Influence of lipoprotein lipids, dietary fat and smoking on macrophage degradation of native and oxidized low density lipoprotein

ATHEROSCLEROSIS ELSEVIER

Atherosclerosis 110 (1994) 13-23

Influence of lipoprotein lipids, dietary fat and smoking on macrophage degradation of native and oxidized low density lipoprotein Jan Regnstrijma,

G&an

Walldius”, Karin Hidella, Jan Johanssona, Anders G. Olssonb, Jan Nilsson*”

Ingar Holmec,

‘King Gustaf V Research Institute and Department of Medicine, Karolinska Hospital, I71 76 Stockholm, Sweden bDepartment of Medicine, University of Linkiiping, Linkijping. Sweden ‘Institute for Medical Statistics, Vllevaal Hospital, Oslo, Norway

(Received 30 December 1993; revision received 5 April 1994; accepted

I1 April 1994)

Abstract Relatively little is known about the biological mechanisms by which lipoproteins promote atherogenesis. It has, however, been shown that structural modification of low density lipoprotein (LDL), such as by oxidation, results in their uptake and degradation by intimal macrophages and consequently leads to formation of lipid-rich atherosclerotic lesions. The aim of the present investigation was to study the influence of dietary intake of fat, lipoprotein lipid composition, smoking and gender on macrophage degradation of LDL before and after oxidation. The study group consisted of 48 males and 56 females with hyperlipidemia taking part in the open prerandomization phase of the Probucol

Quantitative Regression Swedish Trial (PQRST). Analysis including lipoprotein determinations, dietary and smoking habit interviews, LDL degradation by macrophages, LDL receptor binding and LDL thiobarbituric acid reactive substance (TBARS) levels before and after copper ion-induced oxidation was done during the prerandomization phase of the study. Increased plasma and very low density lipoprotein (VLDL) triglyceride levels were associated with an increased macrophage degradation of native LDL, whereas no such association was found after oxidation of LDL. The dietary intake of polyunsaturated fatty acids (PUFA) was also inversely related to the degradation of native LDL by macrophages, but increased the rate at which oxidized LDL was degraded. Smoking and gender did not influence the rate of macrophage degradation of native or oxidized LDL. It is concluded that hypertriglyceridemia is associated with an increased macrophage degradation of LDL. This may represent a mechanism by which hypertriglyceridemia promotes atherosclerosis. Keywords:

Low density lipoprotein;

Oxidation;

Macrophage;

1. Introduction

A high dietary intake of fat, hyperlipidemia and smoking are well-established risk factors for coro* Corresponding author, Tel.: 46 8 7293245; Fax: 46 8 311298. 0 1994 Elsevier Science Ireland 0021-9150/94/$07.00 SSDI 0021-9150(94)05265-K

Hypertriglyceridemia

nary heart disease (CHD) [1,2], but little is still known about the biological mechanisms by which they promote the development of atherosclerotic plaques. Accumulation of lipoprotein-derived cholesterol in intimal macrophages is one of the hallmarks of atherosclerosis and the mechanism

Ltd. All rights reserved

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J. Regnsrriim et al. /Atherosclerosis

responsible for this accumulation is likely to be a key factor in atherogenesis [3]. Macrophages have few receptors for LDL, suggesting that mechanisms other than LDL receptor-mediated uptake of cholesterol may be important in intimal cholesterol accumulation. Structural modification of LDL has been demonstrated to increase its affinity for macrophage cell surface binding sites [4]. The most studied modification in this respect is oxidation, which leads to uptake of LDL in cultured macrophages by specific scavenger receptors [5,6]. By using antibodies against epitopes present on oxidized LDL, it has been shown that much of the lipid present in intimal macrophages is oxidized t7,81. In the present study, which was performed during the open prerandomization phase of the Probucol Quantitative Regression Swedish Trial (PQRST), we investigated the influence of dietary intake of fat, lipoprotein lipid composition, smoking and gender on LDL degradation by macrophages, LDL lipid peroxide content and binding of LDL to the LDL receptor before and after oxidation by copper ions. 2. Methods 2.1. Patients and study design Subjects eligible for inclusion in the PQRST study were men and women below the age of 71, with a total cholesterol above 6.88 mmol/l, LDL cholesterol above 4.53 mmol/l and total triglycerides below 4.0 mmol/l, at the time of referral [9]. Patients were initially given dietary advice (see below) aiming at a reduction of the intake of saturated fat and cholesterol and increasing the intake of mono- and polyunsaturated fat. After three months of diet, patients were given 8-16 g of cholestyramine and probucol placebo for another two months. Patients responding with at least an 8% lowering of total cholesterol on this treatment continued with diet, cholestyramine and probucol O.Sg b.i.d. to replace placebo for another two months. Included in the present study were 104 consecutive patients recruited during the last part of the recruitment phase of PQRST who had reached the diet period of the prerandomization phase of the trial.

110 (1994) 13-23

2.2. Dietary advice and analysis An initial dietary interview based on a five-day registration of the patients dietary habits was done by a dietitian at the beginning of the study period. Dietary advice (equivalent to the American Heart Association Step 1 diet) focusing on a reduction of the intake of total fat, saturated fat and cholesterol was given to the patient. Increased intake of mono- and polyunsaturated fat was also recommended to maintain a polyunsaturated to saturated fatty acids (P/S) ratio of about 0.8. Based on a data program and food tables for basic nutrients of the Swedish National Food Administration, intake of saturated, mono- and polyunsaturated fatty acids was calculated [ 10,111. A second complete dietary analysis was performed seven months after the initial dietary instructions were given. During the study period, 62 of the original 104 subjects were excluded from the study according to the PQRST study protocol [9] and the final dietary analyses were, consequently, only performed on the remaining 42 individuals (22 males and 20 females). These data were related to the functional characteristics of native and oxidized LDL isolated from blood samples drawn after three months of diet. Less extensive dietary interviews were performed every two months during the study period and showed that the change in dietary habits occurred during the three first months of the study and that the habits remained constant after that. Thus, the data obtained at the final dietary interview should be representative also for the time when the blood samples for isolation of LDL were taken. None of the patients used vitamin supplements. 2.3. Smoking habits The patients were interviewed about their smoking habits and divided into three groups; those who never had smoked, former smokers (stopped smoking for at least two months) and present smokers. The number of cigarettes smoked per day, and the estimated ‘smoke-years’ (average number of cigarettes smoked per day times the number of years they had smoked), were other grouping characteristics. Before blood sampling, the patients were instructed to refrain from smoking for at least 12 h.

J. RegnstrBm et al. /Atherosclerosis

2.4. Analysis of the effects of oxidation on LDL lipid TBARS, LDL binding properties and macrophage degradation of LDL

Ten to twelve weeks after the patients had been given dietary instructions, venous blood was drawn after overnight fasting into precooled vacutainer tubes containing 1 mg/ml of disodium EDTA. Plasma was recovered by means of lowspeed centrifugation (1400 g, 20 min) at 1°C and kept at this temperature throughout the separation procedures, LDL (density interval 1.025- 1.050 kg/l) was isolated from plasma by sequential preparative ultracentrifugation in a 50.3 Ti Beckman fixed angle rotor (Beckman L8-80 ultracentrifuge) at 40 000 rev./min for 20 h. The total protein content of the LDL preparation was determined according to Lowry [ 121. LDL was labeled with 125I essentially as described by McFarlane [ 131. The labeling procedure did not result in oxidative modification of LDL as assessed by the LDL content of TBARS or mobility on agarose gel. The iodinated LDL was dialyzed against 0.15 M NaCl/l mM EDTA, pH 7.4, filtered through a 0.45 pm filter and stored at 4°C. The specific activity of the preparations ranged from 400-600 countslminlng protein. Oxidation of the iodinated LDL was done by first dialyzing the LDL extensively against 0.15 M NaCl without EDTA overnight and then 200 pg of LDL was incubated in 1 ml of Ham’s F- 10 medium with the addition of 50 &ml penicillin, 50 U/ml streptomycin and 5.0 PM CuSO, for 18 h at 37°C. Presence of lipid peroxides in LDL before and after oxidation were measured by determining the amount of TBARS as described by Yagi [14] and expressed as malondialdehyde (MDA) equivalents. For analysis of LDL receptor binding, confluent cultures of human lung embryonic fibroblasts or peripheral vein smooth muscle cells (both cell types were found to have equal capacity for binding LDL) grown in 12-multiwell plates were incubated in Ham’s F-12 medium containing 5% human lipoprotein-deficient serum for 24 h. The cultures were then cooled for one h at 4°C and incubated for 2h at 4°C with Ham’s F-12 medium containing 5% lipoprotein deficient serum (LPDS) with the addition of 10 p g/ml of native or oxidized

110 (1994) 13-23

15

‘*‘I-LDL. The specific receptor binding of LDL was determined by measuring the amount of radioactivity released by heparin as described by Brown and Goldstein [ 151. The binding of LDL was expressed as ng LDL per mg cell protein. For analysis of macrophage degradation of LDL, macrophages were isolated from the peritoneal cavity of female NMRI mice and cultured in 1Zmultiwell plates in Ham’s F- 10 medium supplemented with 10% fetal calf serum for 24 h. They were then incubated with Ham’s F-10 medium containing 10 pg/rnl of native or oxidized 1251LDL for 5 h at 37°C. The cells were cooled to 4°C and the amount of degraded ‘251-LDL determined by measuring the trichloroacetic acid-soluble ‘25I in the medium. The LDL degradation was expressed as pg LDL degraded per mg cell protein. A detailed account of the LDL receptor binding assay and the macrophage degradation assay has been given earlier [ 161. All analyses were carried out on individual patients. Analysis of duplicate samples demonstrated an intraassay variation of less than 5% for the macrophage LDL degradation and TBARS assays and less than 20% for the Iibroblast LDL binding assay. The variablity was also evaluated by comparing LDL isolated from 35 individuals with a one year interval. The correlation coefticients between the analyses was 0.68 (P < 0.0005) for macrophage LDL degradation assay, 0.64 (P < 0.001) for the TBARS assay and 0.54 (P < 0.005) for the libroblast LDL binding assay. 2.5. Lipoprotein determinations EDTA and mertiolate were added to the serum to a final concentration of 1.3 mmol/l and 0.25 mmol/l, respectively. Very low density lipoprotein (VLDL) was separated from LDL and high density lipoprotein (HDL) lipoproteins by preparative ultracentrifugation at d = 1.006 kg/l. LDL and HDL were separated by precipitation of LDL in the infranatant. LDL concentrations of triglycerides and cholesterol were calculated by subtraction of HDL from the concentration before precipitation [ 171.Cholesterol was determined according to the method of Zlatkis [18] and triglycerides according to the method of Fletcher [19] in each fraction.

J. Regnstriim et al. /Atherosclerosis

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2.6. Statistics

13-23

exposure to copper, there was a sevenfold increase in the degradation of LDL by the macrophages. Oxidation of LDL also resulted in an approximately 50% reduction of LDL receptor binding and a twenty-fold increase in the LDL TBARS content (Table 2). Subgroup analysis showed that native LDL isolated from males was degraded by macrophages at an approximately 30% higher rate than LDL from females, but this difference did not reach statistical significance (P = 0.0’7). Otherwise, there were no differences in macrophage degradation, LDL receptor binding or TBARS in native or oxidized LDL between males versus females and smokers versus nonsmokers (Table 2). Further evaluation revealed no difference between former smokers and subjects who never smoked before, nor was there any relation between the amount of tobacco consumed among the smokers and any of these variables (data not shown). The age and body mass index (BMI) of the patients did not influence LDL receptor binding, rate of macrophage degradation or the LDL TBARS content. There were significant associations between the plasma triglyceride levels, and also the cholesterol and triglyceride contents of VLDL, and the rate at which native LDL was degraded by macrophages

Conventional methods were used to determine means and standard deviations. Coefficients of skewness and kurtosis were calculated to test deviations from normal distribution. Differences in continuous variables were tested by Student’s unpaired two-tailed t-test. Relations between cell biological and lipid variables were analyzed by computation of correlation coefficients. Multiple stepwise linear regression was performed to analyze the independent relationships. The variable with the highest partial correlation coefficient was entered at each step until no variable remained with an F value (F to enter) of 4 or more. 3. Results The basic clinical characteristics of the study group are presented in Table 1. Plasma triglycerides, VLDL cholesterol and triglyceride levels were higher in males, whereas HDL levels were higher among the females. Exposure of cultured mouse peritoneal macrophages to native ‘251-labeled LDL resulted in a small, but clearly detectable degradation of the labeled LDL. Following oxidation of LDL by

Table 1 Basic patient

110 (1994)

characteristics All (n = 104)

Age (years) BMI (kgim2)

Males (n = 48)

Females

(n = 56)

P

Mean

S.D.

Mean

S.D.

Mean

S.D.

53.5 24.6

9.4 2.8

50.7 24.9

9.4 2.3

55.9 24.4

8.8 3.2

< 0.005 ns

I .44

Total plasma (mmol/l) Cholesterol Triglycerides

9.05 2.16

1.50 0.97

8.92 2.38

1.57 1.11

9.16 I .97

0.78

ns <0.05

LDL Cholesterol Triglycerides

6.56 0.55

1.48 0.17

6.38 0.52

1.64 0.14

6.72 0.57

1.33 0.19

ns ns

VLDL Cholesterol Triglycerides

0.93 1.36

0.51 0.80

I .07 1.59

0.55 0.92

0.81 1.17

0.45 0.63


HDL Cholesterol Triglycerides

1.45 0.18

0.37 0.04

1.34 0.17

0.31 0.04

1.54 0.18

0.33 0.03

<0.005 ns

BMI, body mass index; HDL, high density lipoprotein.

lipoprotein;

LDL. low density

lipoprotein;

ns. non-significant;

VLDL. very low density

LDL, low density

lipoprotein;

0.8 12.1

2.0 40.2 equivalents;

30.6 16.2

0.24 I .46

38.2 20.9

0.40 2.47

MDA eq, malondialdehyde

0.8 12.8

Native Oxidized

2.1 39.6

0.23 0.23

36.2 16.0

0.34 2.31

43.2 Native 21.7 Oxidized TBARS (nmol MDA eq/mg LDL protein)

Native Oxidized B,E-receptor binding (ng LDLimg cell protein)

Males (n = 48) Mean S.D.

by tibroblasts

(n = 104) Mean S.D.

binding Gender

LDL receptor

Total group

by macrophages,

Macrophage degradation (rg LDL/mg cell protein)

Table 2 LDL degradation

0.8 13.0

39.9 15.8

0.22 1.36

thiobarbituric-acid

2.2 38.9

ns ns TBARS,

47.5 23.0

0.35 2.24

substances.

2.0 39.9

0.8 15.4 reactive

30.3 14.5

41.1 21.1 46.3 18.7

0.8 11.3

0.04 1.35

SD.

ns ns

ns ns

ns ns

P

by copper

0.34 2.34

0.22 1.55

No (n = 71) Mean

before and after oxidation

Yes (n = 33) Mean S.D.

ns ns

ns ns

P

content

Smoking

muscle cells and TBARS

ns, non-significant;

2.1 39.2

47.1 22.3

0.31 2.17

Females (n = 56) Mean SD.

and smooth

ions

J. Regnstriim et al. /Atherosclerosis

18

(Table 3). Low HDL cholesterol levels were also associated with an increased macrophage degradation of native LDL. There was a weak inverse relation between the LDL cholesterol levels and macrophage degradation of native LDL. In stepwise regression analysis, VLDL cholesterol entered the equation as first variable and no other variables were subsequently found to be independently related to the rate of macrophage degradation of native LDL. When the patients were divided into a hypertriglyceridemic (plasma triglycerides L 2.3 mmol/l, n = 38) and a normotriglyceridemic group (plasma triglycerides < 2.3 mmol/l, n = 66), the rate of macrophage degradation of native LDL was found significantly increased in the hypertriglyceridemic group (Table 4). Compositional analysis revealed that LDL from hypertriglyceridemic subjects was triglyceride enriched as compared with LDL from normotriglyceridemic subjects (0.64 f 0.21 vs. 0.50 f 0.11 mmol/l, P < 0.001) but contained comparable concentrations of cholesterol (6.35 f 1.39 versus 6.68 f 1.53 mmol/l, P = 0.276). There was no significant difference in alcohol intake between the hyper- and

Table 3 Correlation

coefficients

between lipoprotein Macrophage (pg LDL/mg

Total plasma (mmolil) Cholesterol Triglycerides VLDL Cholesterol Triglycerides

lipid composition degradation cell protein)

110 (1994)

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normotriglyceridemic group (13.3 f 11.9 g/day vs. 12.1 f 12.9 g/day). Following oxidation, LDL was degraded at a similar rate in the two groups. The binding of native and oxidized LDL to receptors on libroblasts and smooth muscle cells was not affected by the lipid composition of the lipoprotein fractions. Except for a weak association between the HDL cholesterol level and the amount of TBARS formed in oxidized LDL, there was no relation between lipoprotein lipid composition and the amount of TBARS in native or oxidized LDL. Following the dietary instructions the patients decreased their daily intake of fat from 81.6 f 30.1 g/day to 65.3 i 20.4 g/day. They also increased their total intake of PUFA from 13.0 * 5.9 g/day to 16.7 g/day and the polyunsaturated/saturated (P/S) ratio increased from 0.45 f 0.22 to 0.83 f 0.32. These dietary changes were associated with a 3% reduction of the plasma cholesterol level and a 10% reduction of the plasma triglyceride level. The relative dietary energy intakes of fat, saturated, mono- and polyunsaturated fatty acids representing three months of dietary treatment were then related to the functional characteristics of native and oxidized LDL.

and the cell biological B,E-receptor (ng LDUmg

properties

binding cell protein)

of dative and oxidized LDL (n=lO4) TBARS (nmol MDA eq/mg LDL protein)

Native LDL

Oxidized LDL

Native LDL

Oxidized LDL

Native LDL

Oxidized LDL

-0.18 0.35***

-0.05 -0.05

0.08 -0.02

0.06 -0.02

0.04 -0.14

-0.06 -0.15

0.36*** 0.39***

-0.03 -0.03

0.00 -0.03

0.06 -0.03

-0.13 -0.16

-0.14 -0.10

-0.01 -0.01

-0.01 0.03

0.12 -0.05

-0.14 -0.19

0.01 0.18

0.09 -0.05

LDL Cholesterol Triglycerides

-0.19* 0.11

0.07 -0.05

HDL Cholesterol Triglycerides

-0.31** 0.06

0.00 -0.13

0.16 0. I4

HDL, high density lipoprotein; LDL, low density lipoprotein; MDA eq, malondialdehyde equivalents; reactive substances; VLDL, very low density lipoprotein. *P < 0.05, **P < 0.005. ***P < 0.0005

TBARS,

0.24* 0.07 thiobarbituric-acid

J. Regnstriim et al. /Atherosclerosis Table 4 LDL degradation by macrophages, triglyceridemic subjects

LDL receptor

Plasma

binding

and TBARS

triglyceride

Macrophage (pg LDL/mg Native Oxidized B,E-receptor (ng LDL/mg Native Oxidized TBARS (nmol MDA Native Oxidized

in normo-

and hyper-

P

~2.3 (n = 38) Mean

S.D.

0.30 2.36

0.21 1.47

SD.

0.43 2.22

0.24 1.31

0.0055 ns

binding cell protein) 45.9 22.0

39.4 14.5

38.7 21.3

30. I 18.4

ns ns

2.2 41.8

0.8 12.7

I.9 35.9

0.9 12.2

ns ns

eq/mg LDL protein)

MDA eq, malondialdehyde

equivalents;

A high intake of PUFA, and a high P/S ratio, was coupled to a low rate of degradation of native LDL by macrophages (Table 5). In contrast, a high intake of PUFA and a high P/S ratio were associated with an increased rate of LDL degradation following oxidation. There were no relations between the dietary intake of fatty acids and LDL

coefficients

between dietary

Macrophage (rg LDLimg

Fat E’%I Vitamin E MUFA E% SAFA E’X PUFA E’%i P/S ratio

before and after oxidation

degradation cell protein)

LDL, low density lipoprotein; stances.

Table 5 Correlation (n = 42)

content

19

level (mmol/l)

<2.3 (n = 66) Mean

I10 / 1994) 13-23

ns, non-significant;

thiobarbituric-acid

reactive sub-

receptor binding or TBARS content of native and oxidized LDL. 4. Discussion To clarify the mechanism by which hyperlipidemia is associated with atherosclerosis, it is

intake of fat and fatty acids and the cell biological

degradation cell proteiu)

TBARS,

B,E-receptor (ng LDL/mg

binding cell protein)

properties

of native and oxidized

LDL

TBARS (nmol MDA eqimg LDL protein)

Native LDL

Oxidized LDL

Native LDL

Oxidized LDL

Native LDL

Oxidized LDL

-0. I I -0.27 -0.02 0.12 -0.31** -0.37**

0.14 0.29 0.01 -0.14 0.39** 0.32*

0.15 -0.33* 0.26 0.23 -0.16 -0.18

0.13 -0.39** 0.19 0.22 -0.10 -0. I8

-0.05 -0.18 0.04 0.09 -0.17 -0.05

-0.24 -0.07 -0. I3 -0. I7 -0.27 0.00

E’X, percent of daily energy intake; LDL, low density lipoprotein; MDA eq, malondialdehyde equivalents; TBARS, thiobarbituricacid reactive substances; MUFA, monounsaturated fatty acids; SAFA. saturated fatty acids; PUFA, polyunsaturated fatty acids: P/S. polyunsaturated/saturated. *P < 0.05, **p < 0.02

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J. Regnsrrb’m et al. /Atherosclerosis

necessary to first identify the biological mechanisms by which lipoproteins induce formation of cholesterol-loaded macrophages, inflammation and muscle cell hyperplasia in the .arterial intima. In its native state, LDL is taken up and degraded by macrophages only at a very low rate. In theory, this restricted ability of macrophages to ingest LDL should protect against the formation of lesions containing lipid-laden macrophages. However, accumulation of cholesterol in intimal macrophages is one of the major characteristics of both the early fatty streak and late stages of atherosclerosis and the processes mediating this uptake are thus likely to play a key role in the formation of atherosclerotic plaques. It was initially shown that acetylation of LDL markedly enhanced its uptake by macrophages and that this uptake was mediated by a specific receptor called the scavenger receptor [4]. It was later shown that also oxidative modification of LDL increases its aflinity for the scavenger receptor pathway [20,21] and several studies have now suggested that LDL may undergo oxidative modification in the arterial wall [5,6]. The results of the present study confirm our earlier observations that oxidation of LDL is associated not only with an increased uptake of LDL by macrophage scavenger receptors but also by a decreased binding to the fibroblast LDL (B,E) receptor [ 161.Moreover, they demonstrate that the macrophage degradation of native LDL occurs more readily with increasing plasma concentrations of triglycerides and with increasing concentrations of VLDL cholesterol and triglycerides. In contrast, high HDL cholesterol levels are associated with a decreased rate of macrophage degradation of LDL, as might be expected from the commonly found inverse relationship between plasma and VLDL triglyceride and HDL cholesterol concentrations. In individuals with triglyceride levels above 2.3 mmolll the average macrophage degradation of native LDL was about 50% higher than that of LDL from normotriglyceridemic subjects. Hypertriglyceridemia has been found to have a profound effect on the structure of all types of lipoproteins [22]. The LDL particle becomes small and dense and on a relative basis the core of the particle is cholesterol-depleted

I10 (I 994) 13-23

and triglyceride-enriched, whereas the surface layer is rich in protein, but contains decreased amounts of phospholipids and free cholesterol. Evidence that these changes alter the metabolism of LDL comes from studies showing that LDL from hypertriglyceridemic patients with coronary heart disease have an increased fractional catabolic rate (FCR) [23]. The increase in FCR is probably not mediated by the LDL receptor pathway since hypertriglyceridemic LDL has been shown to have a decreased affinity for LDL receptor binding [24]. A similar observation was made also in the present investigation, but did not reach statistical significance. Evidence for a genetic defect in the mechanisms regulating triglyceride metabolism and development of an atherogenic lipoprotein phenotype characterized by increased plasma triglyceride levels, increased VLDL mass, low HDL cholesterol and small lipid-depleted LDL particles has recently been presented by Austin and co-workers [25]. The present finding of an association between increased plasma triglyceride, high VLDL levels, low HDL cholesterol on the one hand and an increased macrophage degradation of LDL on the other hand may represent a possible mechanism explaining the atherogenicity of this lipoprotein disorder. Hypertriglyceridemic LDL may also be more susceptible to undergo oxidative modification and may thus contain some oxidized lipids already in its native state, or acquire these during the experimental procedure, leading to an increased uptake of the LDL through the scavenger receptor pathway. Some support for this explanation comes from studies demonstrating that small and dense LDL particles (which are known to accumulate in hypertriglyceridemic states) are more susceptible to oxidative modification than large and light LDL particles [26,27]. The hypertriglyceridemic LDL was not found to contain increased amounts of lipid peroxides as compared to normotriglyceridemic LDL, but it may be that this assay is too insensitive to identify small differences in lipid peroxide levels (Table 4). No associations were found between the plasma or LDL cholesterol levels and the macrophage degradation, LDL receptor binding or TBARS levels of native and oxidized LDL. In contrast,

J. Regnstriim et al. /Atherosclerosis

Lavy et al. [28] found that oxidation of LDL isolated from patients with familial hypercholesterolemia was associated with increased TBARS levels and macrophage uptake as compared with normolipidemic subjects. The inclusion of mainly hypercholesterolemic patients in our study may explain the lack of association between cholesterol levels and macrophage degradation of oxidized LDL. In the present study, we found no differences in LDL TBARS levels between smokers and nonsmokers either before or after copper oxidation. Moreover, there was no difference between smokers and nonsmokers regarding the rate of macrophage degradation of LDL or the binding to LDL receptors before or after oxidative modification These findings suggest that smoking does not influence the functional properties of native LDL and does not lead to an increased oxidative moditication of circulating LDL. Neither does smoking appear to affect the properties and lipid peroxide content of extensively oxidized LDL. Since smokers were asked to refrain from smoking for at least 12 h before blood sampling, the acute effect of smoking could not be evaluated. Smoking has not been found to alter the susceptibility of LDL to copper induced oxidation 1291,whereas extensively oxidized LDL from smokers contained higher amounts of TBARS and promoted cholesterol ester accumulation in a macrophage cell line [30]. The reasons for these contradictory results remain to be explained but they may be due to differences in patient selection, oxidation procedures and assays for macrophage cholesterol uptake. Double bonds of fatty acids are susceptible to oxidation initiated by an attack of free radicals [31]. A diet rich in PUFA increases the susceptibility of LDL to oxidation, whereas dietary substitution with the MUFA, oleic acid, was shown to increase the resistance of LDL to oxidation [32-341. Accordingly, the present investigation demonstrated a positive association between the intake of PUFA and the rate of macrophage degradation of extensively oxidized LDL. An increased amount of substrate for the oxidation process in PUFA-enriched LDL may result in a more profound structural reorganization of the particle when it is exposed to a strong oxidative

110 (1994)

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21

stress as used in our studies. However, it is not clear whether such extensive oxidation may occur in vivo. There was a significant inverse relation between the intake of PUFA and the degradation of native LDL by macrophages, which is more diflicult to understand. One possible explanation for this finding is that increased amounts of antioxidants in polyunsaturated dietary fats leads to a more effective protection against oxidative modification of circulating LDL. Another possibility is that enrichment of PUFA in LDL is accompanied by non-oxidation dependent structural changes in LDL leading to a decreased macrophage degradation. A recent clinical study has shown an association between the susceptibility of isolated LDL to undergo oxidative modification and the severity of coronary atherosclerosis [35]. In that study, the LDL cholesterol level and the LDL oxidation susceptibility were independently related to the severity of coronary atherosclerosis, whereas the association between the LDL triglyceride level and coronary atherosclerosis disappeared after adjustment for LDL oxidation susceptibility. In the present study we have analyzed macrophage degradation, LDL receptor binding and TBARS content of LDL before and after it has been extensively oxidized by exposure to copper ions. This approach measures only the functional characteristics of native and oxidized LDL and is not likely to provide information about the susceptibility of LDL to become oxidatively modified. In the present study we found no relation between LDL lipid levels and the degradation, binding properties or TBARS levels of extensively oxidized LDL. Taken together the findings of these two studies suggest that the lipid composition of LDL plays an important role in determining its proneness to oxidation but has little influence on the functional and biochemical characteristics of the extensively oxidized particle. It should, however, be kept in mind that oxidation of LDL in vivo may be less extensive and associated with different binding and biochemical characteristics than the in vitro oxidized LDL used in the present study. Besides earlier findings of an association between hypertriglyceridemia and decreased insulin

22

J. Regnstriim

et al. /Atherosclerosis

resistance and impaired tibrinolytic function [36,37], the present results demonstrate that hypertriglyceridemia is also associated with an increased affinity of LDL for degradation in macrophages. This may be yet another mechanism by which hypertriglyceridemia may be involved in CHD. The present study further suggests that the functional characteristics of both native and oxidized LDL are influenced by the dietary intake of PUFA, but the relative role of this influence on the atherogenic properties of LDL remains uncertain.

8

9

10 11

Acknowledgments

12

This study was supported by grants from the Marion Merrell Dow Research Inc. (Kansas City, Missouri, USA), the Swedish Medical Research Council (19x-204, 6992 and 83 1l), the Knut and Alice Wallenberg Foundation, King Gustaf V 80th Birthday Fund, the Swedish Heart-Lung Foundation, Nanna Svartz Fund, Svenska Tobaks AB and the Swedish Margarine Industrial Association for Nutritional-Physiological Research. We thank K. Carlson and K. Eriksson for expert technical assistance provided during these studies, and Inger Malmeus at DAFA, Stockholm for help in constructing the data base.

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