the preparative ultracentrifugation were verified and checked by pyknometry. After obtaining the HDL-2 and HDL-3 fractions, both were layered with solutions of NaCl/KBr mixtures having a density of 1.125 and 1.210 respectively, and ultracentrifuged. The cholesterol contents of the HDL-2, HDL-3, in&anate after the second centrifugation of HDL-2, and infranate after the second centrifugation of HDL-3 were totalled. This sum was divided by the amount of C-HDL obtained by the heparin-MnCl* precipitation method [14,17] to obtain an arbitrary estimate of the “recovery” of C-HDL. The supernate obtained after the second ultracentrifugation of HDL-2 and HDL-3 was then dialyzed exhaustively against 0.15 M NaCl in 0.01 M EDTA in distilled water, at pH 7. These fractions were then subjected to the following analyses. Protein concentration was measured according to the method of Lowry et al. [20]; phospholipids according to Sunderman [21], and cholesterol and triglyceride8 following the Lipid Research Clinics Manual [17]. Polyacrylamide gel electrophoreses of constituent apolipoproteins of HDL-2 and HDL-3 were performed using tetramethylurea for delipidation according to the m&hod of Kane [ 221. Statistical comparisons between normal and FHA values were carried out using Student’s t-test [23].


Results As summarized in Table 1, the normal women and women with FHA had normal levels of cholesterol and triglyceride [ 3] ; C-HDL levels were within the normal range for the 4 normals, and were elevated in the 5 women with FHA [ 21. Normal and FHA women had comparable ages. Table 2 summarizes the concentration of the components of HDL-2 and HDL-3 in normal and FHA women. In the HDL-2 fraction, FHA women had nearly twice as much cholesterol, with increments in proteins, triglycerides, and phospholipids, as compared to normals. In comparison of HDL-3 in FHA and normal subjects, FHA subjects had more protein, cholesterol, and phospholipids, and total HDL-3 concentration was also notably higher than in normals (Table 2). The percent composition of HDL-2 and HDL-3 in FHA and normal subjects is summarized in Table 3. The percent composition of proteins, total cholester-



(N) Group





HDL-2 (d 1.063-1.125) (4) Normal a

Proteins(mg/lOO ml)

(5) FHA a

(4) Normal a

46.7 + 11.2 26.8 f 6.2

62.6 + 6.1 43.2 + 3.3 ’

93.8 f 5.3 21.5 f 1.3

Triglycerides (mg/lOO ml) Phospholipids (mg/lOO ml)

7.6 f 1.2 26.0 ?r 6.1

9.6 f 36.4 f

6.8 + 0.6 26.7 f 1.6


f 24.3



HDL-3 (d 1.125-1.210)

Total cholesterol (mg/lOO ml)

Total (mg/lOO ml)


1.6 2.6

+ 10.4


+ 7.2

(5) FHA a 139.1 f 14.7 b 33.8 f 4.1 ’ 8.5+ 41.0 f 222.4

1.5 3.4b

f 22.6 ’

a Duplicate analyses in each subject. b P < 0.01. = P < 0.025. d = density.



(N) Group

Roteti (I) T?+I chq\esterol (%) Mplycerides (sb) PhosphoIipids (96) -I1 : _



(d 1.063-1.125)



HDL-3 (d 1.125-1.210)

(4) Normal a

(5) FHA a

(4) Normal a

(5) FHA a

43.2 24.4 7.7 24.7 N.S.

41.1 28.5 6.3 24.0 N.S.

63.0 14.5 4.6 18.0 N.S.

62.6 16.1 3.8 18.6 N.S.

+ f f +

2.0 1.9 0.6 0.9

f 1.4 + 1.6 f 0.7 ?r 1.2

a Duplicate analyses in each subject. N.2. = no &&cant difference in comparison of normal to FHA. d = density.

f f f +

1.3 0.5 0.2 1.1

f 0.6 f 0.6 +‘D.4 f 0.7


01, triglycerides, and phospholipids in HDL-2 and HDL-3 was nearly the same for FHA and normal subjects. Total HDL cholesterol by ultracentrifugation, divided by CHDL obtained by heparin-MnC& [14,17] provided an arbitrary assessment of “recovery”. Mean (* SD) recovery for the 5 FHA subjects was 91.3 + 7.9, for the 4 normals, 81 + 9.4%. There was no significant difference in the recovery for the two groups, P > 0.1, t = 1.8. Polyacrylamide gel electrophoresis revealed no qualitative differences in band migration and appearance of the HDL-2 and HDL-3 fractions in normal and FHA subjects. Lipoprotein electrophoresis on agarose-agar of the whole plasma in FHA revealed normal electrophoretic patterns except for densely stained alphalipoprotein bands. Electrophoresis of the supernate after heparinMnClz precipitation of all lipoproteins except HDL failed to reveal any beta, pre-beta, or LP(a) lipoproteins which had been incompletely precipitated. Discussion Examination of HDL-2 and HDL-3 in the 5 subjects with familial hyperalphalipoproteinemia revealed increases in the total concentration of HDL-3, increments in cholesterol in HDL-2 and HDL-3, and increments in protein and phospholipid in HDL-3. These findings were consistent with the increase in C-HDL as determined by precipitation with heparin-MnCl, [2,14], and in part validate the identification of FHA by virtue of elevated C-HDL. In FHA the percent composition and qualitative apolipoprotein electrophoretograms were entirely comparable to those in normals, indicating that FHii in these 5 women is characterized by an increased concentration of HDL-2 and HDL-3 of normal composition. Our results for composition of HDL-2 and HDL-3 in normal subjects corresponded very closely with data from other laboratories [24,25], so that the increased concentrations of cholesterol in HDL-2 and HDL-3 observed in the FHA subjects appear to be generalizable in contrast to normals. The finding of an increased concentration of normal HDL-2 and HDL-3 appears to be in accord with the observations of Naito et al. [9] who found no significant alteration in the lipid composition of total HDL in 2 FHA patients (HDL-2 and HDL-3 were not specifically isolated). Our findings are, however, in contrast with those of Avogaro et al. [lo] who reported that the HDL lipid moeity was disproportionately rich in cholesterol in 4 FHA subjects. FHA may be a heterogeneous condition [1,2,9,10] in which subjects may have a quantitative increase in total cholesterol of HDL-2 and HDL-3 and/or a qualitative abnormality in the HDL. Further work in several other kindreds, and in affected male kindred members, is necessary to ascertain the extent of the heterogeneity. Our preliminary observations of an elevated percent composition of triglyceride (12.6%) in HDL-2 from 6 women with FHA [26] were not confirmed in this study, further suggesting possible heterogeneity in HDL lipoproteins in FHA. FHA patients appear to have a family history of longevity, and a decreased rate of ischemic heart disease [2]. If this observation is confirmed with time, it will be important to establish whether any associations can be made between the apparent longevity and certain subgroups of patients with FHA.


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Composition of HDL-2 and HDL-3 in familial hyperalphalipoproteinemia.

131 Atherosclerosis, 25 (1976) 131-136 0 Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands COMPOSITION OF HDL-2 AND HD...
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