Effects of dietary supplementation with marine lipid concentrate on the plasma lipoprotein composition of hypercholesterolemic patients

Atherosclerosis, 79 (1989) 157-166 Elsevier Scientific Publishers Ireland,

ATHERO

157 Ltd.

04391

Effects of dietary supplementation with marine lipid concentrate on the plasma lipoprotein composition of hypercholesterolemic patients Papasani V. Subbaiah, Michael H. Davidson, Mary C. Ritter, W. Buchanan and John D. Bagdade Department

of Medicine, Section of Endocrinology and Metabolism, and Department Rush-Presbyterian-St. Luke’s Medical Center, Chicago, IL (U.S.A.)

of Biochemistry,

(Received 23 November, 1988) (Revised, received 20 May, 1989) (Accepted 1 June, 1989)

Summary Although the triglyceride-lowering actions of n - 3 fatty acids of marine lipids are now well-recognized, their effects on plasma lipoproteins have not been studied systematically in patients with hypercholesterolemia. To address this question, we supplemented the Phase 1 American Heart Association diets of 14 hypercholesterolemic ambulatory outpatients with a commercially available preparation of marine lipid concentrate (SuperEPA) containing 7.5 g n - 3 fatty acids per day and studied their plasma lipids and lipoproteins before and after 30 days of treatment. Both plasma triglyceride and cholesterol levels fell uniformly in all patients while the mean VLDL- and LDL-cholesterol decreased by 58% (P -c0.005) and 13% (P < 0.025) respectively. The decrease in whole plasma cholesterol was significantly correlated with the fall in LDL-cholesterol (r = 0.85, P < O.Ol), and not VLDL-cholesterol (r = 0.39, NS). Among the other potentially beneficial actions observed was an increase in HDL, in all patients (mean increment 41%, P -C0.005) and an increase in the HDLJHDL, ratio (+ 46%, P < 0.001) and decreases in the LDL/HDL ratio ( - 14%, P < 0.005) and in the unesterified cholesterol/lecithin ratio (- 17%; P < 0.001) in plasma. The increase in the unesterified cholesterol/esterified cholesterol ratio in VLDL and HDL, suggested that marine lipid therapy resulted in a reduction in the size of lipoprotein particles in these fractions. Since these changes may reduce cardiovascular risk, these findings suggest that marine lipids may prove useful in the treatment of certain patients with hypercholesterolemia.

Correspondence to: Dr. P.V. Subbaiah. Dept. of Medicine, Section of Endocrinology and metabolism, Rush-PresbyterianSt. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, U.S.A. Tel.: (312) 942-4866. Abbreuiutions: EC. esterified cholesterol; FCH, familial combined hyperhpidemia; FH, familial hypercholesterolemia; HDL. high density lipoproteins; LDL. low density lipopro-

0021-9150/89/$03.50

0 1989 Elsevier Scientific

Publishers

Ireland,

teins; LPC, lysophosphatidylcholine (lysolecithin); PC. phosphatidylchohne (lecithin); PE. phosphatidylethanolamine; PI, phosphatidyhnositol; PS. phosphatidylserine; Sph. sphingomyehn; TC, total cholesterol; TG, triglycerides; TLC, thin layer chromatography; UC. unesterified cholesterol; VLDL. very low density lipoproteins.

Ltd.

158 Key words:

n - 3 fatty acids; Hypercholesterolemia; lecithin ratio; Phospholipids

Introduction Interest in the potential benefits of dietary supplementation with marine lipids on cardiovascular risk have been stimulated by the observation [l] that the population of Greenland Eskimos who consume large amounts of fish experience significantly less coronary heart disease. This reduction in coronary risk is believed to relate to the capacity of the n - 3 fatty acids of marine lipids to lower triglyceride levels, and to decrease platelet aggregation and blood pressure [2]. While the triglyceride-lowering property of marine oils has been well documented [3-71, their effects on plasma lipoproteins in hypercholesterolemic subjects without hypertriglyceridemia have not been systematically studied. Previous studies in which marine lipids were fed to patients with elevated cholesterol levels alone, and to those in whom plasma triglyceride and cholesterol were both increased, have demonstrated inconsistent changes in plasma cholesterol levels associated with significant reductions in plasma triglyceride [5,6]. In studies in ambulatory subjects ingesting normal Western diets, the impact of marine lipids on plasma HDL levels has been similarly inconclusive; one study reported no significant change [S] and another showed a slight increase but no alteration in the LDL/HDL ratio [6]. We have shown earlier in a double blind placebo-controlled study in a mixed group of hyperlipidemic subjects that dietary supplementation with marine lipid concentrate significantly lowered plasma TG and cholesterol levels [8]. Because of the paucity of information about the effects of fish oil on plasma lipoproteins in ambulatory free-living patients with hypercholesterolemia alone, we have now studied patients with normal or only slightly elevated plasma triglyceride levels and quantitated whole plasma and lipoprotein lipids with particular attention to the 2. major subfractions of HDL, namely HDL, and HDL,, the latter of which appears to be a better predictor of cardiovascular risk than total HDL-cholesterol

Lipoproteins;

HDL,;

Unesterified

cholesterol/

[9,10]. In contrast to previous studies, we find significant reductions in whole plasma and LDLcholesterol levels associated with uniform increases in the HDL, subfraction. This combination of changes suggests that marine oils may have application to the treatment of some patients with hypercholesterolemia. Methods

Subjects Fourteen non-obese patients (11 male, 3 female; mean age 59 f 8.5 years, range 44-69) with hypercholesterolemia were recruited for the study from clinics of Rush-Presbyterian-St. Luke’s Medical Center and through public appeal. All were within 15% of their ideal body weight. The majority had positive family histories of premature cardiovascular disease and were presumed to have an inherited basis for their hypercholesterolemia. Nine of the patients exhibited a type IIa phenotype, while the other 5 were of type IIb phenotype. Informed consent was obtained from each subject according to the guidelines established by the Institutional Human Investigation Committee. None were smokers, diabetic, or had liver or renal disease. All subjects had plasma cholesterol levels of at least 232 mg/dl (6 mmol/l), and plasma triglyceride levels no greater than 2 SD of the group mean (204 f 110 mg/dl; 2.31 f 1.24 mmol/l) at the time of initial screening. None was receiving lipid-lowering drugs, 4 were taking /l-blocking agents, and 2 others long-acting nitroglycerin preparations. Nine subjects had angiographic evidence of coronary artery disease, and 5 of these had previous incidence of myocardial infarction. Before the start of the study, the patients had been on American Heart Association Phase I diet for at least 3 months and had stable plasma cholesterol levels as evidenced by 2 determinations done 2-3 weeks apart before the start of dietary supplementation. During the one-month period of study, all patients continued their Phase I American Heart Association diets (adherence to the diet docu-

159 mented using 3-day food recall by registered dietitians) and their medications. There was no significant change in weight during this period in any patient. Lipoprotein separations After a 20-ml basal blood sample was obtained in EDTA following an overnight fast, the patients were given 15 l-g capsules of SuperEPA (donated by Res-Q International, Marlton, NJ and by Pharmacaps Inc., Elizabeth, NJ) daily which contained in total 7.5 g n - 3 fatty acids as methyl esters and 15 mg cholesterol. Analysis of the fatty acid composition of SuperEPA preparation by capillary GC showed the presence of 29% eicosapentaenoic acid (EPA), 19% decosahexaenoic acid (DHA) and 8% saturated fatty acids. Patient compliance was monitored by pill counting and physician interviews. After 30 days of ingesting the fish oil supplement, a second fasting blood specimen (20 ml) was collected. Blood samples before and after treatment were handled in an identical manner as follows. Plasma was obtained immediately by centrifugation at 1500 rpm for 15 min; small aliquots were frozen for subsequent analysis. A larger aliquot (5 ml) of plasma was centrifuged at 4°C in a Beckman 40.3 rotor at 100000 X g for 18 h to separate VLDL from the other lipoproteins. After slicing the tube to remove VLDL (supematant), the LDL, HDL,, and HDL, were separated from the infranatant by differential precipitation according to the method of Warnick et al. [ll]. To 2-ml of the infranatant, 0.2 ml dextran sulfate-Mg*+ solution (obtained by mixing equal volumes of 2% (w/v) dextran sulfate and 1 M MgCl,) was added and the mixture incubated at room temperature for 10 min. Following centrifugation of the LDL-containing precipitate at 1500 X g for 30 mm at 4°C the supematant fraction was removed with a Pasteur pipette. To 1.0 ml of the supematant, 0.1 ml of the HDL,-precipitating reagent (prepared by mixing 1 part 4% (w/v) dextran sulfate with 3 parts 2 M MgCl,) was added and the mixture incubated at room temperature for 15 min. The precipitate formed (HDL2) was centrifuged at 1500 x g for 30 min at 4O C and the supematant containing the HDL, was removed. The LDL and the HDLz precipitates each were resuspended by vortexing

in a volume of 0.15 M NaCl/l mM EDTA, equal to that of the whole plasma from which they were obtained. Lipid analysis Total cholesterol in whole plasma and in each lipoprotein fraction was determined by using a Boehringer-Mannheim kit. To estimate unesterified cholesterol, a similar reagent solution was prepared and employed without cholesterol ester hydrolase but containing 0.2 M potassium phosphate, pH 7.7, 9.8 mM phenol, 1.8 M methanol, 0.98 M aminoantipyrene, 3 mM sodium cholate, 150 mu/ml cholesterol oxidase, 100 mu/ml horseradish peroxidase and 0.1% Triton X-100. Two ml of this solution was added to either the plasma or lipoprotein fraction, incubated for 30 min at room temperature, and the color measured at 500 nm. Lyophilized plasma (Ortho Pharmaceuticals) was used as standard. Crystalline cholesterol (Sigma Chemical Co.) dissolved in isopropanol was also used as calibration standard. The values for the 2 standards agreed within 5% of each other. For the analysis of phospholipids, the plasma fractions were extracted with chloroform/ methanol [12], and the phospholipids separated from each other on silica gel TLC plates (Whatman), with the solvent system chloroform/methanol/acetic acid/O.15 M NaCl (50 : 25 : 8 : 2.5, v/v). The phospholipids were identified with appropriate standards and after exposure to iodine vapor, were scraped from the plate and phosphorus determined by the modified Bartlett’s procedure [13]. Plasma triglyceride and HDL cholesterol were estimated by standardized methods [14]. This separately determined HDL cholesterol value, provided a reference for the cholesterol values which were estimated with the enzymatic method in each of the HDL subfractions isolated by differential precipitation. The mean difference of the values for HDL cholesterol determined in whole plasma by heparin-Mn2+ precipitation [14]. and the sum of cholesterol obtained in HDLz and HDL, by enzymatic methods after dextran sulfate-Mg*+ precipitation was 7%. Apoproteins A-I, A-II and B in whole plasma were measured by radial immunodiffusion in the laboratory of Dr. John Albers, at the University of Washington [15,16].

160 TABLE 1

TABLE 2

LIPIDS OF WHOLE PLASMA MARINE LIPID TREATMENT

BEFORE

AND

AFTER

Mean + SD (n = 14)

APOPROTEIN LEVELS IN HYPERCHOLESTEROLEMIC PATIENTS BEFORE AND AFTER MARINE LIPID TREATMENT Mean + SD (n = 14).

Lipid

TG TC UC LPC Sph PC PI+Ps PE UC/EC ratio UC/PC ratio Sph/PC ratio

pmol/ml

plasma

Before

After

2.307 + 1.245 8.137+ 1.505 2.225 + 0.376 0.158 f 0.050 0.521+0.119 2.133 kO.425 0.070 + 0.029 0.100 + 0.025 0.386 kO.081 1.057 +0.154 0.248 + 0.057

1.239 kO.549 6.693 + 1.409 1.821 kO.386 0.167 + 0.048 0.601+0.168 2.121 kO.532 0.118 +0.038 0.158 + 0.038 0.389 + 0.107 0.874&0.138 0.289 f 0.069

P values

< 0.001 < 0.001 < 0.001 NS < 0.025 NS < 0.025 < 0.001 NS < 0.001 < 0.025

Statistical analysis All values were calculated as mean f SD. The differences in pre- and post-treatment samples were compared by the two-tailed paired t-test. A P value of less than 0.05 was taken as significant. Correlation coefficients (Pearson’s r) were calculated by linear regression analysis.

Results The effect of dietary supplementation with marine lipid concentrate on the whole plasma lipids of these hypercholesterolemic patients is shown in Table 1. As shown previously [3-S], the plasma TG concentration decreased markedly. The magnitude of the decrease in the whole plasma TG was positively correlated with the pre-treatment concentration (r = 0.918; P < 0.01). In addition, a significant decline in the concentrations of TC and UC was observed. However, the decrease in TC of the plasma was not correlated with the initial cholesterol concentration (r = 0.468, NS) or with TG decrease (r = 0.411, NS). Despite the fall observed in plasma TG and cholesterol, however, all the phospholipids in plasma except for LPC and PC actually increased significantly. There was a significant decrease in the UC/PC ratio, but an increase in the Sph/PC ratio. No significant changes were observed in the

Apoprotein Apoprotein A-I Apoprotein A-II Apoprotein B A-I/A-II ratio

Before

After

(mg/dI)

(mg/dl)

138 k21 22 +7 145 f47 7.01 f 2.80

126 +29 18 f 7 146 +40 7.18+ 1.76

P values NS NS NS NS

plasma concentrations apoproteins A-I, A-II or B (Table 2). As expected from the decrease in plasma TG levels, there was a marked decrease in the concentration of total ( - 58%), esterified ( - 61%) and unesterified (-56%) cholesterol and most phospholipids in the VLDL fraction (Table 3). The decrease in VLDL TC was positively correlated with the decrease in plasma TG (r = 0.907; P < 0.01). Similar correlations were seen between the decrement in plasma TG and those observed in VLDL UC and PC (results not shown). However, the components of VLDL did not all decrease uniformly, indicating that the composition of these particles also changed. In addition, UC/EC ratio, an indirect estimate of surface to core volume of

TABLE 3 LIPID COMPOSITION OF VLDL IN HYPERCHOLESTEROLEMIC SUBJECTS BEFORE AND AFTER MARINE LIPID TREATMENT Mean + SD (n = 14). pmol/ml

TC UC EC LPC Sph PC PI+Ps PE UC/PC UC/EC Sph/PC

plasma

0.699 + 0.381 0.364kO.189 0.335 +0.196 0.009 * 0.009 0.046 + 0.031 0.514+0.316 0.013 + 0.010 0.029 + 0.027 0.777 + 0.213 1.162 + 0.339 0.091 f 0.029

After

P values

0.293 k 0.232 0.160 + 0.091 0.132kO.144 0.018 f 0.012 0.036 + 0.015 0.177 + 0.106 0.021+ 0.006 0.026 f 0.009 0.943 f 0.169 2.598 f 1.669 0.231 f 0.074

< 0.005 i 0.005 i 0.005 NS NS < 0.005 =z0.05 NS NS i 0.025 i 0.001

161 TABLE

4

10

LIPID COMPOSITION OF LDL MARINE LIPID TREATMENT

BEFORE

AND

AFTER

Mean + SD (n = 14). pmol/ml

TC UC EC UC/EC LPC Sph PC PI+Ps PE UC/PC Sph/Lec

plasma

Before

After

P values

5.732 1.347 5.047 0.273 0.053 0.321 0.765 0.038 0.034 1.802 0.422

4.967 k 1.482 1.08OkO.184 4.260 f 1.402 0.272 + 0.075 0.090 + 0.033 0.453+ 0.117 0.890 k 0.249 0.048 + 0.027 0.057 f 0.021 1.275 kO.319 0.522 +0.094

< 0.025 i 0.005 < 0.05 NS i 0.01 i 0.001 < 0.05 NS < 0.005 < 0.001 < 0.005

f 1.765 + 0.298 + 1.292 & 0.050 f 0.019 *0.104 + 0.198 f 0.011 + 0.011 kO.348 f 0.089

the particles, increased more than 2-fold following marine lipid supplementation suggesting smaller VLDL particles were formed. Although the Sph concentration of VLDL did not change significantly, the marked decrease in the concentration of PC resulted in an increase in Sph/PC ratios. In contrast to most components of VLDL, there was a significant increase in the PI + PS fraction. The effects of marine lipid supplementation on LDL lipids are shown in Table 4. Here also the mean TC (- 13%) EC (- 16%) and UC (- 20%) decreased significantly, but unlike in VLDL, no change was observed in the UC/EC ratio. Of the 14 patients, ten showed a decrease in LDL cholesterol while 4 showed an increase (Fig. 1). While most phospholipids increased, the UC/PC ratio fell and the Sph/PC ratio increased. The decrease in the TC content of LDL was positively correlated with the decrease in whole plasma TC (r = 0.851; P < 0.01) but not with the decrease in whole plasma TG (r = 0.032, NS). In contrast, the increase in LDL PC and the magnitude of the fall in whole plasma total cholesterol were inversely related (r = - 0.68, P -c0.05). However, the decrease in LDL TC and VLDL TC did not correlate with each other (r = 0.007). In contrast to the TC content of VLDL and LDL which both fell after marine lipid supplementation, increases in TC as well as in UC and PC were observed in HDL, (Table 5), although its

Post

Pre

Fig. 1. Changes in LDL TC in 14 hypercholesterolemic subjects following marine lipid supplementation for 30 days. Mean k SD for pre-treatment values is 5.732 f 1.765, and for posttreatment values 4.967 f 1.482 ( P < 0.025).

Sph content did not change. The increase in TC was found in all 14 patients studied with a mean increase of 41% (P -c0.005). There was a significant decrease in the UC/PC and Sph/PC ratios indicating that HDLz was altered both qualitatively and quantitatively. The decrease in UC/PC of HDL, was positively correlated with the decrease in plasma TG (Fig. 2). Except for an increase in UC, there were no other significant quantitative changes in HDL, (Table 6). As a consequence of this increase in UC, however, the UC/EC (+19%) and UC/PC (+ 8%) ratios both increased. The LDL/HDL cholesterol ratio declined significantly (mean f SD, 5.540 f 1.616 pretreatment, 4.752 + 1.373, post-treatment, P < 0.005) (Fig. 3) after marine lipid supplement, whereas the HDL,/HDL, cholesterol ratio increased (mean + SD. 0.194 +

TABLE

5

LIPID COMPOSITION AND AFTER MARINE

OF HDL, FRACTION LIPID TREATMENT

BEFORE

Mean f SD (n = 14). pmol/mI

TC UC Sph PC UC/PC UC/EC Sph/PC

plasma

Before

After

0.168 0.048 0.015 0.044 1.222 0.555 0.376

0.237 0.066 0.016 0.095 0.723 0.449 0.176

f 0.070 k 0.015 f 0.007 + 0.018 * 0.505 +0.551 & 0.136

P values f 0.104 + 0.023 f 0.007 f 0.029 f 0.251 f 0.149 & 0.077

i 0.005 < 0.005 NS < 0.001 < 0.005 NS < 0.005

162 r=0.687

iP
y=O.41x

to.061 0.60 t

f

0.40

-

0.30

-

P 0.20 0.10

I

I

-1

0

Decrease

3

2

1

in Plasma

TG

(,unoi/mll

Fig. 2. Correlation of the decrease in plasma decrease in VLDL-cholesterol.

TABLE

4

TG with

the

BEFORE

AND

pmol/ml

Discussion

plasma

Before

After

0.872 f 0.101 0.166 kO.069 0.093 + 0.041 0.068 +0.019 0.439 + 0.092 0.024+0.012 0.027 kO.016 0.258 kO.167 0.380 f 0.120 0.156 -f 0.042

0.823 0.189 0.086 0.070 0.457 0.028 0.021 0.308 0.412 0.157

0.283 f 0.113 posttreatment,

AFTER

Mean + SD (n = 14)

Sph PC PI+Ps PE UC/EC UC/PC Sph/PC

Fig. 4. Changes in the ratio of HDL,/HDL, TC after marine lipid treatment in individual hypercholesterolemic subjects. The mean+ SD for pre-treatment values is 0.194&0.085, and for post-treatment values 0.283 +0.113 (P i 0.001).

0.185 pretreatment, P < 0.001) (Fig. 4).

6

LIPID COMPOSITION OF HDL, MARINE LIPID TREATMENT

TC UC LPC

-

P values f f f f f f f f f k

0.093 0.059 0.020 0.019 0.072 0.004 0.010 0.125 0.097 0.042

??

NS < 0.01 NS NS NS NS NS < 0.025 < 0.05 NS

Post

Fig. 3. Changes in the ratio of LDL/HDL TC following marine lipid supplementation in individual hypercholesterolemic subjects. The mean f SD for pre-treatment values 5.540 f 1.616, and for post-treatment values, 4.752 f 1.373 (P -C 0.005).

While the triglyceride-lowering effects of n - 3 fatty acids are well documented [3-71, no prior studies have discerned clearly whether these polyunsaturated fatty acids have specific independent actions on the cholesterol-rich LDL in hypercholesterolemic patients without hypertriglyceridemia. Decreases in plasma cholesterol after dietary supplementation with n - 3 fatty acids have been demonstrated, but in most cases, the fall has been linked primarily to the decline occurring in VLDL [2,6,17,18], the neutral lipid core of which contains about 25% cholesterol. Furthermore, the majority of past studies in which changes in plasma cholesterol were monitored were performed either in normal volunteers or in patients hospitalized on metabolic wards where they were fed drastically altered diets [2,17]. In this study, we find that supplementation of a standard American Heart Association Phase I diet in ambulatory free-living hypercholesterolemic patients with one commercially available preparation of marine lipid concentrate results in a significant reduction of plasma cholesterol within 1 month. Since the patients had been on this diet for at least 3 months prior to the study and had stable plasma cholesterol levels and were monitored closely during the study, it is unlikely that the results obtained can be attributed to the abrupt alterations

163 in the diet. While a decrease was found in the cholesterol concentrations of both LDL and VLDL, these decreases were not correlated with each other (r = 0.007) and the decline in whole plasma cholesterol level correlated only with the fall in LDL cholesterol (r = 0.85). These findings indicate that the cholesterol-lowering effects of marine oil on whole plasma and LDL cholesterol in these patients were unrelated to changes in the plasma triglyceride levels, and suggests that their fall in plasma cholesterol instead was closely linked to alterations in LDL concentration. In the absence of evidence that marine oils accelerate the removal of either VLDL or LDL [5,17], it is likely that the changes we observed here involve a reduction in hepatic lipoprotein production or secretion. Indeed, the uniform fall we observed in TG levels is consistent with previous demonstrations [17,18] that marine oil inhibits the hepatic synthesis of VLDL. How then might n - 3 fatty acids reduce LDL largely independent of changes in VLDL? One possible explanation for our finding this effect on LDL is that the majority of the hypercholesterolemic patients selected for study may have had forms of hyperlipidemia such as familial combined hyperlipidemia (FCH), and familial hypercholesterolemia (FH), in which the de novo synthesis of LDL is known to be increased [19-211. It is, therefore, possible that marine lipids may have inhibited the de novo synthesis of LDL in these patients, since it is known that they inhibit hepatic lipid and apoprotein B synthesis in normal subjects [18,22]. However, our failure to demonstrate any change in plasma apoprotein B levels which agrees with the findings of Failor et al. [23] in FCH patients, suggests that either the synthesis of apoprotein B is not inhibited or its catabolism slowed down following marine lipid treatment in our patients. Recent preliminary studies show that unlike in FCH, there was a significant reduction in LDLcholesterol in FH patients [24,25] and therefore it is possible that our study group included some patients with FH. Our results showing that a reduction in LDL-cholesterol but not apoprotein B suggest that the LDL particles in our patients may have become relatively cholesterol-depleted. Our finding that HDL-cholesterol was unchanged after feeding marine lipid concentrate is

consistent with other studies [3,5,7]. However, when HDL subfractions were examined separately, we found that HDL, levels increased (by an average of 41%) in all subjects, although the absolute increment was insufficient to significantly alter total HDL-cholesterol because of the variable responses observed in HDL,. Our findings contrast with those previously reported by Sanders et al. [5] in similarly treated patients with hypertriglyceridemia. Epidemiological studies imply that the increase we find in the HDL,/HDL, ratio may be desirable and reduce cardiovascular risk [9,10]. It seems unlikely that the changes we have observed in HDL, are methodologic, since the precipitation method we employed to separate HDL, from HDL, has been shown to give results comparable to those obtained with an ultracentrifugation technique [ll]. While incorporation of n - 3 fatty acids into the lipoproteins could theoretically alter their precipitability and contribute to the increased HDL, levels we have found in our study, there is no evidence that fatty acid composition plays any role in the precipitation by dextran sulfate [26]. Precisely how marine oils increase HDL, is unclear. This subfraction of HDL normally increases during the catabolism of triglyceride-rich lipoproteins because VLDL provide a number of surface constituents essentially for its formation [27]. Kinetic studies by both Nestel et al. [18] in hypertriglyceridemic patients and Illingworth et al. [22] in normal volunteers indicate that n - 3 fatty acids lower triglyceride by decreasing production rather than enhancing VLDL degradation. If their conclusions are applicable to our hypercholesterolemic patients, then it is unlikely that the increase we have found in HDL, results from accelerated VLDL catabolism. The second possibility, namely that marine oils decrease the activity of hepatic lipase, also requires further scrutiny, though preliminary data obtained by Illingworth et al. [17] in normal subjects indicate that n - 3 fatty acids have little effect on either hepatic or lipoprotein lipase measured in vitro. Furthermore, the possibility that the in vitro activities of these enzymes are adversely affected because n - 3 fatty acids alter either their membrane or lipoprotein fluidity [28] cannot be excluded. We have recently shown that the transfer of EC from

164 HDL ‘to VLDL and LDL is decreased significantly after marine lipid treatment [28]. This may indicate yet another possible mechanism for the increased level of HDL, found in the present studies because more of the EC formed by lecithin : cholesterol acyltransferase reaction may remain in HDL particles. While we found a small but significant decrease in LDL-cholesterol, several others reported no change or even an increase in LDL following marine lipid administration [6,30-321. There are several possible explanations for this discrepancy. (1) It is possible that the majority of the patients studied here have different forms of hypercholesterolemia compared to the above studies. The response to n - 3 fatty acid feeding is dependent on the underlying cause of hyperlipidemia. Thus, Phillipson et al. [33] reported a decrease in LDLcholesterol in type IIb patients, but an increase in patients with type V phenotype. Similarly, the recent preliminary studies by Friday et al. [24] and Hatcher et al. [25] show that the LDL levels are significantly lowered in normal subjects and in patients with FH, but not in patients with FCH after’ marine lipid feeding. In our patients we found varying levels of decrease in 10 out of 14 patients, while the other 4 showed an increase, and the mean LDL-cholesterol level of all patients significantly decreased. (2) The amount of cholesterol present in the SuperEPA preparation used in our study was 1 mg/capsule and the total amount administered was 15 mg/day. In contrast, the preparations of MaxEPA used in the majority of above studies contained 6 mg cholesterol/capsule [6]. To provide the same amount of n - 3 fatty acids (7.5 g/day) as in our study, one has to administer MaxEPA containing about 150 mg cholesterol/day. (3) The percentage of saturated fatty acids in SuperEPA was found to be less than 10% whereas the MaxEPA preparation contained about 27% saturated fatty acids [32]. The combination of higher cholesterol and saturated fatty acid content may have adversely affected the LDL levels in the previous studies. (4) The SuperEPA preparation is supplied as methyl or ethyl esters of fatty acids whereas the MaxEPA is in the form of triglyceride. The possible differences in the efficacy of the 2 chemical forms of n - 3 fatty acids have not yet been established.

In addition to determining the neutral lipid concentrations before and after marine lipid treatment, we studied the composition of phospholipids in all lipoprotein fractions because a high percentage of n - 3 fatty acids are incorporated into plasma phospholipids [7]. Despite the decrease in neutral lipids, we found a significant increase in PI + PS as well as PE of whole plasma, VLDL and LDL. Since these phospholipids are known to incorporate relatively more of the polyunsaturated fatty acids than PC, those results suggest that the increased pool size of the polyunsaturated fatty acids stimulated the synthesis of these phospholipids. It would be of interest to know if this change in phospholipid composition of lipoprotein results in any functional alterations. Our results also show that marine lipid treatment increased the ratio of free cholesterol/ esterified cholesterol in VLDL and HDL,. Since free cholesterol is present mainly on the surface of lipoproteins and esterified cholesterol is in the core, the increased surface/core ratio we have found in these lipoproteins suggests that fish oil administration is associated with the formation of smaller particles. A reduction in the size of VLDL particles after feeding marine lipids has also been reported in hypertriglyceridemic subjects by Sullivan et al. [30] who suggested that such a change in VLDL particle size may lead to increased formation of LDL, although our results do not show an increase in LDL levels. The decrease we have found in the unesterified cholesterol/lecithin (UC/PC) ratio of the plasma lipoproteins following the fish oil supplement is a theoretically beneficial change because Kuksis et al. [34] have shown in a large Lipid Research Clinic study that an increased UC/PC ratio is an independent risk factor for coronary heart disease. Changes in the phospholipid and UC content appear to influence the capacity of lipoproteins to participate normally in the transfer of cholesterol ester among lipoproteins [35]. In the case of HDL, an increase in the UC/PC ratio appears to be a critical determinant of the bidirectional flux of tissue and lipoprotein cholesterol [36]. Consequently, our findings suggest marine oil feeding may also improve cardiovascular risk by favorably affecting these important factors in reverse cholesterol transport.

165 In summary, supplementation of AHA diets of hypercholesterolemic patients with a lowcholesterol, low-saturated fat-containing n - 3 acid preparation resulted in several potentially beneficial effects with regard to cardiovascular risk: a reduction of VLDL and LDL, an increase in HDL,, and a reduction in UC/PC ratio. These results suggest that marine lipids may be beneficial in the treatment of certain types of hypercholesterolemic subjects.

11

12

13 14

Acknowledgment 15

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