High density lipoprotein distribution

161

Atherosclerosis, 29 (1978) 161-179 0 Elsevier/North-Holland Scientific

HIGH DENSITY Resolution Population

LIPOPROTEIN

and Determination Sample

D.W. ANDERSON

I, A.V.

Dormer Laboratory, Cal. 94720 (U.S.A.) (Received (Revised, (Accepted

Publishers,

DISTRIBUTION of Three Major Components

NICHOLS

Lawrence

Ltd.

*, S.S. PAN and F.T. LINDGREN

Berkeley

25 April, 1977) received 21 November, 1 December, 1977)

in a Normal

Laboratory,

University

of California,

Berkeley,

1977)

Summary In an earlier report we identified at least 3 major components within the serum total HDL distribution of normal subjects. In the present study, the serum concentrations of these components, which we designate HDLzb, d 1.063-1.100 g/ml; HDL2,, d 1.100-1.125 g/ml; HDLa, d 1.125-1.200 g/ml are determined for 160 clinically-screened subjects from a normal population sample. This determination involves graphically fitting reference schlieren patterns for each of the three components to the subjects’ total HDL schlieren patterns with the aid of a computer. The mean error of fitting all three reference patterns was low (9 of 8% (SD) of total area). Mean serum levels of HDLzb and HDLs, were significantly higher in females than in males for each of the four age decades surveyed (27-36, 37-46, 47-56 and 5766 years). Mean HDL3 serum levels were slightly higher in males than in females for the population as a whole. Regression of HDL2,,, HDLz,, and HDLa levels on each other and on total HDL serum concentration revealed 2 sex-independent trends: (1) HDL3 levels are negatively correlated (r = 0.315) with HDLzb levels, uncorrelated with HDLz, levels, and are relatively constant (mean level 158 f 30 mg/dl (SD)); (2) HDLZb and HDL2, levels are highly correlated (r = 0.725) and display a progressive build up with increasing total HDL levels. Since HDLzb This from

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from

1 Present tutes * To

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Training

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all correspondence of California,

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to:

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94720

Dormer

(U.S.A.)

162

and HDLz, levels account in major part for the differences in total HDL levels observed, we propose that they may be the critical HDL components determining the reported inverse correlation of HDL cholesterol with coronary heart disease. Key words:

HDL distribution Three component

- GDL,,, analysis -

HDLJ - Normal population HDL,,, Ultracentrifugal schlieren pattern

sample

-

Introduction Analytic ultracentrifugation has been extensively employed in the determination of serum concentrations of human serum total high density lipoproteins (HDL) [l-4]. This procedure usually provides a schlieren pattern of the total HDL which can be further analysed for serum levels of HDL components. Two major components have been defined in terms of their associated flotation rate intervals, the Fy.*,, 3.5-9.0 * and Fy.20 O-3.5 components. These correspond to but are not exactly equivalent to the HDL components, HDL2 and HDL3, which have generally been defined in terms of the density intervals used in their ultracentrifugal isolation, d 1.063-1.125 g/ml and d 1.125-1.200 g/ml. Because of these differing definitions, HDL component analysis of ultracentrifugal schlieren patterns as usually performed results in an overestimation (HDL3) and underestimation (HDL,) of serum concentrations of the densitydefined components. Based on equilibrium density gradient ultracentrifugal studies [ 51, we have reported the presence of at least 3 major components in the HDL distribution which fall within the following size ranges: I (10.8-12.0 nm in diameter), II (9.7-10.7 nm in diameter), and III (8.5-9.6 nm in diameter) **. Since the particle hydrated density ranges for HDL from size range I (d 1.063-1.100 g/ml) and from size range II (d 1.100-1.125 g/ml) comprise the HDL, particle density range (d 1.063-1.125 g/ml), these two components are designated in this report as HDL components HDLzb (HDL from size range I) and HDLz, (HDL from size range II). The particle hydrated density range of HDL from size range III (d 1.125-1.200 g/ml) is identical to that of HDL3 given above. The values of the molecular properties (peak Fy.,, rate, particle diameter, and molecular weight) for the HDLsb, HDLz,, and HDLs components were found to be independent of sex and varied little in the HDL from of these three components eight normal subjects [ 51. The serum concentrations did vary between males and females, however. A preliminary comparison of male and female schlieren patterns indicated that the percent total HDL associated with the HDL,, component was greatly increased in female HDL while that of the HDL, component was increased in male HDL. This report describes a procedure for reconstructing the standard total HDL ultracentrifugal schlieren pattern from reference patterns of the three compo*

E-0 is the HDL flotation rate, expressed in svedbergs (10-l 3 cm/s/dyne/d), fully corrected for conce%r~tion dependence and to standard conditions of 0.195 m NaCl and 2.762 m NaBr at 26’C (d2b = 1.200 g/ml). ** Size ranges were determined by electrophoresis of total HDL on porosity gradient polyacrylamide gels calibrated for Stokes’ diameter as a function of migration distance.

163

nents noted above. This procedure is applied and evaluated in the determination of HDLsi,, HDL2,, and HDLs levels within the total HDL schlieren patterns of 160 normal individuals surveyed in a study reported elsewhere [6]. From these results, male and female serum levels of the three components are compared. In addition, the relationship between the percent of total HDL distributed among HDLz,,, HDLs,, and HDLs to the total HDL serum concentrittion is described. Materials and Methods (1) The three component approach to HDL schlieren pattern analysis Analysis of ultracentrifugal patterns of lipoproteins in normal sera have generally assumed a continuum of lipoproteins with respect to flotation rate [ 71. In such analyses, the sum of the concentrations of the lipoprotein continuum between 2 arbitrary flotation rate limits is determined. For example, the total HDL schlieren pattern area between Fy.,,, rates 9.0 and 3.5 was set by Lindgren [l] as representative of the concentration of the HDL;? density class (d 1.0631.125 g/ml) while the area between Fy.,, rates 3.5 and 0 was considered as representative of the concentration of the HDLJ density class (d 1.125-1.200 g/ml) (Fig. la). As stated above, we have reported evidence for the existence in the HDL distribution of three major HDL components, distinct in particle size, hydrated density, and peak Fy.,, rate. We have found that standard total HDL

b

Fig. la. Representation

of atotal

HDL schlieren

pattern

and indication

of Fy,,,

3.5-9.0

and Fy.20

o-3.5

components.

Fig. lb. Representation of a total HDL schlieren corresponding F(: .* o rates, [Fi], i = 1, 2, _.., 173.

pattern

as an array

of heights.

[Hi],

i = 1. 2, . . . . 1’73 at

164

ultracentrifugal schlieren patterns can be analysed as a summation of 3 separate HDLat,, HDLs,, and HDLs reference patterns after adjustment of each to an appropriate concentration. In particular, we have analysed the total HDL corrected schlieren patterns of 160 subjects, ages 27-66 years, surveyed in a normal population study (Modesto Study) conducted in Modesto, California by Lindgren et al. [6]. In this study a random subpopulation of 20 subjects in each decade of each sex was selected from 774 clinically screened normal volunteers, All schlieren patterns in this report were obtained by procedures [8] which corrected for the concentration dependence of Fy.*,, rate and the Johnston+)gston effect. Final computer-derived patterns were plotted in terms of 173 heights, represented by the array of heights [Hi], i = 1, 2, . . . . 173, at corresponding values of Fy_,, rates, represented by the array of Fy.*,, rates of the corrected schlieren [Fi], i = 1, 2, . . . . 173 (Fig. lb). This representation pattern in terms of 173 heights along the Fy,,,, rate scale is used in this report to describe the operations involved in the three component analysis. A description of the reference HDLsb, HDL2,, and HDLs schlieren patterns and their application to the three component analysis of the total HDL pattern follows. (2) Reference HDL,,, HDL2,, and HDL3 schlieren patterns Reference schlieren patterns of the HDLsb, HDL2,, and HDLs components are used in the present analysis to reconstruct the standard total HDL pattern. The reference patterns are the mean corrected schlieren patterns obtained by separately averaging the HDLzb, HDL2,, and HDLs patterns from four normolipemic males and four normolipemic females (age 25-34 years) *, and are shown in Fig. 2. The reference patterns in Fig. 2 were used to obtain the average serum concentrations of the three components from the four males and four females. The molecular properties and compositions of the three HDL components are given in Table 1. F” 1.20

9.0 0.0

.

PEAK SERUM

fi

7.0 6.0 5.0

.

.

* 1

F,fzo RATE:

4.0

3.0

2.0

. 1 1

1.0

5.37

CONC : 75 mg/dl

HD’-2, PEAK SERUM

FGo

RATE:

3.15

CONC.:

112 mg/dl

F,I;o RATE:

1.56

CoNc.:

154mgldl

HDL, PEAK SERUM

Fig. 2. HDL2b. HDL2,. ing component patterns

and HDL3 reference from 4 normolipemic

schlieren patterns. Each pattern is the mean of correspondmales and 4 normolipemic females.

* Corrected schlieren patterns of the three HDL components fractions of the eight subjects as previously described [51.

were obtained

from density

gradient

sub-

1

for pre-

HDL

the

samples.

x

Fy.20

Fy_,,

Peak

rate

rate

(dadtons)

InoIecuIar

weight

4

isolation

Peak

lo-

fugal

(g/ml)

5.37

r 0.30

pattern

* 0.17

5.34

Reference

i

4.10

0.20

k 0.5

5.50

4.29 r 0.25

? 0.21

3.15

r 0.06

pattern

r 0.04

t 0.10

Reference

3.14

2.62

+ 0.3 2.64 3.16

? 0.10 r 0.05

? 0.11

* 0.09

* 0.3

1.56

? 0.13

Reference

1.56

1.77

9.2

10.1

range

10.2

1.145

Size

range.

11.1

size

11.0

+ 0.3

in each

calculated

from

pattern

the

1.56

1.17

9.3

* 0.07

+ 0.09

? 0.2

The

Svedberg f 51.

Female

gels

III (HDL3)

polyacrylamide

(do) g (nm)

II (HDL2&

of HDL

were

gradient

weights

on porosity

Molecular

1.125-1.200

range

patterns

its migration

Malt?

1.110

Size

schlieren

from

Female

r 0.6

the corrected

as determined

1.100-1.125

over

particle

III 4 female

Male

1 (HDLZb)

averaging

of

and

I, II, AND samples

Female

range

by

value

4 male

RANGES

1.063-1.100

1.090

Size

calculated

diameter

for

SIZE

errors

FROM

standard

Male

were

Stokes’

OF

and

Sex

ultracentri-

range

parative

Density

position

(g/ml)

banding

density

Mean

the

are means

PROPERTIES

patterns

represents

(dO)gg

the reference

presented

Values

MOLECULAR

TABLE

Fy.,,,

equation peak

rates

151

of

and

:. ,f’ Go

6.0 2.0 4.0 3.0 20

9,O 6.0 70

------

TOTAL

-.-.-.-.-

DIFFERENCE

-

REFERENCE

---~~-~~--~~~~~ FITTED

I.0

HOL PATTERN

HOL

HOLs

FITTING ERROR AREA

HDLg

FITTING ERROR AREA

PATTERN HDL COMPONENT COMPONENT

m

HDLzb FITTING ERROR AREA

3. Three component analysis scheme. Step 1: (a) The corrected schlieren pattern of total HDL is divided into 173 values of the ordinate height. This pattern can then be represented by the array of heights [HiIT paired with corresponding values of the Fy 2. rate scale, [Fi] , i = 1, 2, . . . . 173 (see Fig. 3a). (b) The reference corrected schlieren patterns of the thr& HDL components are likewise divided into 173 values of the ordinate height. These patterns can values then be represented by the arrays of heights: [hi]2b, [hi]2a. and [hi] 3 paired with corresponding Fig.

of the F”, 2. rate scale, [Fi], i = 1, 2. . . . . 173. NOTE: The corrected schlieren patterns in both (a) and(b) are obtained by the method Step2: The fitting factor, f3, for the component HDL3 is computed (Fig. 3b):

of Ewing et al. [I].

($(Hj)T/(hj)3) f3

=

]=a

(b-a++) where b = 27, a = 12 as seen from Fig. 3b. to yield the Step 3: The reference schlieren pattern of HDL 3, [hi]3, is fitted to the total Pattern, [HiIT. fitted HDL3 pattern, fg . [hi]3 (Fig. 3b): f3 . [hi]3 = f3 . [hi]3 for all i = 1, 2. . . . . 173. Step 4: The fitted HDL3 pattern. f3 . [hi]3. is subtracted from the total HDL pattern, [HiIT, to yield a difference pattern. [HiIT - f3 . [hi]3 (Fig. 3~): [HiIT - f3 . [hi]3 = (Hi)T - f3 . (hi)3 for all i = 1, 2, . . . . 173. Step 5: The fitting factor. fp,, for the component HDL2, is computed: (5

f

(Hj)T-f3

2a=

‘(hj)l)

(hj)2a

j=a (b-a+

1)

where b = 60. a = 28 as seen from Fig. 3c. Step 6: The reference schlieren pattern of HDL2,, f3[hil3 1, 2, . . . . Step 7: [hi13 to

[hi]2a,

is fitted

to the difference

Pattern

[HiIT-

to yield the fitted HDLz~ pattern, fpa . thil2a (Fig. 3~): fpa. [hi]2a= fpa .(hi)2a for all i = 173. fp a . [hi]2a is subtracted The fitted HDLz~ pattern. from the difference pattern, [HiIT - f3 yield a second difference pattern, [HiIT - 13 . [hi]3 - f2a. [hi]2a (Fig. 3d).

167

(3) Quantitative analysis of total HDL schlieren patterns from the Modesto Study in terms of three components The design of this analysis is to successively fit the contours of each of the three reference patterns to that of the total HDL pattern. The successive fitting procedure is based on the fact that in a system of more than one component undergoing ultracentrifugal flotation, the contour of the corrected schlieren pattern will be the sum of the contours of the component corrected patterns. In Fig. 3 a description of the analysis scheme is given. The first step is to fit the HDL3 reference pattern to the total HDL pattern. This is done by determining the factor f3 (see Fig. 3, Legend, Step 2), by which every HDL3 reference pattern height (hi)39 in the flotation rate interval Fy.,, 0.50-1.30 *, is multiplied to equal the corresponding total HDL pattern height (Hi)T in that interval. This particular Fy.,, rate interval (0.50-1.30) is chosen in order to fit the HDL3 reference pattern to the interval of the total HDL pattern where there are no contributions from the HDLzb or HDL2, reference patterns (both HDLzb and HDL2, have higher flotation rates). Each of the 173 heights of the HDL3 reference pattern are then multiplied by f3 to generate an HDL3 pattern fitted to the total HDL pattern. This fitted pattern is designated by the expression f3 . [hi], (see Fig. 3b). Since both the initial total HDL pattern and the reference HDL component patterns have been corrected to standard conditions, as described above, their contours are not differentially distorted as a function of lipoprotein concentration. Thus the multiplication of all 173 heights of a reference HDL component pattern by a single factor to adjust its area to represent a higher or lower serum concentration is justified. The fitted HDL3 pattern, f3 - [hiIs, is then subtracted from the total HDL pattern, designated as the array [Hi]r, to yield a difference pattern, ([Hi]r f3 * [hiIs) (Fig. 3~). In practice, each of the heights of the fitted HDL3 pattern is usually not exactly equal to heights at corresponding Fy.,, rate values of the initial total HDL pattern in the Fy.,,, O-1.30 interval. Hence, subtraction of the fitted HDL3 pattern from the total HDL pattern yields a positive or negative difference which can be used as a measure of the error of fit (Fig. 3~). The reference HDLz, pattern, [hi]aa, is then fitted to the difference pattern, ([HiIT - f3 * [hiIs) between the flotation rates Fy.,, 1.35-3.00 ** in the manner described above (see Fig. 3, Legend, Steps 5 and 6). The Fy.,, rate interval (1.35-3.00) is chosen in order to fit the HDLza reference pattern to the interval of the difference pattern where there are no contributions from the HDLSi, 2.

Step 6: The serum fitted patterns as:

concentrations

of HDLZb.

and FI is as follows:

HDL2,.

and HDL3

Fy.Z,,

are calculated

HDL2b

= A.

AF . C i=60

Fy,20

from

their respective

(Hi)T-

f3

. (hi)3

-

fpa.

(hi)za,

cont.

HDLza

= A . AF . C i=l

173 and cont.

HDL3

= A

’AF

where A = high density

0.50 = F, *,

173

173 Cont.

0 = F,,

C

i=l

lipoprotein

f3

. (hi)3 concentrationjschlieren

pattern

area and AF = 0.05 cm.

f2a.

(hi)za*

168

reference pattern (HDLa,, has higher flotation rates and the fitted HDLs pattern has already been subtracted). This yields the fitted HDLza pattern designated by the array faa * [hi]aa. The fitted HDLa, pattern is then subtracted from the difference pattern ([Hi]r - f3 . [hiIs) to yield a second difference * [hi]aa) (Fig. 3d). As in the fitting of the pattern, ([Hi].-ff,. [hi],-fa, HDL, pattern, the fitted HDLa, pattern heights will not be exactly equal to the corresponding heights of the difference pattern ([Hi]r - f3 . [hi]J) in the 1.35-3.00 interval and a fitting error (positive or negative area) can be FY.20 determined from the differences. For the case given in Fig. 3d, overfitting occurred and is seen as a negative area in the interval Fy.,, 1.35-3.00. The reference HDLzb pattern, [h,]1 2b, can now be fitted to the second difference pattern ([Hi]r - fs * [hi]3 - faa * [hi]za) between the flotation rates F 1.20 3.05-5.00 * in the same manner as described above. This yields the fitted HDLzb pattern, designated by the array fsb * [hilab (Fig. 3d). Subtracting the fitted HDLzb pattern from the second difference pattern then gives the fitting error for the interval F,.,, 3.05-9.00. At this point, the analysis has been completed and the serum concentrations of each component (HDL2b, and HDL,) are given by the values of their fitted schlieren patterns. In HDLa,, view of the near identity in most samples between the second difference pat-

a

9.0

A.C?

CONC.

HDL,,

CONC

176

HDL,,

CONC.

207

HDL,

CONC.

88

TOTAL

6.0

20

60

F’1.20

5.0

4.0

3.0

2.0

1.0

0

16 n

HDL CONC. 0

PEAK

Fl.20

RATE

b A.C?

CONC.

HDL,,

CONC.

80 mgib., 267

” II

HDL,,

CONC.

I42

HDL,

CONC

91

HDL

CONC.

TOTAL PEAK

” 580

F,T2,, RATE

mg/dl

I

5.49 ‘_.’

*A.C.

= ADDITIONAL

(HDL)

COMPONENTS

Fig. 4a. Mean schlieren pattern (2 female total HDL concentrations greater than 475 Fig. 4b. Total HDL schlieren pattern from tional components (A.C.) not fitted by the

*

3.05 FP.*O

= Fe

],

Fy.20

5.00

= Fg7.

and 2 male cases) representative of Modesto Study cases with mg/dl. subject with highest concentration in Modesto Study of addireference HDL2b pattern.

169

tern,([&IT -f3

-faa.

* [hi13 [hil2a), and the adjusted HDLzb pattern, f sb . [hilab (Fig. 3d), the area under the second difference pattern can be usually taken to represent the serum concentration of the HDL2,, component without detailed fitting of the reference HDL 2b component pattern. However, in czxs where the HDL2b serum levels are approximately 150 mg/dl or greater, the reference HDLlb pattern consistently underfits the second difference pattern (Fig. 4). The positive error area generated increases with increasing HDL2b concentration and will be discussed in the Results section. Results (1) Determination of fitting error in three component analysis for serum levels and HDL, in the Modesto Study of HDL,b, HDL,,, The effectiveness of the three component analysis in describing the total HDL pattern was evaluated in terms of its ability to account for the total area of that pattern. As described in Materials and Methods, Part (3), fitting errors are encountered during adjustment of each of the reference component patterns to the total HDL pattern. The mean value of the error in fitting the HDL3 reference pattern was 3.5 + 1.2% (SD) when expressed as percent of the total HDL serum concentration. In virtually every case, the error was due to an overfitting * of the total HDL pattern contour by the HDL3 reference pattern contour (Fig. 3d). The fitting errors in the determination of HDLzb and HDL2, represent both underfitting and overfitting of the total HDL pattern contour by their reference patterns and had mean values of 3.2 ? 6.1% (SD) and 2.8 + 5.2% (SD), respectively. Taken together these errors can be used as a measure of the effectiveness of the three component analysis to quantitatively describe the total HDL pattern. For the 160 patterns analysed, the mean value of the total error in fitting the HDL2b, HDL2a, and HDLs reference patterns was 9.5 ? 8.5% (SD) when expressed as percent of the total HDL serum concentration. The value of the inherent uncertainty of measurement of the total HDL concentration is +5% [ 11. If the total fitting error is corrected for the estimated contribution due to sedimenting protein * then the fitting error for the total three component analysis would be approximately 7.5 * 8.1% (SD). Furthermore, it should be noted that in schlieren patterns with high levels of HDLzb (>150 mg/dl), such as those in Figs. 4a and 4b, a significant amount of pattern area between Fy.,, rates 5.0 and 9.0 can not be completely fitted by the HDL2b reference pattern. In the present study, however, only about 18% (14 cases) of the Modesto females and 1% (1 case) of the males showed HDLat, serum levels of 150 mg/dl or greater. In 14 of these cases the area not completely fitted by the reference HDLzb pattern was only 9% or less (Fig. 4a) of the fitted HDL2b pattern area. Only one case exhibited a larger discrepancy (30%) in fit and its pattern is shown in Fig. 4b. Since insufficient data were * Contributing to the overfitting of the HDL3 reference pattern is a reduction in the total HDL pattern heights in the HDL3 region due to the presence of a small amount of sedimenting protein remaining in the d < 1.21 g/ml fraction after a 24 h preparative ultracentrifugation. When total HDL fractions were analysed after a 48 h ultracentrifugation at d 1.20 g/ml, no reduction of pattern heights due to sedimating protein was observed and the fitting error for HDL3 was smaller (2.0 f 0.8% of the total HDL concentration, for 10 samples).

years

years

years

years

years

27-36

47-56

57-66

27-66

decade

in mg/lOO

3746

Age

given

presented

Values

2

SERUM

MEAN

TABLE

ml.

80

20

20

20

20

n

are means

OF

AND

HDL3 female

HDL3 r 26

in each

COMPONENTS samples

FOR of the

270

287

283

275

235

f 38

f 81

? 17

t 84

f 55

12

25r

33

27

29

* 21

17

f 40

k 37

* 31

70

81

82

82

88

r 37

r 22

? 40

f 47

t 52

153

164

172

174

158

? 14

k 26

+ 32

f 26

342?

385?

413

402

382 r 46

90

i 104

? 116

42

HDL

85

108

74

96

63

k 34

k 72

+ 45

_+ 90

138

148

144

164

145

f 28

? 22

? 47

* 55

r 45

152

161

164

141

141

HDL3 ~~

Modesto

DECADES in the

HDL2a

__~______~_

? 39

HDLzb

AGE

surveyed

STUDY

age decades

MODESTO four

Total

HDL2a

and

Total

male

HDL2,. for

HDLzb

errors

HDLZb,

F.?lIl& __-.

HDL

standard

Male

and

CONCENTRATIONS

? 14

+ 36

_+ 26

+ 25

+ 26

Study.

All

concentrations

are

0

-3

171

available for delineation of the number of contours of possible reference components in this flotation rate region, material with Fy_,, rates between 5.0 and 9.0, which was not fitted by the HDLzb reference pattern, was included as part of the HDLZb component concentration. The error in the determination of levels due to such pooling was minimal in this population since the HDL,, fitting error for the reference HDL 2b pattern was only 3.2 + 6.1% (SD). In studies on specific subpopulations exhibiting elevated levels of HDLZb, such as subjects with hyperalphalipoproteinemia, the amount of this material may be substantial and separate analysis in terms of appropriate reference components would be required. In preliminary studies, we have isolated material in this flotation range by equilibrium density gradient ultracentrifugation [9] and have determined its mean particle hydrated density (1.075 g/ml), approximate particle diameter as measured by electron microscopy (13-14 nm), mean peak mass ratio (0.31) [9]. FY.*cl rate = 6.87 and total protein to total lipoprotein Further characterization of this material with respect to particle size and hydrated density distribution will be important in determining the number of components it represents. The establishment of reference schlieren patterns for each component will then enable the determination of their serum concentrations from total HDL schlieren patterns, in accord with the present three component analysis. (2) Serum levels of the HDLzb, sex and age

HDL2,,

and HDL3 components

as a function

of

The mean serum concentrations of the HDLz,,, HDLa,, and HDLa components for the Modesto Study population are presented according to age and sex in Table 2. For both males and females the progressive increase in mean total HDL concentration from one decade to the next is not statistically significant (P > 0.05). However, the mean total HDL concentration increase between the 27-36 and 47-56 or 27-36 and 57-66 year decades is significant (P < 0.05). A similar trend is seen in the HDL3 levels of both males and females. The levels of both HDLa,, and HDL2, in female HDL are significantly higher (P< 0.05) than in male HDL for each of the four age decades. The mean female HDL3 level is actually lower than the mean value for all corresponding age decades and the difference of their mean levels is significant for the population as a whole (P < 0.05). The fine structure of the differences between female and male patterns of the Modesto Study was also investigated independent of the three component analysis. Difference patterns (female mean total HDL pattern minus male mean total HDL pattern) for each of the four age decades were obtained by computer and are shown in Fig. 5. All four patterns clearly demonstrate the presence of peaks in the female-male difference patterns which are localized within specific flotation regions of the total HDL pattern. As indicated in the figure, these flotation regions are those that are associated with the major reference components (HDL2,,, HDL2,, HDLs) used in the present analysis. When the population difference patterns are analysed via the three component fitting procedure, the fit of the three reference patterns to the difference pattern contour is excellent, strongly suggesting that the reference patterns are representative of subspecies within the HDL distribution whose serum levels may

172

DIFFERENCE

DIFFERENCE

27-36

FEMALE - MALE,

years ,

DIFFERENCE

PATTERN

FEMALE - MALE, 37-46 0 9.0 0.0 I I

DIFFERENCE

PATTERN

FEMALE-MALE,

F1.20 ?;O 6;O 5.0 40 ,I I

3.0 2.0 I.8

1.0 I,

1

57-66

years

F%o , 9.0

-

, , 0.0 10

I., 60

,.I 5.0 4.0 3.0

AHDL SUM

I 2.0

I. 1.0

_ I

I

ATOTAL HDL

------

47-56years

PATTERN

FEMALE - MALE,

years

PATTERN

COMPONENTS OF A HDL

COMPONENTS

Fig. 5. Total HDL difference patterns (mean female minus mean male patterns) for the four age decades Mean total HDL schlieren patterns of both males and females were fitted of the Modesto Study ( -). and HDLQ fitted patterns of males by the three component analysis scheme. Separate HDL2b. HDLz,, were subtracted from corresponding female fitted patterns for each age decade to generate the component difference patterns (- - -). The sum of the three separate component difference patterns (. . . . .) closely approximates the total HDL difference pattern in each age decade.

reflect component-specific metabolic control. The relationship between HDL2b and HDL2, levels among the 160 cases is shown in Fig. 6a. A polynomial regression curve of the data shows a nearly linear relationship (r = 0.726, P < 0.001) between both male and female levels of these components. Based on values of the slope and intercept of the regression curve, the average mole ratio of HDLza to HDLab in HDL from this population is approximately 2 : 1. In addition, at HDL2, levels of 40 k 10 mg/dl or less, no HDLab can be detected by this analysis. The regression of HDL2b serum levels on corresponding HDL3 serum levels (Fig. 6b) reveals a small but significant negative correlation (r = -0.315, 0.005 > P> 0.001) whereas the regression of HDL,, serum levels on corresponding HDL3 serum levels (Fig. 6c) shows no significant correlation (r = 0.105, P > 0.25) and suggests that HDLza serum levels for both males and females may be independent of HDL3 levels. It should be noted, however, that the range of HDL3 serum levels (Fig. 6d) among the 160 cases of the Modesto Study extends only from 100 mg/dl to 220 mg/dl with a standard deviation of the mean of 30 mg/dl (or 19% of the mean). The standard deviations of the mean HDL ab and HDL2, levels (257 mg/dl or 106% of the mean for HDLab and k55 mg/dl or 48% of the mean for HDLa,) indicate

Fig. 6a. Regression of HDLZb on HDL2a concentration. For Figs. 6a. b. and c, the solid squares (m) represent female values, empty squares (0) represent male values, and solid line represents the second degree polynomial regression line. The HDLZb, HDLza, and HDL3 components for the 80 male and 60 female subjects in the Modesto Study were compared under the assumption that the values of all three components have a multivariate normal distribution with the same unknown covariance matrix. Fig. 6b. Regression of HDL2b on HDL3 concentration. Fig. 6~. Regression of HDL2, on HDL3 concentration. Fig. 6d. Distribution of percent of total number of Modesto Study cases within specific concentration ranges of HDLZb, HDL2,. and HDL3.

that both HDLzb and HDLza component levels exhibit a substantially wider range of biological variation in the Modesto Study population than do HDL3 component levels. (3) The relationship of serum levels of the HDLzb, nents to total HDL concentration

HDL2,,

Serum levels of both HDLzb and HDL2, show strong total HDL concentration (r = 0.872, P < 0.001 and r HDL2b and HDL2, in Figs. 7a and b, respectively). In curve of HDL3 serum concentration versus total HDL Fig. 7c is essentially flat at an HDL3 level approximating

and HDLS compo-

positive correlations

to for contrast, the regression serum concentration in the mean HDL3 serum

= 0.926,

P < 0.001

Fig. la. Regression of HDLZb on total HDL concentration. For Figs. 7a. b. c, d. e, and f the solid squares (m) rePreSent female values. the empty squares (0) male vabes, and the solid line represents the second degree polynomial regression line. See further bottom p. 175.

175

level of the Modesto population (158 mg/dl). The correlation of HDL3 serum levels on total HDL concentration was low order and not significant (r = 0.106, P> 0.25). The regression curves of percent, HDLPb, HDLz,, and HDLs versus total HDL concentration can be used to predict the minimal total HDL concentrations at which HDLPb and HDLs, can be demonstrated in serum samples from the Modesto population. The regression curve in Fig. 7d predicts detection of HDLzb only for those samples in which the total HDL concentration is greater than 200 * 8 mg/dl. Likewise the regression in Fig. 7e predicts measurable HDLz, levels only in samples with total HDL concentrations greater than 100 f 5 mg/dl. This value agrees closely with that of the total HDL concentration (115 f 7 mg/dl) at which the regression curve of percent HDL3 versus total HDL concentration in Fig. 7f approaches the 100% HDL3 value. Based on these regression data (Figs. 7d and 7e), samples with total HDL concentrations of less than 100 f 5 mg/dl would be expected to exhibit negligible levels of both HDLPb and HDLz,. (4) The relationship of lipoprotein composition to total HDL concentration In a previous study [5] we reported the lipid and protein compositions of the three reference components HDLsb, HDLz,, and HDLs isolated from 8 normolipemic subjects. These lipid and protein compositions (Table 3) can be used with values of the regression coefficients (Figs. 7d, e, and f) of percent HDLz,,, to estimate the variation of HDLz,, and HDLs on total HDL concentration each compositional parameter as a function of total HDL concentration (Fig. 8). It is assumed that the lipid compositions of each HDL component do not vary with increasing serum levels of these components throughout the Modesto population. This assumption is supported by the recent work of S. Mendoza et al. [lo] who found that 5 familial hyperalphalipoproteinemic women with TABLE

3

LIPID AND PROTEIN Values presented

COMPOSITIONS

are means and standard

OF HDL COMPONENTS deviations

HDLzb Percent Percent Percent

protein a phospholipid unesterified

37.0 39.6

k 2.9 + 4.2

for four male samples and four female samples. HDLza 41.7 36.6

t 0.9 ? 4.5

HDL3 54.5 23.0

+ 2.0 r 3.1

cholesterol Percent cholesteryl

6.8 + 0.7

3.2 t 0.3

3.0 k 0.2

ester Percent triglyceride

14.6 ? 2.0 2.0 * 0.2

11.4 + 0.8 1.1 * 0.1

17.9 ? 0.6 1.6 ? 0.2

a Percent

denotes

percent

of total lipoprotein

weight.

Fig. lb. Regression of HDL2, on total HDL concentration. Fig. 7~. Regression of HDL3 on total HDL concentration. Fig. 7d. Regression of weight percent HDL2b on total HDL concentration. For Figs. 7d, e, and f the standard deviation of the data about the fitted standard deviations of the three polynomial coefficients. Fig. le. Regression of weight percent HDL2, on total HDL concentration. Fig. 7f. Regression of weight percent HDL3 on total HDL concentration.

curve was calculated

from the

176

0

200

so0

TOTAL

400

HDL

500

605

CONCENTRATION

100

img/dll

Fig. 8. Estimated variation of protein and lipid composition of total HDL with increasing total HDL concentration. Values of the compositional parameters for HDLZb, HDL2,. and HDL3 (Table 3) were used with the fitted polynomial coefficients from Figs. 7d. e. and f to calculate these curves.

increased concentrations of HDLz (d 1.063-1.125 g/ml fraction) and HDL3 (d 1.125-1.200 g/ml fraction) showed normal lipid and protein compositions for each fraction. The data in Fig. 8 indicate that the relative amounts of protein and cholesteryl ester decrease while those of phospholipid, unesterified cholesterol, and triglyceride increase with increasing total HDL concentration. Discussion This report describes a three component (HDLzb, HDL2,, and HDLs) analysis of analytic ultracentrifugal schlieren patterns of total serum HDL. The procedure employs reference schlieren patterns which were obtained by averpatterns from 4 normolipemic aging HDLzb, HDL2,, and HDL, component males and 4 normolipemic females. In cases with HDLzb serum levels greater than 150 mg/dl, a significant amount of schlieren pattern area between FP.,, rates 5.0 and 9.0 is not fitted by the reference HDLzb component, indicating the presence of an additional component or components with greater size and lower weight percent protein than HDL2b. Evaluation of HDL2b, HDLz,, and HDL3 serum levels as a function of age for both males and females failed to establish any statistically significant trends, although a tendency of mean total HDL and HDL3 levels to increase with age was observed (Table 2). In general two trends among the serum levels of the three HDL components in the Modesto population sample were ob-

177

served: (1) The HDL3 serum levels show little variation with age and only slight variation with sex, with an average increase of 10 mg/dl in the male mean value over the female mean value for each age decade. HDL3 serum levels show no correlation with HDL2, levels and only a small negative correlation with HDLzb serum levels. (2) The HDL2,, and HDL,, serum levels exhibit a wide range of values (O-250 mg/dl). Elevations in mean serum levels of the HDLzb and HDLs, components in female samples account for the differences in mean total HDL levels between females and males. The results of the three component analysis of total HDL (Figs. 7d, e, and f) indicate that in cases where the total HDL concentration ranges between 100 mg/dl and 600 mg/dl, the distribution of the total HDL concentration among the three HDL components can be correlated with the total HDL concentration. In cases with total HDL concentrations close to 100 k 5 mg/dl, over 90% of the total HDL concentration is represented by HDLs. At higher total HDL concentrations (100 f 5 mg/dl to 200 mg/dl) the increase in concentration is observed primarily as a build-up in HDLs, level with little change in HDL3 and no demonstrable HDLzb. At still higher total HDL levels (200 + 8 mg/dl to 475 f 20 mg/dl *), the increase in concentration is reflected in further build-up with the appearance and build-up of HDLsb, and no of HDLs,, accompanied change in mean HDL3 level. Finally at total HDL concentration of 475 + 20 mg/dl and above, the increase in concentration includes not only a further build-up of HDLzb and HDLza levels but also the appearance of additional faster-floating components (Figs. 4a and 4b); HDL3 levels remain relatively unchanged. The regression curves of percent HDLsb, HDL2,, and HDLs (Figs. 7d, e, and f) can further be used with the lipid and protein compositions of the three HDL components (Table 3) to predict, for the high density lipoproteins as a whole, a progressive increase in mean particle size, weight percent phospholipid, and weight percent unesterified cholesterol with increasing total HDL serum concentration. In the same manner, a decrease in the mean particle hydrated density, weight percent protein, and weight percent cholesteryl ester is predicted with increasing total serum concentration. It is interesting that the LCAT * * substrates phospholipid and unesterified cholesterol increase in weight percent whereas the product of LCAT activity, cholesteryl ester, decreases in weight percent with increasing total HDL levels. levels and Further investigation of HDLzb, HDLs,, and HDLs component compositions with increasing total HDL serum concentration in other normal populations is required to determine whether the present observation can be generalized to other populations. Moreover, it will be important to establish if the above distribution of total HDL concentration among the three HDL components is observed when total HDL concentrations are altered in individuals by metabolic and/or environmental factors. Will the interrelationships established in Figs. 7d, e, and f for the 160 Modesto cases hold in correlating HDL component levels with total HDL concentration for a given normal individual as his total HDL serum level is progressively altered? Do currently

* 475 k 20 mg/dl corresponds to the total levels reach 150 mg/dl. ** Lecithin:cholesterol acyltransferase.

HDL serum concentration

in Fig. 7d at which HDL2b

serum

178

known factors which alter total HDL serum levels (environmental and metabolic) affect component levels in a manner consistent with the Modesto population interrelationships? Thus, estrogen administration to human subjects leads to an increase in total HDL concentration and androgen administration results in a considerable decrease in the total HDL concentration [ll]. Data are not currently available to determine whether these changes in total HDL serum concentration with gonadal hormone administration translate themselves into according to changes in levels of the HDLsb, HDL,,, and HDLs components the interrelationships seen in Figs. 7d, e, and f. If confirmed, such an influence of gonadal hormones on HDL component levels may be one factor influencing their variation in a normal population. Recently several population studies have demonstrated a significant inverse relationship between serum HDL cholesterol levels and the prevalence of coronary heart disease [ 12,13, and 141. Our results indicate that the major differences in serum total HDL as well as in serum HDL cholesterol levels, within a normal population such as the Modesto sample, arise predominantly from differences in serum levels of the HDL sa and HDLsb components. Hence, it is most likely that the above two HDL subclasses are the major contributors to the inverse correlation of HDL cholesterol with the prevalence of coronary heart disease. Since low total HDL levels are associated with the presence primarily of the HDL3 component, it would appear that, at the concentrations naturally encountered, this component’s contribution to any “protective” effect with respect to atherosclerosis is minimal. Further investigation into the role of HDL components in atherosclerosis-related lipid metabolism is clearly indicated. Acknowledgements The authors gratefully acknowledge the role of Dr. Peter Wood in the original Modesto Study and his consent to our use of the data. The assistance of Jerry Adamson and Robert Wills in the preparation of data is also most appreciated. References Ewing, A.M.. Freeman. N.K. and Lindgren. F.T.. The analysis of human serum lipoprotein distributions. In: R. Paoletti and D. Kritchevsky (Eds.). Advances in Lipid Research, Vol. 3, Academic Press, New York, N.Y., 1965. pp. 2541. DeLalla, O.F.. Elliott. H.A. and Gofman, J.W., Ultracentrifugal studies of high density serum lipoproteins in clinically healthy adults. Amer. J. Physical., 179 (1954) 333. Barclay. M.. Barclay. R.K. and Skipski, V.P., High density lipoprotein concentrations in men and women. Nature (Land.), 200 (1963) 362. Nichols, A.V., Human serum lipoproteins and their interrelationships. In: J.H. Lawrence and J.W. Gofman (Ed%). Advances in Biological and Medical Physics, Vol. 11. Academic Press, New York, N.Y.. 1967. pp. llO---156. Anderson, D.W., Nichols, A.V., Forte, T.M. and Lindgren. F.T., Particle distribution of human serum high density lipoproteins, Biochim. Biophys. Acta, 493 (1977) 5548. Lindgren, F.T.. Adamson, G.L., Jensen, L.C. and Wood, P.D., Lipid and lipoprotein measurements in a normal adult American population. Lipids, 10 (1975) 750. DeLalla, O.F. and Gofman, J.W., Ultracentrifugal analysis of serum lipoproteins. In: D. Glick (Ed.), Methods of Biochemical Analysis, Vol. 1. Interscience Publishers. New York, N.Y., 1954, PP. 459478.

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F.T., Jensen,

L.C. and Hatch, F.T.. The isolation

and quantitative

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12 13

14 15

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D.W., Forte, S., Lutmer,

T.M. and Nichols, R.F.,

Glueck,

A.V.,

C.J., Chen,

Unpublished C.-Y.,

analysis of serum lipoproComposition,

and Metab-

results.

Steiner.

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M.L.,

Composition of HDL-2 and HDL-3 in familial hyperalphalipoproteinemia, Atherosclerosis, 25 (1976) 131. Furman, R.H., Alaupovic, P. and Howard. R.P.. Effects of androgens and estrogens on serum lipids and the composition and concentration of serum lipoproteins in normolipemic and hyperlipidemic states. In: D. Kritchevsky. R. Paoletti and D. Steinberg (Eds.), Progress in Biochemistry and Pharmacology, Vol. 2. Karger. Bawl, 1967. pp. 215-249. Miller, G.J. and Miller, N.E., Plasma-high-density-lipoprotein concentration and development of ischemic heart disease, Lancet, 1 (1975) 16. Castelli, W.P., Doyle, J.T., Gordon. T.. Hames, C., Hulley, S.B.. Kagan, A., McGee, D., Vicic, W.J. and Zukel. W.J., HDL cholesterol levels in CHD -A cooperative lipoprotein phenotyping study. Circulation, II-52 (1975) 97. and coronary heart disease in a Rhoads. G.G., Culbrandsen, C.L. and Kagan. A., Serum lipoproteins population study of Hawaii Japanese men. New Engl. J. Med., 294 (1976) 293. Hatch, F.T. and Lees, R.S., Practical methods for plasma lipoprotein analysis. In: R. Paoletti and D. Kritchevsky (Eds.), Advances in Lipid Research, Vol. 6, Academic Press, New York, N.Y., 1968. PP. 148.