Quantitation of High Density Lipoproteins JOHN J. ALBERS, G. RUSSELL W A R N I C K , and M A R I A N C. C H E N N G ,

Northwest Lipid Research Clinic, Department of Medicine, School of Medicine, University of Washington, Seattle, Washington ABSTRACT

The demand for high density lipoprotein (HDL) quantitation has dramatically increased with the renewed awareness of the importance of HDL as a negative risk factor for coronary heart disease. HDL is usually estimated by specific precipitation of the non-HDL apoB-containing lipoproteins by polyanions and divalent cations followed by measurement of cholesterol in the supernatant. A common procedure involves precipitation with sodium heparin at 1.3 mg/ml and MnC12 at 0.046 M (final concentrations). This method is appropriate for serum but less than ideal for plasma because of incomplete precipitation and sedimentation of the apoB-containing lipoproteins. A two-fold increase in Mn 2+ to 0.096 M improves precipitation of the apoB-associated lipoproteins from plasma without excessive precipitation of HDL. This modified heparin-Mn2+ procedure gives results nearly identical to the results with the ultracentrifugal reference method (cholesterol in the d> 1.063 fraction corrected for losses, and the presence of apoB-associated cholesterol). The dextran sulfate 500-Mg 2+ and the sodium phosphotungstate-Mg 2+ procedures give results consistently 2-4 mg/dl lower than does the reference method. In contrast, a heparin-Ca 2+ method gives results 5-8 mg/dl higher than does the reference method. Immunochemical analysis of apoA-I in the precipitate and apoB in the supernatant indicates that lower values for the phosphotungstate-Mg2+ procedure is due to partial precipitation of the A-I-containing lipoproteins, while higher values by the heparin-Ca 2+ method are due to incomplete precipitation of the apoB-containing lipoproteins. Quantitation of the principal apoproteins of HDL, A-I and A-II, represent an important additional index of HDL concentrations and composition. Quantitation of plasma A-I and A-II concentrations by radial immunodiffusion indicates that women generally have higher HDL concentrations than men (women, A-I, 135 9 25, A-II, 36 • 6; men, A-I, 120 • 20, A-II, 33 • 5; mean • S.D., in mg/dl). A-I and A-II do not increase with age in men but show a slight increase with age in women. Estrogen increases HDL cholesterol and protein and may in part account for the higher HDL in women. The lighter density HDL subclass has a higher A-I/A-II ratio than the denser HDL subclass, with women generally having significantly more of the lighter HDL subclass. Density-gradient ultracentrifugation in CsC12 gradients indicates that HDL contains subpopulations of differing hydrated density which vary in the A-I]A=II ratio. Immunoassay of A-I and A-II when used in combination with HDL cholesterol analysis is a powerful tool for studies of HDL structure, epidemiology and metabolism. INTRODUCTION

High density lipoproteins ( H D L ) are generally defined in t e r m s of o n e or m o r e of t h e i r p h y s i c o - c h e m i c a l p r o p e r t i e s ; i.e., h y d r a t e d density, f l o t a t i o n characteristics in t h e analytical u l t r a c e n t r i f u g e , electrophoretic m o b i l i t y , and a p o l i p o p r o t e i n c o m p o s i t i o n . T h e f r a c t i o n of p l a s m a l i p o p r o t e i n s w i t h d 1.063-1.21 has b e e n o p e r a t i o n a l l y defined as t h e high d e n s i t y l i p o p r o t e i n s (1,2). Since H D L have alpha-elect r o p h o r e t i c m o b i l i t y , t h e y are a f t e n a l t e r n a tively calied a l p h a l i p o p r o t e i n s . More precisely, H D L can be d e f i n e d in t e r m s of a p o p r o t e i n c o m p o s i t i o n as t h e a p o l i p o p r o t e i n A ( a p o A ) c o n t a i n i n g l i p o p r o t e i n s w h i c h are free of apol i p o p r o t e i n B ( a p o B ) (3). H o w e v e r , t h e H D L u l t r a c e n t r i f u g a l f r a c t i o n c o n t a i n s some apoBc o n t a i n i n g l i p o p r o t e i n s , p r i n c i p a l l y t h e Lp(a) lipoprotein found primarily in the d 1 . 0 5 0 - 1 . 0 9 0 p l a s m a f r a c t i o n (4,5) a n d m a y also c o n t a i n s o m e n o n - L p ( a ) a p o B - c o n t a i n i n g lipop r o t e i n s (6). T h u s , if we define H D L (or m o r e a p p r o p r i a t e l y t h e a l p h a l i p o p r o t e i n s ) in t e r m s of t h e i r a p o p r o t e i n c o m p o s i t i o n , t h e n t h e 926

a p o B - c o n t a i n i n g lipoproteins in t h e d 1.063-1.21 f r a c t i o n m u s t be e x c l u d e d f r o m o u r d e f i n i t i o n of HDL. F o r t h e p u r p o s e of this treatise, H D L will generally be c o n s i d e r e d s y n o n y m o u s w i t h the a l p h a l i p o p r o t e i n s , b u t special e m p h a s i s will be placed u p o n t e c h n i q u e s w h i c h o p t i m i z e t h e q u a n t i t a t i o n of l i p o p r o t e i n s t h a t c o n t a i n a p o A a n d are free of apoB. HDL CHOLESTEROL QUANTITATION

With the n e w awareness of t h e i m p o r t a n c e of H D L as a negative risk f a c t o r for c o r o n a r y h e a r t disease (7-9), the d e m a n d for H D L q u a n t i t a t i o n has d r a m a t i c a l l y increased. H D L is generally e s t i m a t e d b y m e a s u r i n g its c h o l e s t e r o l c o n t e n t . A practical a p p r o a c h w h i c h avoids u l t r a c e n t r i f u g a t i o n is t h e selective p r e c i p i t a t i o n of t h e n o n - H D L apoB-associated l i p o p r o t e i n s with sulfated polysaccharides and divalent cations a n d t h e m e a s u r e m e n t of c h o l e s t e r o l in the s u p e r n a t a n t s o l u t i o n . A c o m m o n p r o c e d u r e involves p r e c i p i t a t i o n of t h e a p o B - a s s o c i a t e d l i p o p r o t e i n s w i t h s o d i u m h e p a r i n at 1.3 m g / d l

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TABLE 1 Heparin-Mn 2+ Precipitation Procedure Standard Method 1. 3 ml plasma 2. 0.12 ml 5000 units heparin/ml

3. 0.15 ml 1 M MnCI2.4 H20 4. Incubate 30 min at 4 C

Modified Method 1. 2 ml plasma 2. 0.2 ml combined heparin-Mn 2+ reagent (0.6 ml 40,000 units heparin]ml, 10 ml 1.06M MnC12 .4 H20 ) 3. Incubate 10 min at 23 C 4. Centrifuge 1500 X g for 30 rain at 4 C

5. Centrifuge 1500 X g for 30 min at 4 C

and MnC12 at 46 m M (10-12). This m e t h o d (Table I), originally applied to serum (10), appears r e a s o n a b l y specific a n d n o t o f t e n s u b j e c t to large errors, p r e c i p i t a t i n g generally less t h a n 2% of t h e H D L ( 1 3 , 1 4 ) , yet p r o d u c i n g n e a r l y c o m p l e t e r e m o v a l of t h e apoB-associated l i p o p r o t e i n s . However, if this p r e c i p i t a t i o n m e t h o d is applied to p l a s m a cont a i n i n g E D T A , it is less t h a n ideal, since a b o u t 10% of t h e p l a s m a samples so t r e a t e d have supernatants with obvious turbidity, indicating incomplete sedimentation. Furthermore, about half of t h e samples w i t h n o n t u r b i d s u p e r n a t a n t s still c o n t a i n ca. 2 m g / d l apoB, leading t o slight o v e r e s t i m a t i o n of H D L cholesterol. A two-fold increase in Mn 2+ c o n c e n t r a t i o n to 92 m m o l M n 2 + / l i t e r i m p r o v e s p r e c i p i t a t i o n of the a p o B , w i t h o u t s u b s t a n t i a l p r e c i p i t a t i o n o f HDL, a n d s e d i m e n t s b e t t e r t h e apoB associated l i p o p r o teins f r o m h y p e r t r i g l y c e r i d e m i c p l a s m a (14). T h e r e f o r e , a m o r e a c c u r a t e and m o r e conv e n i e n t m o d i f i e d version o f t h e h e p a r i n Mn 2+ p r e c i p i t a t i o n t e c h n i q u e is r e c o m m e n d e d for plasma, n a m e l y a smaller sample v o l u m e , 2 m l i n s t e a d o f 3 ml, a t w o - f o l d increase in t h e M n 2+ r e a g e n t c o n c e n t r a t i o n , a d d i t i o n of h e p a r i n Mn 2+ as a c o m b i n e d reagent, f o l l o w e d b y incubation at r o o m t e m p e r a t u r e for 10 m i n i n s t e a d of 30 rain at 4 C (Table I). A collaborative s t u d y b y eight l a b o r a t o r i e s to evaluate t h e modified heparin Mn 2 + precipitation procedure also shows t h e m o d i f i e d p r o c e d u r e to be b o t h m o r e specific and m o r e c o n v e n i e n t . By t h e s t a n d a r d m e t h o d , 32 of 375 (9%) of t h e samples h a d t u r b i d s u p e r n a t a n t s , a n d 72% of t h e samples h a d d e t e c t a b l e a p o B in n o n t u r b i d samples. O n t h e o t h e r h a n d , of the p l a s m a samples p r e c i p i t a t e d b y t h e m o d i f i e d p r o c e dure, only 2% h a d t u r b i d s u p e r n a t a n t s a n d o n l y 15% h a d d e t e c t a b l e apoB in n o n t u r b i d supern a t a n t s (15). With hypertriglyceridemic samples, t h e density Of t h e h e p a r i n - M n 2+ l i p o p r o t e i n aggre-

TABLE II Methods for HDL Estimation in Hypertrtglyceridemic Samples 1. Centrifuge plasma 105,000 x g for 18 hr. Do precipitation on the d > l . 0 0 6 fraction. 2. Dilute plasma with an equal volume of isotonic saline before precipitation. 3. Perform precipitation. Centrifuge turbid supernatants 12,000 X g for 10 min. Determine cholesterol on clear subnatant solution. 4. Perform precipitation. Filter turbid supernates with 0.22 #m filter protected by two depth prefilters. Determine cholesterol on clear filtrate.

gate f r e q u e n t l y is t o o low for it to s e d i m e n t . These l i p o p r o t e i n s t h u s r e m a i n s u s p e n d e d in the solution, producing obvious turbidity. A n u m b e r of p r o c e d u r e s have b e e n d e s c r i b e d for c i r c u m v e n t i n g t h e p r o b l e m of t u r b i d supern a t a n t s (Table II). M e t h o d 1 is expensive a n d t i m e c o n s u m i n g . M e t h o d 2 magnifies t h e i m p r e c i s i o n of t h e m e a s u r e m e n t of c h o l e s t e r o l in t h e h e p a r i n - M n 2+ s u p e r n a t a n t . M e t h o d 3 is s a t i s f a c t o r y for t h e m a j o r i t y o f samples, b u t requires h i g h s p e e d c e n t r i f u g a t i o n (14). M e t h o d 4 r e m o v e s essentially all of t h e a p o B - a s s o c i a t e d lipoproteins without removing appreciable a m o u n t s o f H D L (16). It appears to be t h e simplest a n d m o s t c o n v e n i e n t p r o c e d u r e for t h e clinical l a b o r a t o r y . Since Mn 2+ i n t e r f e r e s w i t h t h e e n z y m a t i c cholesterol procedure, other polyanion-cation c o m b i n a t i o n s i n c l u d i n g d e x t r a n sulfate 500 ( P h a r m a c i a , P i s c a t a w a y , NJ 08854)-MGC12 (17), sodium p b o s p h o t u n g s t a t e - M g C 1 ; (18), a n d heparin-CaC12 (19), along w i t h an u l t r a c e n t r i fugal m e t h o d at d 1.063 were e v a l u a t e d (Table III). T h e u l t r a c e n t r i f u g a l a p p r o a c h was imp r o v e d as a r e f e r e n c e m e t h o d b y c o r r e c t i n g the c h o l e s t e r o l of t h e d ~ 1 . 0 6 3 g / m l f r a c t i o n for losses (5%) and t h e p r e s e n c e o f a p o B - a s s o c i a t e d c h o l e s t e r o l (average 5.7 m g / d l , as d e t e r m i n e d LIPIDS, VOL. 13, NO. 12

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JOHN J. ALBERS, G. RUSSELL WARNICK, AND MARIAN C. CHENNG TABLE Ili Comparison of Mean HDL Cholesterol by Several Techniquesa

Samples

Source

n

Corrected Heparin_Mn2+ d>1.063 fraction b .046 Mc .092 Md

Dextran Sulphate e 500 Mg2+

Phosphotungstatef Mg2+

Heparing Ca2+

Plasma Women Men Children

30 27 8

61.7 44.4 52.2

63.9 45.7 53.0

60.7 44.1 50.8

58.1 43.6 48.0

57.4 42.8 49.0

66.7 51.5 57.2

All Subjects

65

53.4

55.0

52.6

50.9

50.3

- 59.2

Serum Women Men

10 10

70.0 44.7

70.5 44.2

66.9 42.8

62.4 40.0

64.7 41.6

75.0 53.1

All Subjects

20

57.4

57.4

54.8

51.2

53.2

64.0

aResults expressed in mg/dl * bcorrected for losses of cholesterol during ultracentrifugation and the presence of apoB~ CAccording to the Lipid Research Clinics Procedure (12). dAccording to Warnick and Albers (14). eAccording to Kostner ( 17~ fAccording to Lopes et al. (18). gAccording to Srinivanson et al. (19). TABLE IV Rid Assay for Human Plasma A-I and A-II 1. 2. 3. 4. 5.

50 ~1 plasma or test sample. 50 /al tetramethylurea, mix. 400 #1 8 M urea, 10 mM Tris pH 8.0, mix. Incubate 30 min. Add 4/*1 of the mixture to the antibody-containing Agarose plate. 6. Incubate 24-72 hr. 7. Measure diameter of precipitation rings. TABLE V Cholesterol and A-I and A-II in Ultracentrifuged Subfractions of HDL a d 1.063- 1.10

Cholesterol A-I A-II A-I/A-II

Men 9.9 10.3 2.0 5.1

Women 18.6 23.8 3.9 6.1

d 1.10- 1.21 Men 32.3 97.2 26.6 3.7

Women 36.1 104.4 27.4 4.0

aResults expressed in mg/dl. Each number is a mean and represents the analysis of six ultracentrifuged fractions from six plasma pools. Each pool consisted of plasma samples from three or four healthy normolipidemic adults. b y radial i m m u n o d i f f u s i o n w i t h a n t i - a p o B ) . T h e d e x t r a n - s u l f a t e 500-Mg 2+ m e t h o d consist e n t l y gave H D L c h o l e s t e r o l values o n p l a s m a s a m p l e s 2-4 m g / d l l o w e r a n d o n s e r u m s a m p l e s LIPIDS, VOL. 13, NO. 12

cholesterol.

4-8 m g / d l l o w e r t h a n t h e H D L values e s t i m a t e d b y c e n t r i f u g a t i o n . Similarly, t h e s o d i u m p h o s p h o t u n g s t a t e - M g 2+ p r o c e d u r e c o n s i s t e n t l y gave l o w e r r e s u l t s t h a n did t h e u l t r a c e n t r i f u g a l m e t h o d . In c o n t r a s t , t h e h e p a r i n - C a 2+ m e t h o d gave r e s u l t s 5-8 m g / d l h i g h e r t h a n t h e centrifugal r e f e r e n c e m e t h o d . A n a l y s e s o f a p o A - I in t h e p r e c i p i t a t e s a n d a p o B in t h e s u p e r n a t a n t fluid b y i m m u n o a s s a y s u g g e s t t h a t lower values b y t h e p h o s p h o t u n g s t a t e - M g 2 + p r o c e d u r e are d u e in p a r t to i n c r e a s e d p r e c i p i t a t i o n o f t h e a p o A - I - c o n t a i n i n g l i p o p r o t e i n s , while h i g h e r values b y t h e h e p a r i n - C a 2+ m e t h o d are d u e t o incomplete precipitation of the apoB-containing lipoproteins. On plasma samples, the modified heparin Mn 2+ m e t h o d , w h i c h u s e s 0 . 0 9 2 M M n 2+, w a s in e x c e l l e n t a g r e e m e n t w i t h t h e e s t i m a t i o n o f HDL cholesterol by the ultracentrifugal method, while t h e Lipid R e s e a r c h Clinic h e p a r i n Mn2+ m e t h o d u s i n g 0 . 0 4 6 M M n 2+ gave 1-to-3 m g / d l h i g h e r values. H o w e v e r , o n s e r u m s a m p l e s , t h e h e p a r i n M n 2+ p r o c e d u r e u s i n g 0 . 0 4 6 M M n 2+ agreed t h e b e s t w i t h t h e ultracentrifugal estimation of HDL cholesterol. T h u s , 0 . 0 4 6 M Mn 2+ a p p e a r s a d e q u a t e f o r prec i p a t i o n of H D L f r o m s e r u m b u t a h i g h e r M n 2+ c o n c e n t r a t i o n is n e e d e d f o r c o m p l e t e p r e c i p i t a tion of apoB-containing lipoproteins from p l a s m a , p r o b a b l y b e c a u s e M n 2 + is b o u n d b y E D T A ( 1 4 , 1 5 ) . Y e t , E D T A - t r e a t e d p l a s m a is p r e f e r r e d to s e r u m f o r lipid a n d l i p o p r o t e i n d e t e r m i n a t i o n s since, d u r i n g clot f o r m a t i o n ,

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TABLE VI Mol Wt, Apolipoprotein and Cholesterol Analyses on HDL Subfractions Isolated by Density Gradient Centrifugation a Density of Subfraction (Male) 1.151-1.200 1.119-1.150 1.106-1.118

1,093-1.10S 1.083-1,092 1.077-1.082 1.063-1.076 (Female) 1.151-1,200 1.119-1.150 1.106-1.118 1.093-1.105 1.083-1.092 1.077-1,082 1,063-1,076

10-Sx Mol Wt

A-I (mg/dl)

A-II (mg/dl)

CH (mg/dl)

A-I/A-II CH/(A-I+A-II) (Molar Ratio) (Wt. Ratio)

1.2 1.4 2.0 2.4 3.0 3.3 3.7

16.2 29.3 23.4 14.6 9.9 7.3 3.9

3.9 8.4 6.3 3.4 1.4 0.8 0.4

4.1 11.9 11.4 7.4 5,9 4.9 3.5

2.6 2.2 2.3 2.7 4,4 5.7 6.0

0.21 0.32 0.38 0.41 0.53 0.63 0.82

1.2 1.4 2.0 2.4 3.0 3.3 3.'/

13.2 27.9 22.6 14.4 11.1 8.4 4,2

3.1 7.9 6.7 3.0 1.5 0.9 0.5

3.0 10.7 11.3 6.5 5.9 5.6 3.5

2.6 2.2 2.1 2.9 4.6 5.5 5,3

0.19 0.30 0,38 0.38 0.47 0.61 0.75

aMean values expressed in mg/dl from six healthy normolipidemic adult volunteers (three male and three female). Mol wt estimated by density gradient electrophoresis. A-I and A-II determined by radial immunodiffusion. HDL of d 1,063-1.25 was placed in a CsC1 gradient and centrifuged in a SW 41 rotor at 40,000 rpm at 16 C for 72 hr. c h y l o m i c r o n s can be r e m o v e d b y t h e clot, a n d changes m a y o c c u r in t h e l i p o p r o t e i n distribution. F u r t h e r m o r e , s e r u m c o n t a i n s h e a v y m e t a l s t h a t are k n o w n to p r o m o t e a u t o - o x i d a t i o n , whereas E D T A in p l a s m a chelates m e t a l ions, minimizing auto-oxidation. Therefore, the most a p p r o p r i a t e p r o c e d u r e for q u a n t i t a t i o n o f H D L c h o l e s t e r o l is p r e c i p i t a t i o n of the a p o B - c o n taining l i p o p r o t e i n s f r o m E D T A - p l a s m a b y the m o d i f i e d h e p a r i n - M n 2+ m e t h o d and d e t e r m i n a t i o n of t h e c h o l e s t e r o l in the s u p e r n a t a n t fraction. Since Mn 2+ i n t e r f e r e s w i t h t h e e n z y m a t i c cholesterol procedure, other polyanioncation c o m b i n a t i o n s n e e d to be c o n s i d e r e d for laboratories using the enzymatic cholesterol procedure. A P O L I P O P R O T E I N A-I A N D A-II Q U A N T I T A T I O N

H D L is usually e s t i m a t e d in t e r m s of its lipoprotein cholesterol content. The cholesterol m o i e t y is, h o w e v e r , only 15-20% of t h e t o t a l weight o f HDL. T h u s , a change in t h e H D L c h o l e s t e r o l c o u l d reflect a change in H D L comp o s i t i o n or a change in w h o l e H D L c o n c e n t r a tion, or b o t h . A s s e s s m e n t of t h e p r i n c i p a l a p o p r o t e i n s o f HDL, a p o p r o t e i n s A-I a n d A-II, t o g e t h e r c o m p r i s i n g 50% of t h e w e i g h t of HDL, thus r e s p r e s e n t s a n i m p o r t a n t a d d i t i o n a l i n d e x of a l t e r a t i o n in H D L c o n c e n t r a t i o n or c o m p o sition. We d e v e l o p e d a simple y e t precise a n d a c c u r a t e radial i m m u n o d i f f u s i o n assay for h u m a n p l a s m a A-I a n d A-II ( T a b l e I V ) ( 2 0 , 2 1 ) . T h e d i a m e t e r of t h e p r e c i p i t a t i o n rings are

m e a s u r e d in 0 . 1 - m m units, using a c a l i b r a t e d viewer. Since usually less t h a n 1% of t h e t o t a l plasma A-I a n d A-II levels are f o u n d in t h e d(1.063 p l a s m a f r a c t i o n , q u a n t i f i c a t i o n of t o t a l p l a s m a A-I a n d A-II is r e p r e s e n t a t i v e o f t o t a l H D L A-I a n d A-II levels (21). It has b e e n e s t i m a t e d t h a t ca. 10% of t h e p l a s m a A-I a n d 3% of t h e p l a s m a A-II is in t h e d > l . 2 1 f r a c t i o n (21). However, it is likely t h a t m o s t of t h e A a p o l i p o p r o t e i n s f o u n d in the d > l . 2 1 f r a c t i o n were t h o s e t h a t h a d b e c o m e dissociated f r o m H D L d u r i n g u l t r a c e n t r i f u g a t i o n . Analysis o f u l t r a c e n t r i f u g e d s u b f r a c t i o n s o f t h e H D L preparations from normolipidemic adult subjects i n d i c a t e s t h a t m e n a n d w o m e n have similar A-I, A-II a n d H D L levels in the d 1.10-1.21 subf r a c t i o n ( T a b l e V). H o w e v e r , w o m e n have a p p r o x i m a t e l y twice as m u c h A-I, A-II a n d H D L c h o e l s t e r o l in t h e d 1.063-1.10 f r a c t i o n as m e n . Also for b o t h m e n a n d w o m e n t h e A-I/A-II m o l a r ratio of t h e l i g h t e r d e n s i t y H D L s u b f r a c t i o n was significantly greater ( p < 0 . 0 1 ) t h a n t h a t in t h e heavier d e n s i t y H D L s u b f r a c tion. It is clear, t h e r e f o r e , t h a t a p o p r o t e i n c o m p o s i t i o n s differ significantly w i t h i n t h e H D L h y d r a t e d d e n s i t y subclasses. F r a c t i o n a t i o n o f H D L o n t h e basis of h y d r a t e d d e n s i t y b y CsC1 density-gradient centrifugation and subsequent A-I a n d A-II analysis shows t h a t t h e A-I/A-II r a t i o varies w i t h the l i p o p r o t e i n h y d r a t e d d e n s i t y ( T a b l e VI). T h e A-I/A-II m o l a r r a t i o o f HDL lipoproteins banding between d 1 . 1 0 6 - 1 . 1 5 0 was nearly c o n s t a n t at 2.2 + 0.2. In t h e d e n s i t y range 1.51-1.25, the A-I/A-II LIPIDS, VOL. 13, NO. 12

930

JOHN J. ALBERS, G. RUSSELL WARNICK, AND MARIAN C. CHENNG TABLE VII Plasma A-II in Normal Subjects (mean • S.D., mg/dl) Number Age

Men

20-29 30-39 40-49 50-59

Plasma A-II Women

27 53 55 35

Men

82 33 44 27

34 33 34 33

• • • •

Women

4 5 5 5

35 35 37 38

• • • •

6 7 5 6

TABLE VIII Plasma A-I Levels in Normal Subjects (mean • S.D., mg/dl) Plasma A-I

Number Age

Men

Women

20-29 30-39 40-49 50-59 60-65

50 77 77 55 4

114 39 62 37 5

Men 117 117 120 125 126

• • • • •

Women

18 19 10 22 20

132

135 137 140 168

-+ 26 -+ 26 • 22 • 32 • 23

TABLE IX Plasma HDL-Cholesterol, A-I and A-II Levels in Normal Subjects a Subject group All m e n All w o m e n Women taking no estrogen b Women on estrogen Women on estrogen and progesterone

n

HDL CH

A-I

A-II

192 188 92 19 56

45 55 54 61 54

120 135 130 149 140

33 36 34 39 39

aNormal subjects refers to a subset of an industrial population who were selected independently of their lipid levels. Results expressed as mean levels in mg/dl. bRefers to a subset of w o m e n from the population who had taken no medication for 2 weeks before blood drawing. r a t i o i n c r e a s e d as t h e d e n s i t y i n c r e a s e d . H o w ever, in t h e d e n s i t y r a n g e b e t w e e n d 1 . 0 7 7 a n d d 1 . 1 0 5 , t h e A - I / A - I I r a t i o i n c r e a s e d as t h e d e n s i t y d_ecreased. T h e c h o l e s t e r o l / ( A - I + A - I I ) r a t i o d e c r e a s e d as t h e d e n s i t y i n c r e a s e d in all six H D L s a m p l e s e x a m i n e d . G r a d i e n t gel electrophoresis of the density-gradient fractions s h o w e d t h a t as t h e d e n s i t y o f t h e H D L p a r t i c l e increased, the apparent mol wt decreased. Thus, HDL contains subpopulations which differ not o n l y in m o l e c u l a r w e i g h t b u t also in t h e A-I/A-II molar ratio. HDL subfractions with the same hydrated density had comparable A-I/A-II and cholesterol/protein ratios whether isolated from men or from women. These results suggest that the differences between HDL concent r a t i o n s in m e n a n d t h o s e in w o m e n p r i m a r i l y r e f l e c t d i f f e r e n c e s in t h e r e l a t i v e p r o p o r t i o n s o f HDL subclasses rather than intrinsic malef e m a l e d i f f e r e n c e s in H D L s t r u c t u r e ( 2 2 ) . LIPIDS, VOL. 13, NO. 12

T a b l e V I I p r e s e n t s t o t a l p l a s m a A - I levels b y age d e c a d e in t h e p o p u l a t i o n t e s t e d . A m o n g t h e m e n , t h e o l d e r age g r o u p s ( 5 t h t o 7 t h d e c a d e s ) h a d s l i g h t l y h i g h e r A-I v a l u e s . T h e w o m e n also h a d s l i g h t l y h i g h e r A-I v a l u e s in t h e o l d e r age groups, with an average annual increment of 0 . 3 3 m g / d l / y r . A - I I d i d n o t i n c r e a s e w i t h age in m e n , b u t s h o w e d a s l i g h t i n c r e a s e w i t h age in women ( T a b l e VIII). H D L c h o l e s t e r o l w a s h i g h l y c o r r e l a t e d w i t h A-I ( I = 0 . 7 2 ) a n d A - I I ( R = 0 . 6 0 ) ; also, A-I w a s h i g h l y c o r r e l a t e d w i t h A-II ( r = 0 . 7 2 ) . W o m e n h a d s i g n i f i c a n t l y h i g h e r H D L c h o l e s t e r o l a n d a p o l i p o p r o t e i n A-I a n d A-II levels t h a n d i d m e n ( T a b l e IX). W o m e n t a k i n g e s t r o g e n - c o n t a i n i n g m e d i c a t i o n h a d signif i c a n t l y h i g h e r H D L c h o l e s t e r o l a n d A-I c o n centrations and somewhat higher"A-II concentrations than those not taking estrogen. The observation that women taking estrogen had s i g n i f i c a n t l y h i g h e r H D L c h o l e s t e r o l levels a n d

HDL QUANTITATION s o m e w h a t h i g h e r A-I/A-II ratios suggests t h a t estrogen p r e f e r e n t i a l l y increases t h e lighter d e n s i t y HDL-2 subclass. O n t h e o t h e r h a n d , women on combination contraceptives had the same H D L c h o l e s t e r o l c o n c e n t r a t i o n s as t h o s e o n n o m e d i c a t i o n , b u t s o m e w h a t h i g h e r A-I a n d A-lI levels, suggesting t h a t p r o g e s t i n s m o d i f y the estrogen r e s p o n s e a n d p r o m o t e a n increase in t h e heavier d e n s i t y HDL-3 subclass (21). T h e w o r k of Krauss et al. c o n f i r m s this suggestion (23). F o u r t e e n w o m e n s t u d i e d b e f o r e a n d a f t e r 14 days of e s t r o g e n t h e r a p y ( e t h i n y l estradiol 1 pg/kg/day) h a d a m e a n A-I increase of 24%, whereas t h e H D L c h o l e s t e r o l increased b y an average o f 19%, s u p p o r t i n g t h e c o n c e p t t h a t estrogen is in p a r t r e s p o n s i b l e for t h e h i g h e r H D L 2 levels o b s e r v e d in w o m e n ( 2 0 , 2 1 ) . T h e r e are m a n y c o n d i t i o n s or diseases in w h i c h H D L c o n c e n t r a t i o n s or c o m p o s i t i o n s are altered; e.g., p e r s o n s w i t h e x i s t i n g c o r o n a r y h e a r t disease a n d t h o s e w i t h m a n y of t h e c o n d i tions associated w i t h i n c r e a s e d risk of c o r o n a r y h e a r t disease have r e d u c e d c o n c e n t r a t i o n s of H D L c h o l e s t e r o l (24-26). Q u a n t i t a t i o n of A-I a n d A-II in 90 male survivors of m y o c a r d i a l i n f a r c t i o n (MI) s a m p l e d at least 3 m o n t h s a f t e r an a c u t e MI i n d i c a t e d that M I survivors h a d significantly l o w e r ( p < 0 . 0 1 ) A-I ( 1 1 2 + 2 m g / d l , m e a n -+ S.E.M.), A-II (29 -+ 1 m g / d l ) a n d H D L CH (39 -+ 1 m g / d l ) t h a n did a p l a s m a - c h o e l s t e r o I a n d triglyc e r i d e - m a t c h e d c o n t r o l group, a n d the H D L c h o l e s t e r o l of the MI survivors was significantly related to log triglyceride (4 = -0.442 lipid m a t c h e d c o n t r o l s , r = - 0 . 5 2 0 MI survivors). These results are c o n s i s t e n t w i t h a relative decrease of H D L in MI survivors over a n d a b o v e t h a t a t t r i b u t a b l e to t h e i r increased triglyceride levels. F u r t h e r m o r e , the low H D L c h o l e s t e r o l / p r o t e i n ratio o b s e r v e d in t h e H D L of MI survivors suggests a relatively greater decrease of the c h o l e s t e r o l - r i c h H D L 2 subclass t h a n t h a t of HDL3 (24). CONCLUSIONS R e c e n t e p i d e m i o l o g i c a l studies have e m p h a sized t h e i m p o r t a n c e of H D L as a negative risk f a c t o r (7-9). As a result, clinical l a b o r a t o r i e s have e x p e r i e n c e d an increased d e m a n d for q u a n t i t a t i o n o f this l i p o p r o t e i n . H D L is usually e s t i m a t e d b y q u a n t i t a t i o n of its c h o l e s t e r o l . P r e c i p i t a t i o n t e c h n i q u e s for e s t i m a t i n g H D L c h o l e s t e r o l are used widely. H o w e v e r , t h e y d o n o t give e q u i v a l e n t results. T h u s , careful validation of specific p r e c i p i t a t i o n c o n d i t i o n s is necessary in o r d e r t o e n s u r e an a c c u r a t e estim a t e of H D L cholesterol. Q u a n t i t a t i o n of t h e p r i n c i p a l a p o l i p o p r o -

931

teins o f HDL, A-I a n d A-II, t o g e t h e r c o m p r i s i n g ca. 50% of t h e weight of HDL, r e p r e s e n t s a n i m p o r t a n t a d d i t i o n a l i n d e x of a l t e r a t i o n s in H D L c o n c e n t r a t i o n or c o m p o s i t i o n . T h e radial i m m u n o d i f f u s i o n assays for A-I a n d A-II do n o t require p r i o r e x t r a c t i o n of lipids, a n d use o n l y 50 pl o f plasma. T h e s e assays t h e r e f o r e are suitable for mass screening. T h e r e f o r e , t h e q u a n t i t a t i o n of A-I a n d A-II shows p a r t i c u l a r p r o m i s e as m e t h o d s for c o m p l e m e n t i n g t h e H D L cholesterol p r o c e d u r e . T h e c o n c e n t r a t i o n , c o m p o s i t i o n a n d subclass d i s t r i b u t i o n of H D L changes in r e s p o n s e t o a variety of physiological, pharmacological, pathological and dietary perturbations. Thus, t h e a p p l i c a t i o n of assay m e t h o d s for H D L are n u m e r o u s . T h e f u n c t i o n a l role o f H D L in h e a l t h a n d disease will b e c o m e clearer as a c c u r a t e a n d precise assays for H D L b e c o m e widely available. ACKNOWLEDGMENT This work was supported by Contract NIHV 12157A (Lipid Metabolism Branch). The authors wish to thank the staff of the Northwest Lipid Research Clinic for assistance in these studies. REFERENCES 1. Hatch, F.K., and R.S. Lees, in "Advances in Lipid Research," Vol, 6, Edited by R. Paoletti and D. Kritchevsky, Academic Press, New York, 1968, p. 1. 2. Scanu, A.M., in "Advances in Lipid Research," Vol. 3, Edited by R. Paoletti and D. Kritchevsky, Academic Press, New York, 1965, p. 63. 3. Kostner, G., and P. Alaupovic, Biochemistry 11:3419 (1972). 4. Albers, J.J., C. Chen, and F. Aladjem, Biochemistry 11:57 (1972). 5. Albers, J.J., and W.R. Hazzard, Lipids 9:15 (1974). 6. Alaupovic, P., D.M. Lee, and W.J. McConathy, Biochim. Biophys. Acta 260:689 (1972). 7. Rhoads, G.G., C.L. Gulbrandsen, and A. Kagan, New Engl. J. Med. 294:293 (1976). 8. Castelli, W.P., J.T. Doyle, and T. Gordon, C.G. Haines, G. Curtis, M.C. Hjortland, S.B. Hulley, A. Kagan and W.J. Zukel, Circulation 55:767 (1977). 9. Miller, N.E., O.H. F~rde, D.S. Thelle, and O.D. Mj~s, Lancet i:965 (1977). 10. Burstein, M., and J. Samaille, Clin. Chim. Acta 5:609 (1960). 11. Fredrickson, D.S., R.I. Levy, and F.T. Lindgren, J. Clin. Invest. 47:2446 (1968). 12. Manual of Laboratory Operations, Lipid Research Clinics Program, Lipid and Lipoprotein Analysis, DHEW Publ. No. (NIH) 75-628 (1974). 13. Bachorik, P.S., J.J. Albers, and P.D.S. Wood, Clin. Chem. 23:912 (1977). 14. Warnick, G.R., and J.J. Albers, J. Lipid Res. 19:65 (1978). 15. Albers, J.J., G.R. Warnick, D. Wiebe, P. King, P. Steiner, L. Smith, C. Breckenridge, A. Chow, K. Kuba, S. Weidman, H. Arnett, P. Wood, and A. LIPIDS, VOL. 13, NO. 12

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Shlagenhaft, 24:853 (1978). 16. Warnick, G.R., and J.J. Albers, Clin. Chem. 24:900 (1978). 17. Kostner, G.M., Clin. Chem. 22:695 (1976). 18. Lopes-Virella, M.F., P. Stone, S. Ellis, J.A. Colwell, Clin. Chem. 23:882 (19"/7). 19. Srinivasan, S.R., R.R. Frerichs, L.S. Webber, G.S. Berenson, Circulation 54:309 (1976). 20. Albers, J.J., P.W. Wahl, and V.G. Cabana, W.R. Hazzard and J.J. Hoover, Metabolism Clin. Exp. 25:633 (1976). 21. Cheung, M.C., and J.J. Atbers, J. Clin. Invest. 60:43 (1977). 22. Cheung, M.C., and J.J. Albers, J. Lipid Res.

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(1979) (In press). 23. Krauss, R.M., F.T. Lindgren, A. Silvers, R. Jutager and D.D. Bradley, Clin. Chem. Acta 80:465 (1977). 24. Albers, J.J., M.C. Cheung, and W.R. Hazzard, Metabolism Clin. Exp. 27:479 (1978). 25. Brunzell, J.D., J.J. Albers, L.B. Haas, A.P. Goldberg, L. Agadoa, and D.J. Sherrard, Metabolism Clin. Exp. 26:903 (1977). 26. Bagdade, J.D., and J.J. Albers, N. Engt. J. Meal. 226:1436 (1977).

[Received June 5, 1978]

Quantitation of high density lipoproteins.

Quantitation of High Density Lipoproteins JOHN J. ALBERS, G. RUSSELL W A R N I C K , and M A R I A N C. C H E N N G , Northwest Lipid Research Clinic...
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