Age related changes in the lipoprotein substrates for the esterification of plasma cholesterol in rats

Mechanisms of Ageing and Development, 61 (1991) 85-98

85

Elsevier Scientific Publishers Ireland Ltd.

AGE RELATED C H A N G E S IN THE L I P O P R O T E I N S U B S T R A T E S FOR THE ESTERIFICATION OF P L A S M A C H O L E S T E R O L IN RATS

S U N - M I N LEE*, B H A L C H A N D R A J. K U D C H O D K A R and A N D R A S G. L A C K O Department of Biochemistry. Texas College of Osteopathic Medicine, University of North Texas, Fort Worth, Texas 76107 (U.S.A.)

(Received January 13th, 1991) (Revision received April 23rd, 1991)

SUMMARY The activity of the enzyme lecithin:cholesterol acyltransferase (LCAT) and the properties of its lipoprotein substrates have been investigated in 6- and 19-month-old Fischer-344 rats. These studies were carried out to determine the nature of the relationship between the observed hypercholesterolemia and the age-related decrease in the fractional rate of lipoprotein cholesterol esterification. The distribution of L C A T activity of plasma fractions was determined following gel chromatography and ultracentrifugation respectively. L C A T activity was found to be associated with the high density lipoprotein ( H D L ) fraction when rat plasma was passed through a BioGel A-5 M column. U p o n density gradient ultracentrifugation for 24 h it was found associated with H D L fraction; d = 1.125--1.21 g/ml. However, following prolonged ultracentrifugation (40 h), the majority of the L C A T activity was displaced into the lipoprotein-free infranatant (d > 1.225 g/ml). The dissociation of L C A T from its complex with H D L occurred to a smaller extent in aged rat plasma than in young rat plasma. Substrate specificity studies indicated that H D L was a considerably better substrate for L C A T than very low density lipoproteins (VLDL) in both young and aged rats. In addition, H D L from young rats was a better substrate for L C A T than the H D L from aged rats. Incubation experiments followed by the isolation of Correspondence to: Andras G. Lacko, Ph.D., Department of Biochemistry/TCOM, 3500 Camp Bowie Boulevard, Fort Worth TX 76107-2690, U.S.A. *Pre-doctoral fellow of the Robert A. Welch Foundation. Abbreviations: LCAT, lecithin:cholesterol acyltransferase; CETP, cholesteryl ester transfer protein; TC, total cholesterol; FC, free (unesterified) cholesterol; CE, cholesteryl ester; HDL, high density lipoprotein(s); LDL, low density lipoprotein(s); VLDL, very low density lipoprotein(s); TG, triglyceride.

0047-6374/91/$03.50 Printed and Published in Ireland

© 1991 Elsevier Scientific Publishers Ireland Ltd.

86 lipoproteins and the subsequent analyses of their cholesterol contents revealed that the age-related hypercholesterolemia was mainly due to an increase in the cholesterol carried lay lipoprotein fractions d = 1.025 -1.07 g/ml (LDL + HDL1). These and other low density lipoproteins (d < 1.025 g/ml) were poor substrates for LCAT. However, these lipoproteins could provide free cholesterol for esterification by first transferring it to H D L (d = 1.07--1.21). The H D L isolated from the plasma of aged rats was enriched with apolipoprotein (apo) E and these iipoprotein particles were found to be inferior substrates for LCAT. These data suggest that the decreased fractional rate of esterification observed in aged rats is due to the slower utilization of the H D L lipid substrate pool by the enzyme LCAT as a result of the accumulation of unfavorable substrates (compositionally altered HDL particles) for the LCAT reaction.

Key words." Ageing; Rats; Plasma LCAT; Lipoproteins; Cholesterol INTRODUCTION Earlier studies have revealed age-related hyperlipidemia and decrease in the rate of endogenous cholesterol esterification in rats [1,2], However, neither the mechanism of the decreased fractional rate of cholesterol esterification (FRE) in older rats nor its relationship to the development of age-related hypercholesterolemia has been clarified. This study was carried to determine the role(s) that LCAT/lipoprotein interactions may play in the mechanisms underlying the age-related decrease of the FRE and the hypercholesterolemia observed in rats. The substrate specificity of L C A T using both artificial and lipoprotein substrates have been shown to require at least one polypeptide cofactor for the full expression of its activity [3] and that H D L was preferred over other lipoproteins as a substrate [4]. The fractional rate of endogenous plasma cholesterol esterification (FRE; % cholesterol esterified/time) has been shown to decrease in human subjects and in animals coinciding with increases in the size of the plasma cholesterol pool [5,61. However, the characteristics of the lipoprotein substrates for LCAT during hypercholesterolemia have not been fully described. The data provided by this research show that H D L is a better substrate for LCAT and that the H D L isolated from the plasma of young rats is a better substrate for LCAT than the H D L from aged rats. Overall, these studies indicate that changes in the composition of the H D L complexes are responsible for its decreased reactivity with LCAT in aged rats. MATERIALS AND METHODS

Animals Male Fischer-344 rats (young; 6 months and aged; 19 months) were obtained from

87 Charles River Breeding Laboratories (Wilmington, MA). They were housed two to a cage at 25°C with 12-h light/dark cycles and given Purina chow (22% protein, 4% fat) and water ad libitum. The animals were sacrificed following an 18 h fast, approximately 15 days after their arrival. Blood was drawn from the inferior vena cava under light ether anesthesia and was collected into chilled tubes containing EDTA (ethylene diamino tetra acetic acid) at a final concentration of 4.5 mM. Plasma was obtained by low speed centrifugation at 4°C. Aliquots of plasma were processed immediately to isolate the plasma lipoproteins. Lipoprotein fractions were stored at 4°C prior to lipid analyses.

Distribution lipoproteins

of lecithin:cholesterol

acyltransferase

( LCA 7/') activity

among

Bio-Gel A 5 M chromatography and ultracentrifugation were used in order to establish the distribution of the LCAT containing lipoprotein fraction(s) in the plasma. Bio-Gel A-5 M chromatography. Two mililiters of rat plasma (pooled from six animals in each group) was labeled with [3H]cholesterol (0.01 mCi/ml) for 4 h at 4°C and was applied to the Bio-Gel A-5 M column (1.5 × 90 cm; equilibrated with a 0.01 M Tris--HC1 buffer (pH 7.4) containing 0.01% EDTA and 0.15 M NaCI) and chromatographed with a flow rate of 20 ml/h; 2-ml fractions were collected. Bio-Gel A-5 M chromatography was also used to fractionate plasma lipoproteins (d < 1.225 g/ml) isolated from a 2-ml sample of labeled plasma by preparative ultracentrifugation as described by Rudel et al. [71. The absorbance (280 nm) and the radioactivity of each fraction was monitored to establish elution pattern of lipoproteins (VLDL, LDL and HDL), and LCAT activity was assayed in alternate fractions according to Chen and Albers [8]. Ultracentrifugation. Rat plasma was also fractionated by density gradient ultracentrifugation essentially as described by Terpstra et al. [9]. Two milliliters of pooled plasma, adjusted to a density of 1.25 g/ml with KBr, was sequentially layered under NaC1-KBr salt solutions of densities 1.225 g/ml (2 ml) and 1.10 g/ml (4 ml) and the tube was filled with distilled water. The tubes were placed in Sorvall SW-40 Ti rotor and centrifuged at 39 000 rev./min (200 000 × g) for 24 h in a Sorvall OTDB ultracentrifuge at 15°C. The procedure was calibrated in our laboratory b.y repeatedly determining the density of successive 1 ml fractions that were removed from the top of the tubes by a narrow-bore Pasteur pipette. The activity of LCAT was determined [8] in each fraction.

Lipoprotein substrate specificity of L C A T Partial purification of rat LCAT. The infranatant obtained from preparative ultracentrifugation of rat plasma was concentrated to 5 ml with Aquacide and dialyzed exhaustively against 0.01 M Tris--HCl (pH 7.4) containing 0.15 M NaCI and 0.01% EDTA and used as a source of LCAT activity.

88

Preparation oflipoprotein substrates. Lipoproteins were isolated using preparative ultracentrifugation and Bio-Gel A-5 M chromatography as described above [7] from pooled rat plasma in which the endogenous LCAT activity was previously inactivated by diiosopropyl fluorophosphate (DFP, 1 raM). The fractions containing H D L and VLDL were pooled respectively and concentrated to 3 ml using Aquacide. Following dialysis against 0.01 M Tris--HCl (pH 7.4) containing 0.15 M NaC1 and 0.01% EDTA, the pooled lipoprotein fractions were analysed with regard to chemical composition and used as substrates for LCAT. In addition, the HDL fractions obtained as apo A-I and apo E enriched components by Heparin-sepharose chromatography [10] were also used as LCAT substrates. Measurement o[" LCA T activity using isolated lipoproteins as substrates. The rate of free cholesterol (FC) esterification of the individual [3H]cholesterol labeled VLDL and HDL fractions was determined [11] following incubation (37°C) of the respective lipoproteins with partially purified LCAT. The incubation time and the amount of enzyme in the reaction mixture were selected to obtain a linear rate of cholesterol esterification The rate of cholesterol esterification (nmol/ml per h) was calculated by multiplying the fractional rate (% cholesterol esterification/time) by the free cholesterol concentration of each sample. The distribution of cholesterol in lipoproteins Aliquots (2 ml) of the [3H]cholesterol labeled plasma samples obtained from both age groups were incubated at 37°C for 0 and 2 h, respectively, to study the consequences of the LCAT reaction. Following the incubation the lipoprotein fractions were isolated by density gradient ultracentrifugation as described earlier. Four fractions were collected: (1) d < 1.006 g/ml (1 ml); (2) d = 1.006--1.025 g/ml (2 ml); (3) d = 1.025--1.07 g/ml (2 ml) and (4) d = 1.07--1.21 g/ml (6 ml) and the total, free and esterified cholesterol content of each fraction was determined. The pooled lipoprotein fractions 3, (LDL + HDLI) and 4, (HDLI + HDL2) were subjected to gel chromatography on Biogel A-5 M to determine their respective elution positions and to heparin-sepharose chromatography to determine the heparin bound portion of each density class. The relative contribution of the apo AI containing (HDL-A) and the apo E enriched (HDL-E) lipoprotein species were determined by estimating the area under the absorbance (A280) upon heparin-sepharose chromatography of HDL.

Characterization of lipoprotein substrates for LCAT Analysis of the composition of lipoprotein substrates. The total cholesterol (TC) was measured by the method of Parekh and Jung [12] and the FC was determined by an enzymatic method [13]. Esterified cholesterol (CE) contents were calculated by subtracting the FC values from the TC values. Triglycerides (TG) were determined by an enzymatic method and phospholipids (PL) were measured according to

89

Raheja et al. [14]. Total PL of the plasma HDL, isolated by the combined techniques of ultracentrifugation and gel chromatography [7], were subfractionated by thin layerchromatography using the solvent system described by Gilfillan et al. [15]. The phospholipid bands corresponding to known standard zones of sphingomyelin (SM), phosphatidyl choline (PC), phosphatidyl inositol plus phosphatidyl serine (PI + PS), and phosphatidyl ethanolamine (PE) were scraped into tubes and the phospholipids content determined [14]. Protein determinations were carried out according to Markwell et al. [16] using bovine serum albumin as standard. Polyacrylamide gel electrophoresis (PAGE) was performed in the presence of 0.1% (w/v) SDS, according to the method of Laemmli [18] to establish the apoprotein profile of the isolated HDL fractions. The relative proportion of the apo-E and apo-AI in HDL was determined by densitometric scanning of the SDS-PAGE gel pattern.

Statistical methods The means and the standard errors of the means (S.E.M.) were determined for those sets of data that were subjected to statistical analysis (Tables III and IV). Subsequently the Student's t-test was performed to determine the significance of the differences between the values obtained for young and aged rats. A probability of 0.05 or less was accepted as statistically significant. RESULTS

Distribution of LCA T activity There were no marked changes in the distribution of LCAT in rat plasma with age. Most of the LCAT activity was found associated with HDL or the HDL fractionation of d = 1.125--1.21 g/ml following the fractionation of the plasma by either gel chromatography or 24-h density gradient ultracentrifugation. When the plasma was fractionated by prolonged (40 h) preparative ultracentrifugation, more than 90% of the LCAT activity was displaced into the lipoprotein free infranatant (data not shown). Fractionation of the ultracentrifugally (40 h) isolated lipoproteins (d < 1.225 g/ml) by gel chromatography showed that the LCAT activity was associated only with HDL and that the amount of HDL-associated LCAT activity was considerably higher in the samples obtained from aged rats as compared to those from young rats. The original amounts of enzyme in the respective whole plasma samples were nearly the same (Fig. I). Lipoprotein substrate specificity of LCA T Figure 2 shows the reactivity of the VLDL and HDL fractions, isolated by ultracentrifugation and Bio-Gel A-5 M chromatography from the plasma of young and aged rats, with partially purified LCAT. The rate of cholesterol esterification was substantially higher with HDL from young rats than the HDL from aged rats

90

young 1.2

VLDL

1.0'

aged rats

rats

1 O0

HDL

I

HDL

80

,¢ .¢

0.8'

I¢ ¢J 60

l',t

I

v,o,

>. 40


> (J

0.4

I20 0.2

0.0

"

0

-

0

100

200

Fig. 1. Absorbance and LCAT activity profiles of rat plasma lipoproteins (d < 1.225g/ml) following prolonged (40-h) ultracentrifugation and Bio-Gel A5 M chromatography. Panels A and B show the distribution of A280readings ( o - o - o ) and LCAT activity ( • - • - • ) for samples from young (A) and aged rats (B). The HDL associated LCAT activity was higher in aged rats (panel B).

(especially at higher free cholesterol concentrations). The results were the same whether the L C A T originated from the plasma of y o u n g or aged rats. VLDL, whether from y o u n g or aged rats had only marginal reactivity with LCAT.

Characterization of lipoproteins from young and aged rats W h e n the L D L + H D L I a n d the H D L j + HDL2 fractions, isolated from the plasma of aged rats, were c h r o m a t o g r a p h e d on Bio-Gel A5 M, they eluted in the same position as 'whole H D L ' (isolated by the c o m b i n a t i o n of 40 h ultracentrifugation a n d gel c h r o m a t o g r a p h y ) (Fig. 3). W h e n equivalent a m o u n t s of whole HDL, L D L + H D L I a n d H D L I + HDL2 fractions were c h r o m a t o g r a p h e d on heparinsepharose, the a m o u n t of material retained on the c o l u m n was considerably larger in the samples o b t a i n e d from aged rats as c o m p a r e d to those from y o u n g rats (data

tJ ,..I

91

I

I

I

I

I

I

I

I

2.0 ! o i

O C

i

>

IO ki

I I

O

•

Q . . . . - ~ . . . . -Q . . . . - o - . . . . o . . . . .

.J

o

0

I

I

l

12.3

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36.9

FREE

[]. . . . . . . . . . . I

49.2

CHOLESTEROL

0 -0- ...........

o-O--

I

I

I

I

61.5

73.8

86.1

98.4

(nMoles/ml)

Fig. 2. R e a c t i v i t y o f L i p o p r o t e i n s i s o l a t e d f r o m the p l a s m a o f y o u n g a n d a g e d r a t s with p a r t i a l l y p u r i f i e d L C A T . T h e r a t e o f c h o l e s t e r o l esterification w a s h i g h e r w h e n H D L f r o m y o u n g r a t s ( • - • - • ) w a s used as s u b s t r a t e c o m p a r e d to H D L f r o m a g e d r a t s ( • - • - n ). V L D L w h e t h e r f r o m y o u n g ( o - o - o ) o r a g e d r a t s ( []- o - n ) r e a c t e d o n l y m a r g i n a l l y with L C A T .

not shown). The peak area ratio of HDL-A/HDL-E was considerably lower for the samples from aged rats (-2.3) as compared to the HDL from young rats ( - 10.2). Similarly, the ratio of apo-Al/apo-E determined by densitometric scanning of the SDS-PAGE patterns of HDL apolipoproteins (Fig. 4) was higher for the samples of young rats (7.6) than in the samples from aged rats (1.2). Thus the change during aging in the HDL-A/HDL-E ratio (-4.5) was comparable to the change in the ApoAI/Apo°E ratio ( - 6.3). The reactivity of HDL subfractions (HDL-A and HDLE) with partially purified LCAT was determined. The V/K of HDL-A (0.0323 ± 0.0015) was significantly higher (P < 0.025) than that of HDL-E (0.0252 ± 0.0022). The chemical composition of VLDL and HDL, isolated from the pooled plasma of young and aged rats is shown in Table I. The relative proportion of the lipid components of HDL was similar for the two age groups except for the lower amount of triglycerides in the HDL of aged rats. However, considerable differences were found in the relative proportion of some of the HDL phospholipids. The relative pro-

92

r Elution volume of HD L1

A

E t-

I

O

¢q

o Z

< m

0.1

n-

0

CO rn
o.0 30

40

50

60

FRACTION

70

80

90

NUMBER

Fig. 3. Bio-GelA5 M chromatographypatterns of lipoprotein fractions isolated from the plasma of aged rats by density gradient ultracentrifugation. The LDL + HDL1(d = 1.025-1.07 g/ml) fraction ( o - o - o ) and the HDL1 + HDL2 fraction (d = 1.07-1.21 g/ml) fraction ( • - •- • ) eluted in the same position as 'whole HDL' isolated by the combination of prolonged (40-h) ultracentrifugation and gel chromatography (see Fig. 1). portions of the phospholipid components for young vs. aged rats were: PC (77.2 vs. 69.4); SM (14.3 vs. 24.1); PI + PS (4.9 vs. 4.0) and PE (3.5 vs. 2.5). The PC/SM ratio in H D L was 5.4 for young rats and 2.9 for aged rats.

The distribution of cholesterol among lipoproteins The plasma TC was significantly higher (P < 0.05) in aged rats (70.4 ± 1.3 mg/dl) as compared to those in young rats (54.5 4- 2.5 mg/dl). The most significant increase in TC with age was seen in the fractions o f d = 1.025--1.07 g/ml. The relative amount of cholesterol (% of the total pool) was significantly lower in the fraction of d = 1.07--1.21 g/ml ( H D L l + HDL2) from aged rats as compared to young rats (P < 0.05). There was no significant difference in the percentage of CE among the isolated lipoprotein fractions. However, the CE content of d < 1.006 g/ml fraction of aged rats tended to be somewhat higher (P < 0.1) than the same fraction from young rats (Table II). All the CE formed during the 2-h incubation was recovered in the lipoprotein fraction of d = 1.07--1.21 g/ml. Furthermore, the amount of CE formed exceeded the original FC content of the same fraction indicating a net transfer of FC from the lower density fractions d < 1.07 g/ml) to the higher density fraction d = 1.07--1.21 g/ml). This net transfer of FC from lower to high density lipoproteins was about the same in both age groups. The CE increased during incubation by 6.2 mg/dl in young rat plasma and by 4.5 mg/dl in aged rat plasma (Table III).

93

2

4

3

Phosphorylase B Bovine serum albumin

W ApoAIV

0velbumln

Apo E Carbonic anhydrase ApoAI Soybean trypsin Inhibitor Apo Cs

(~.lactalbumin

Fig. 4. SDS-PAGE (IIY¼,) of HDL apoproteins from young and aged rats. Acrylamide gels were loaded with 50/zg of HDL-protein. Lanes I and 3 represent molecular weight standards as shown on the left margin. HDL from aged rats (lane 4) is enriched in apo E compared to the HDL from young rats (lane 2).

TABLE I COMPOSITION OF LIPOPROTEINS ISOLATED FROM THE PLASMA OF YOUNG AND AGED RATS (wt%) Component

Protein Phospholipids Cholesteryl Esters Free cholesterol Triglycerides

HDL

VLDL

Young rat

Aged rat

Young rat

Aged rat

29 37 24 8.3 1.9

27 36 27 9.8 0.3

I1 8.4 7.3 5.4 68

6.0 9.9 6.1 5.6 72

Lipoproteins (d < 1.225 g/ml) were isolated from plasma (pooled from 6 animals) and fractionated on a Bio-Gel A-5 M chromatography.

94

TABLE II DISTRIBUTION OF FREE A N D E S T E R I F I E D CHOLESTEROL A M O N G THE LIPOPROTEIN FRACTIONS OF Y O U N G A N D A G E D RATS

Buoyant density ( g/ml )

Cholesterol fraction

Experimental group

< 1.006

Total cholesterol (mg/dl)

1.006-1.025

Esterified cholesterol (%) Total cholesterol (mg/dl)

1.025-1.07

Esterified cholesterol (%) Total cholesterol (mg/dl)

1.07-1.21

Esterified cholesterol (%) Total cholesterol (mg/dl) Esterified cholesterol (%)

Young rats

Aged rats

10.1 (18.5 59.4 2.4 (4.5 65.0 9.7 (17.8 74.2 32.3 (59.1 80.5

14.0 (19.6 63.8 3.7 (5.2 58.5 18.8 (26.3 76.2 33.9 (47.5 78.0

+ ± 4+ 44444444-

1.50 2.12) 3.75 0.22 0.55) 3.88 0.71 1.83) 3.27 2.23 2.92) 1.98

± 444444+ 4± ± 4-

2.53 3.46} 3.32 0.44 0.64) 3.46 0.82* 1.34)* 2.30 0.51 0.85)* 1.50

Data represent mean cholesterol concentrations + S.E.M. for four animals in each group. Numbers in parentheses represent the mean + S.E.M. value for the percentage distribution of cholesterol among the different density fractions. *Denotes significant difference (P <0.05) between young and aged groups. DISCUSSION

The specific roles for HDL, LCAT and the HDL/LCAT complexes in reverse cholesterol transport have been proposed to include the conversion of FC to CE in HDL, rendering the HDL particle capable of acquiring more FC from peripheral tissues including the arteries, and thus prevent accumulation of cholesterol in those tissues [18,19]. The lower rate of esterification of HDL-FC in aging, could impede TABLE II1 CHANGES IN THE CHOLESTEROL C O N T E N T OF LIPOPROTEINS D U R I N G THE INCUBATION OF RAT PLASMA OF Y O U N G A N D A G E D RATS (mg/dl)

Buoyant Density (mg/dl)

Young rats 0ti

< 1.006 1.006~1.025 1.025--1.07 1.07 1.21

Aged rats 2It

2 h~) h

O It

2 h

FC

CE

FC

CE

FC

CE

FC

CE

FC

4.1 0.8 2.5 6.6

6.0 1.6 7.2 26

2.7 0.3 1.2 4.3

6.4 0.9 6.8 32.2

-1.4 -0.5 -1.3 -2.3

0.4 -0.7 -0.4 6.2

5.1 1.5 4.5* 7.5

8.9 4.0 2.2 I.I 14.3" 3.2 26.5 6.4

2 h 4) h CE

FC

CE

9.1 1.7 13.9 31

-1.1 0.2 -0.4 -0.5 - I . 3 -0.4 -I.1" 4.5*

Data represent the mean value for four animals in each group. For visual clarity S.E.M. values are not shown. *Denotes significant difference (P < 0.05) between young and aged groups.

95 this 'reverse cholesterol transport' process and thus may have a significant impact on cholesterol homeostasis. These current studies were undertaken to examine the relationship between the key components of reverse cholesterol transport (LCAT and HDL) in rats where moderate hypercholesterolemia accompanies aging [1,2]. Our results show that in rats, most of the plasma LCAT activity was associated with the HDL fraction except when the plasma was subjected to more prolonged (40 h) ultracentrifugation, which resulted in the displacement of more than 90% of the LCAT activity into the lipoprotein depleted infranatant (d > 1.225 g/ml). These observations are similar to those made earlier [20,21] and suggest that prolonged ultracentrifugation results in the dissociation of the enzyme from its complex with HDL. Interestingly, the amount of the lipoprotein-associated LCAT activity that remained with the HDL fraction following the 40 h ultracentrifugation run, was considerably higher in the samples from aged rats as compared to those of young rats (Fig. 1). These data suggest that LCAT may be more tightly bound to HDL in the plasma of aged rats than in young rats. Whether this 'tighter' binding of LCAT to HDL in aged rats, is related to the decreased fractional rate of esterification (FRE) of FC with age [2], remains to be determined. Although plasma HDL is known to be the physiological substrate for LCAT, VLDL and LDL are also known to contribute lipid substrates for the reaction by first transferring them to HDL [18, 19]. The data presented in Table III indicate that in vitro net transfer of FC from lower density fractions (d < 1.07 g/ml) to higher density fraction (d = 1.07--1.21 g/ml) provides a major portion of the FC substrate for esterification in rat plasma during prolonged incubation and that this process is not substantially affected in aging. In accord with other studies [18,19], HDL was found to be a much better substrate than VLDL for LCAT. However, the HDL from young rats was a better substrate than HDL from aged rats especially at higher free cholesterol concentrations (Fig. 2). The results were the same regardless whether the LCAT was prepared from the plasma of young or aged rats. These data suggest that the decreased FRE observed in aged rats [2] is not a direct consequence of FC pool size or some defect in the LCAT enzyme itself but it is likely to be due to changes in the nature of the substrate lipoproteins. The decreased reactivity of LCAT towards the HDL substrate from aged rats may be due to changes in substrate composition or to the accumulation of product lipoproteins or both [22,23]. HDL represent a family of particles, differing in composition, size and density due to extensive remodeling during its maturation [23,24]. Although the relative proportion of total protein and lipid components of HDL (isolated according to Rudel et al. [7]) was the same in young and aged rats (Table I), several significant differences between properties of the lipoproteins of the two age groups were found. In accord with previous observations [25], our data showed that the relative amount of sphingomyelin increased in HDL and consequently the ratio of phosphatidyl choline/sphingomyelin, an important parameter for LCAT substrates [26], was lower in aged rats.

96 Another marked, age-related change observed during this study was in the apo E content of H D L in aged rats (Fig. 4). These findings are in good agreement with the large increases found in the lipoprotein fraction of d = 1.025--1.07 g/ml in aged rat plasma (Table II) as most of the apo E rich HDLI has been shown to be present in this zone of buoyant density [24]. High molecular weight, apo E containing H D L species ( H D L 0 isolated in the same density range, have been found to accumulate in response to a number of metabolic and nutritional conditions which are known to be associated with decreases in the F R E of FC [27--29]. 'Nascent' apo E-rich HDL containing mainly phospholipid and FC are superior substrates for LCAT [30]. However, these discoidal (nascent) lipoprotein particles circulate only for a very brief period [31] as they are rapidly converted to spherical (mature) H D L via the action of LCAT. Consequently, the nascent H D L species have only been found in significant amounts in the plasma of patients with liver disease or with inherited LCAT deficiency [18, 32]. Our data show that mature H D L - E isolated from the rat plasma has a lower substrate potential for LCAT compared to HDL-A from rat plasma. This is in agreement with earlier findings using human lipoproteins [33]. Consequently, the increased levels of the HDL-E subclass in aged rats may also contribute to the decreased substrate potential of the H D L pool. This concept is supported by the findings that both lipoprotein fractions, isolated by density gradient ultracentrifugation (at d = 1.025--1.07 g/ml and 1.07--1.21 g/ml) elute in the same position as whole H D L (used as a substrate for LCAT) when subjected to gel chromatography (Fig. 3). The data presented in Table III suggest that lipoproteins of d = 1.025--1.07 g/ml (LDL + HDLI) are relatively poor substrates for LCAT as there is no change in the CE content of this fraction upon incubation of the whole plasma. Only the FC derived from the lipoprotein fraction of d = 1.07--1.21 g/ml provides the substrate (FC) for LCAT. The decreased F R E in the plasma of aged rats may thus be due to the increased contribution of those components (LDL + HDLI) to the lipoprotein pool that provide inferior substrates for the enzyme. These lipoprotein species (LDL + HDLI) contribute 38% of the total FC pool in aged rats while only 27% in young rats. The data presented in Table III shows that the main H D L component of d = 1.07--1.21 g/ml from aged rats is also a relatively poor substrate for LCAT. The decrease of FC in this fraction upon incubation of the whole plasma was 2.3 mg/dl in young rats and only 1.1 mg/dl in aged rats even though the initial amount of FC was comparable in both pools (Table III). The H D L fraction (d = 1.07--1.21 g/ml) in aged rats was enriched in apoE and thus may lead to decreased reactivity with LCAT. Further support for this mechanism comes from preliminary studies of young and aged rats treated with ethinyl estradiol. In the plasma of these animals there was no difference in the FRE of FC apparently because ethinyl estradiol treatment markedly reduced apo E containing H D L in aged rats [34]. Swaney et al. have shown [35] that upon incubation of rat plasma, the H D L became enriched in core lipid components in addition to apo E and apo A-IV. They reasoned that the increased amounts of apo E would allow a more efficient receptor mediated removal of the

97 ' p r o d u c t ' H D L f r o m the c i r c u l a t i o n . In this study, the H D L i s o l a t e d f r o m a g e d rats h a d an i n c r e a s e d a p o E c o n t e n t . C o n s e q u e n t l y , the a c c u m u l a t i o n o f this a p o E rich H D L in the p l a s m a o f a g e d rats suggests t h a t the r e c e p t o r m e d i a t e d u p t a k e o f these l i p o p r o t e i n s m a y be d e c r e a s e d d u r i n g a g i n g [36]. ACKNOWLEDGEMENTS This research was supported

by f u n d s f r o m the N a t i o n a l I n s t i t u t e o f H e a l t h

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