ACAT activity in freshly isolated human mononuclear cell homogenates from hyperlipidemic subjects

ACAT Activity

in Freshly

Isolated Human Hyperlipidemic

Mononuclear Subjects

Cell Homogenates

From

Jean Dallongeville, Jean Davignon, and Suzanne Lussier-Cacan Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the esterification of cholesterol in human mononuclear cells (MNC). In order to assess the relationship between lipid levels and ACAT activity in circulating MNC, we measured the rate of [W]oleoyl-CoA incorporation into cholesterol ester in freshly isolated MNC homogenates from hyperlipidemic subjects. Baseline, off-treatment results obtained in 14 hypertriglyceridemic subjects (eight type IV and six type Ill) and seven subjects with familial hypercholesterolemia (FH) due to the same deletion of greater than 10 kb on the low-density lipoprotein (LDL)-receptor gene were compared with values determined in 12 healthy normolipidemic subjects. The rate of cholesterol esterification was 45 + 28 pmol/5 min/mg cell protein in healthy normolipidemic controls. This rate was significantly higher in type IV subjects (84 ? 52 pmol/5 min/mg cell protein, P < .05) and FH subjects (87 + 25 pmol/5 min/mg cell protein,P < .05). The values were more dispersed in type Ill subjects; the mean value for the group (72 f 48 pmoU5 min/mg cell protein) was not statistically different from the control. Hypertriglyceridemic patients were then treated with 5 g/d of w-3 fatty acids. This resulted in a significant reduction in plasma total triglycerides and very-low-density lipoprotein (VLDL)-cholesterol in both type Ill subjects (-57% and -51%. P < .05) and type IV subjects (-82% and -82%, P < .Ol). The reduction in VLDL concentration was associated with a significantly lower ACAT activity in MNC homogenates from type IV subjects (from 84 -C 52 to 80 2 38 pmol/5 min/mg cell protein, P < .05), but not from type Ill hypertriglyceridemic subjects (from 72 2 48 to 73 of:38 pmol/5 min/mg cell protein). In conclusion, we found that cholesterol esterification in human MNC is elevated in hyperlipidemic subjects and can be decreased with normalization of lipid levels. However, ACAT activity changes occurring with treatment are heterogeneous among hyperlipidemic subjects, suggesting that factors other than plasma lipid level reduction affect ACAT activity in vivo. Copyright 0 1992 by W.B. Saunders Company

A

CYL-COENZYME A:cholesterol acyltransferase (ACAT; EC 2.3.1.26) catalyzes the esterification of cholesterol in mammalian cells.’ This enzyme has been studied extensively in hepatic and intestinal cells in which nutritional and pharmacological regulating factors have been identified. ACAT activity is elevated in the vessels of experimental animals with atherosclerosis.’ Conversely, the inhibition of ACAT by pharmacological treatment has been shown to prevent atheroma formation,3 suggesting that ACAT might play a significant role in atherogenesis.4 The ability of blood mononuclear cells (MNC) to ester@ free cholesterol with long-chain fatty acids has been previously demonstrated?’ This process is stimulated by lowdensity lipoprotein (LDL)5.6 and by B-very-low-density lipoprotein (p-VLDL)8,9 in macrophages. In vivo, the activities of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA R) and of the LDL receptor in human MNC were found to depend on LDL-cholesterol levels” and to be regulated by cholesterol ingestion”.” or pharmaceutical treatment.“.14 These observations suggest that lipoprotein levels may determine the intracellular cholesterol homeostasis in the circulating MNC. To our knowledge, such a

From the Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, Quebec, Canada; and the Departments of Medicine and Nutrition, University of Montreal, Quebec, Canada. Supported by grants from the Heart and Stroke Foundation of Quebec, the Medical Research Council of Canada ICiba Geigy Canada Universityllndustryprogram (U-0029), and by la Succession J.A. DeSeve. Address reprint requests to Jean Dallongeville, MD, Clinical Research Institute of Montreal, I10 Pine Ave, U: Montreal, Quebec, Canada, H2WlR7. Copyright 0 1992 by W.B. Saunders Company 0026-0495/92/4102-0010$03.00f0 154

regulation for ACAT activity has not yet been evaluated in humans. Therefore, the purposes of this study were to assess the influence of circulating lipoprotein concentrations on the regulation of cholesterol esterification in humans and to evaluate changes in ACAT activity occurring during a dietary trial. MATERIALS

AND METHODS

Subjects

Blood samples were obtained from 12 fasting normolipidemic and 21 hyperlipidemic subjects. All gave their informed consent and the protocol was approved by the institutional ethics committee. Nomolipidemic

Controls

Twelve normolipidemic, healthy volunteers (six men and six women, aged 23 to 42 years) constituted the control group. All had plasma cholesterol levels less than 200 mg/dL, triglyceride levels less than 150 mg/dL, and LDL-cholesterol levels less than 160 mg/dL on repeated measurements. They all had the apolipoprotein (apo) E phenotype E 3/3, with two exceptions; one subject had apo E phenotype E 312 and another had E phenotype E 413. These subjects consumed their usual diet during the experiment and were not receiving any drug that might affect lipid metabolism. Type III and Type IV Hypertriglyceridemic

Subjects

Six men with type III dysbetalipoproteinemia and eight type IV hypertriglyceridemic subjects (seven men, one woman) were investigated. Type III was diagnosed on the following criteria: plasma cholesterol levels greater than 200 mg/dL, triglyceride levels greater than 200 mg/dL, the presence of B-VLDL on agarose gel electrophoresis, and the apo E 212 phenotype. Type IV hypertriglyceridemic individuals had triglyceride levels greater than 200 mg/dL, LDL-apo B levels less than 110 mg/dL, and the apo E 313 phenotype. AI1 subjects were free of secondary causes of hypertriglyceridemia such as diabetes, obesity ( > 120% ideal body weight), and excessive alcohol ingestion. They were not receiving any drug

Metabolism, Vol41, No 2 (February), 1992: pp 154-159

ACAT ACTIVITY

155

IN HYPERLIPIDEMIA

known to affect lipid metabolism for at least 8 weeks before blood sampling and were on a diet low in saturated fat and cholesterol. These subjects entered a treatment protocol in which they received 6 g/d of o-3 fatty acids (Promega, Parke-Davis, WarnerLampert Canada, Scarborough, Ontario; 12 capsules/d) for 4 weeks. Blood samples were taken before and after supplementation with the fish oils to measure lipid concentration, ACAT activity, and cellular cholesterol content. Simultaneously, the same measurements were performed at 4-week intervals in 12 normolipidemic subjects (while on their usual diet) in order to control for variation due to time.

reaction was stopped by the addition of 4 mL of chloroform/ methanol (2:l). Cell lipids were extracted twice with chloroform/methanol (21) and separated by thin-layer chromatography on polysilicic acid gel-impregnated sheets (ITLC, Gelman Sciences, Montreal, Canada) using heptane/diethyl ether/acetic acid @X5:15:2)as the solvent system. Spots corresponding to cholesterol esters were collected into scintillation vials. Ten milliliters of scintillation liquid (Opti phase III, LKB Scintillation Products, Loughborough, England) was added and radioactivity was counted on an LKB Wallac 1217 Rack B-liquid scintillation counter.

Hypercholesterolemic Subjects

Other Methods

Seven subjects (five men, two women) with familial hypercholesterolemia (FH) were investigated. The diagnosis of FH was based on LDL-cholesterol levels greater than 190 mg/dL and the presence of tendinosum xanthoma in the subjects or in first-degree relatives. The clinical diagnosis was confirmed by the presence of a greater than 10 kb deletion on the LDL receptor gene.” Subjects were not receiving any lipid-lowering drug for at least 8 weeks before blood sampling, and none had been previously treated with probucol.

Lipoproteins were separated under standard conditions by a combination of ultracentrifugation (at d = 1.006 g/mL) and heparin-manganese precipitation of the apo B-containing lipoproteins in the d > 1.006 infranatant, according to the Lipid Research Clinics protocol.‘* Plasma total and lipoprotein cholesteroP9 and triglycerides” were measured enzymatically, on an automated analyzer (Abbott Bichromatic Analyzer 100, Abbott Laboratories, Pasadena, CA). Cellular cholesterol was measured enzymatically (Boehringer Mannheim, Germany) according to De Hoff et al.” The identification of cell populations was performed by diffraction (FAC Scan, Becton Dickinson, Mississauga, Ontario, Canada). Cell homogenate protein concentrations were measured according to the method of Lowry et al, with serum bovine albumin as standard.” The apo E phenotype was determined by isoelectric focusing of VLDL apoproteins as previously described.z3

Isolation of MNC MNC were isolated following a slight modification of the method described by Boyum.16A 40-mL blood sample was centrifuged at 1,700 g, 4°C for 10 minutes. Plasma was kept for lipid measurements. White blood cells were collected and resuspended in 5 mL of .9% sterile saline. The cell suspension was layered onto 5 mL of Ficoll-Paque (Pharmacia, Piscataway, NJ) in 15mL polystyrene tubes (Falcon, Becton Dickinson, Fullerton, CA) and centrifuged at 400 g for 30 minutes. MNC were collected at the saline/FicollPaque interface and washed once with Hanks’ balanced salt solution and TRIS- NH&l (TRIS-base, 1.21 g/L, NH&l, 8.7 g/L, pH 7.6), and again with Hanks’ balanced salt solution. Cells were then suspended in a O.l-mmol/L phosphate buffer, pH 7.4, and kept in liquid nitrogen for less than 6 weeks. This method yielded an average of 75% f 12% lymphocytes, 15% 2 8.6% monocytes, and 10% 2 7% granulocytes (n = 64).

Cholesterol Estetification Assay The assay was adapted from Stange et al.” It consisted of the determination of the esterification rate of endogenous cholesterol with exogenous radiolabeled oleoyl-CoA. Standardization of the assay was performed on pools of cells from three to five normolipidemic or hyperlipidemic subjects.

Cells were thawed at room temperature and immediately homogenized in a nitrogen cell disruption chamber (Parr 4635, Parr Instrument, Moline, IL). The cell suspension was brought to 15 kPa in 5 seconds and kept at this pressure for 30 minutes and brought back to ambient pressure in 10 seconds. The resulting cell homogenate contained no intact cells. The reaction substrate was prepared from [‘4C]oleoy1-CoA (52.3 mCi/mmol; Amersham Chemicals, Arlington Heights, IL) and cold oleoyl-CoA (Sigma Chemicals, St Louis, MO) in O.l-mmol/L phosphate buffer. In the standard assay, 5 nmol of oleoyl-CoA (60 dpm/pmol) were added to the cell homogenates. The final assay was performed as follows: 200 kg of cell homogenate protein was preincubated for 10 minutes at 37°C in a phosphate buffer (0.1 mmol/L, pH 7.4), in the presence of 0.4 umol of glutathione (Sigma Chemicals) and five nmol of delipidated albumin (Sigma Chemicals). Cholesterol esterification was measured in the presence of 5 nmol [‘4C]oleoyl-CoA (60 dpm/pmol) during a 5-minute incubation in a final volume of 200 FL. The

StatisticalAnalysis The Mann-Whitney CJtest was used to compare the mean values of the hyperlipidemic groups with those of the control group, and the Wilcoxon matched-pairs, signed-ranks test was used to evaluate the effects of treatment.24 RESULTS

Cholesterol Estetification The optimal conditions for assay were determined. Figure 1A shows the effect of increasing [‘4C]oleoyl-CoA concentrations in the presence of a fixed amount of albumin (25 kmol/L) during 5 minutes of incubation with 200 pg of cell protein (mean of three experiments). The rate of oleate incorporation into cholesterol ester increased progressively up to 25 pmol/L of [Vloleoyl-CoA in the medium and then decreased. In the presence of a constant (1:l) oleoylCoA to albumin ratio (Fig lB), there was a significant increase in cholesterol esterification without apparent saturation up to 250 p,mol/L of oleoyl-CoA. Figure 1C presents the effect of different cell homogenate protein concentrations, and Fig 1D presents the effect of the time of incubation on cholesterol esterification. Cholesterol esterification was linear with respect to cellular protein concentrations up to at least 300 kg (Fig 1C; mean of two experiments). Oleate incorporation was also linear as a function of time for 5 minutes and then reached saturation (Fig 1D). In the standard assay, 200 pg of cell homogenate protein was preincubated for 10 minutes in the presence of 2 mmol/L glutathione and 25 kmol/L albumin and then incubated for 5 minutes with 25 pmol/L of oleoyl-CoA and endogenous cholesterol. Under these conditions, the intraassay variability was 7.9% (mean of three assays in quintupli-

DALLONGEVILLE, DAVIGNON, AND LUSSIER-CACAN

20

40

60

80

100

100 150 200 Oleoyl CoA (pM)

Oleoyl CoA (j.tM)

12-

c

.

lo-

iv, , , ,

864-

oy. 8 0 50

8

100

’

r

a

150 200 250 Cell protein (pgg)

1

300

8

350

0

5

10 Minutes

15

20

Cholesterol esterification rate as a function of (A) oleoyl-CoA in the Presence of a fixed amount of albumin (25 pmol I L); (B) oleoyl-CoA in the presence of a constant ratio of albumin/oleoyl CoA (1:l); (C) cellular protein concentration; (D) time.

cate) and the between-assay variability was 5.5% (mean of 19 assays in duplicate). The ACAT activity in frozen cell homogenates was twice the value measured in unfrozen tissue and was stable during storage in liquid nitrogen for up to 6 weeks. After 12 weeks of storage, a 20% to 50% reduction of activity was observed. Therefore, all measurements were completed within six weeks after blood sampling. Comparison of Cholesterol Esterification Among Hyperlipidemic Subjects

Mean lipid and lipoprotein concentrations and ACAT activity for control and hyperlipidemic subjects are presented in Table 1. As expected, subjects with type III and type IV hyperlipidemias had significantly higher plasma triglyceride levels (P < .05, P < .OOl) and VLDL-cholesterol levels (P < .OOl, P < .OOl) and FH subjects had higher plasma cholesterol (P < .OOl) and LDL-cholesterol (P < .OOl) concentrations than control subjects. The mean ACAT activity in cell homogenates (76% lymphocytes, 17% monocytes, 7% polynuclear) from the 12 control subjects was 45 2 28 pmoli5 min/mg of cell protein (range, 17 to 94). In type IV subjects, ACAT activity ranged from 23 to 15.5, with a mean value of 84 t 52 pmol/5 min/mg cell protein, and was significantly higher than that of the control group (P < .05). ACAT activity in type III hyperlipidemic subjects ranged from 38 to 165 pmol/5 min/mg of cell protein. Five subjects had values between 38 and 68 pmol/5 min/mg of cell protein and one outlier had a value of 165 pmol/5 min/mg of cell protein. As a conse-

quence, mean levels in type III hyperlipidemic subjects (72 2 46 pmol/5 min/mg cell protein) were not statistically different from those of the control group. The rates of cholesterol esterification were less dispersed in FH subjects than in the control and other hyperlipidemic groups (range, 39 to 103 pmoli5 min/mg of cell protein), with a mean value of 67 + 25 pmol/5 min/mg of cell protein, significantly higher than the control value (P < .05). The distribution of Table 1. Mean Values (SD) of Plasma Lipoprotein Concentrations, ACAT Activity, and Cellular Cholesterol in MNC Homogenates

of

Normolipidemic Controls and Type IV, Type Ill, and FH Subjects COlWOlS

TypelV

Type III

(n = 12)

(n = 8)

(n = 6)

FH (n = 7)

160 (13)

238 (53)t

384 (145)*

339 (78)t

61 (22)

478 (147)t

675 (660)*

175

ImgtdL) LDL-cholesterol

13 (4)

95 (46)t

231 (170)t

ImgidL) HDL-cholesterol

92 (12)

111 (26)”

123 (46)*

273 (85)t

(mg/dL) ACAT (pmol/5

52 (13)

31 (7.6)

29 (10)t

30 (8)t

45 (28)

84 (52)*

72 (46)

70 (25)’

49 (13)

49 (11)

43 (7.4)

57 (9.4)

45 (12)

45 (13)

40 (10)

50 (7.8)

Cholesterol (mg/dL) Triglycerides (mg/dL)

(58)t

VLDL-cholesterol 35 (12)t

min/mg cell protein) Cell total cholesterol (pgimg cell protein) Cell free cholesterol (pg/mg cell protein)

Statistically different from control group (*P < .05, tP i ,001) by the Mann-Whitney Utest.

157

ACAT ACTIVITY IN HYPERLIPIDEMIA

cell populations in the intact samples was similar among groups. There were no statistically significant differences in the cellular content of total and free cholesterol between the control group and hyperlipidemic subjects.

noll5minlmg

cell protein

Type

III

Type

IV

Effect of Fish Oil Treatment on Cholesterol Estertfication in Hypertriglyceridemic Subjects

Table 2 shows the effect of o-3 fatty acid treatment on lipid, lipoprotein, and cholesterol esterification activity, or cellular cholesterol content of control and hyperlipidemic subjects. Values are presented for hypertriglyceridemic subjects at baseline and after 4 weeks of o-3 fatty acid treatment (6 g/d); for control subjects, values are presented at baseline and after 4 weeks on their usual diet. There were no significant changes in plasma lipid, ACAT activity, or cellular cholesterol content in the control group after 4 weeks. Conversely, plasma cholesterol, triglycerides, and VLDL-cholesterol were significantly lower after o-3 fatty acid treatment in the hypertriglyceridemic subjects. In addition, ACAT activity was significantly reduced after fish oil treatment in type IV subjects (P < .05), but not in type III hyperlipidemic subjects. Cholesterol esterification decreased in all type IV hypertriglyceridemic subjects but one. The response was more heterogeneous in type III subjects, with a reduction in three of them and an increase in the three others (Fig 2). There was no significant correlation between changes in plasma lipids and ACAT activity in type IV or type III subgroups. DISCUSSION

The goals of our study were, first, to assess the influence of plasma lipid levels on the rate of cholesterol esterification in circulating MNC, and second, to evaluate whether changes in plasma lipid levels would affect ACAT activity in these cells. To answer these questions, we developed an assay of ACAT activity in human MNC homogenates. The cells had been frozen in liquid nitrogen immediately following their isolation, in order to maintain a representation of in vivo conditions. The homogenates were obtained in a nitrogen cell disruption chamber to avoid the use of detergent that may affect enzyme activity.‘,” In a recent report, Einarsson et al demonstrated that the tissue cholesterol of frozen liver homogenates is a saturating source of cholesterol for microsomial ACAT. Similarly, we used endogenous cholesTable 2. Effect of w-3 Fatty Acid Supplementation

I

I

Baseline

I

I

Treatment

I

I

I

Baseline

Fig 2. Changes in levels of ACAT activity after fish oil supplementation (6 g/d) in human MNC homogenates in eight subjects with type IV and six patients with type Ill hyperlipidemia.

terol and [‘4C]oleoyl-CoA as substrates for cholesterol esterification. The fatty acid to albumin ratio is critical for ACAT activity determination. In our experiment, the rate of cholesterol esterification peaked when the ratio was equal to 1 and then decreased progressively. This is in agreement with previous observations27828 of rat and human liver microsomes. Cholesterol esterification ranged from 16 to 94 pmoli5 min/mg of cell protein in MNC homogenates from normolipidemic subjects. These values are five times lower than that reported for frozen human hepatic tissue.26,2R Individually, there was large scatter in the values of normolipidemic and hyperlipidemic subjects. This heterogeneity persisted in normolipidemic subjects when values were measured at 4-week intervals, with less than 11% variation between measurements. Intragroup variability was not accounted for by differences in lipid levels, lipoprotein levels, cell cholesterol content, or cell population distribution, but may have been related to differences in diet or interaction with lipoproteins and hormonal factors.’ As a consequence, values tended to overlap between groups. Nonetheless, the cholesterol esterification activity tended to be higher in hyperlipidemic subjects than in controls. The higher levels obtained in FH heterozygotes, compared with those of normolipidemic individuals, are consistent with the previously reported observation of a reduced HMG-CoA R

on Plasma Lipid and Lipoprotein Concentrations,

ACAT Activity,

and Total and Free Cellular Cholesterol Controls (n = 12) Before

After

160 (16)

163 (26)

61 (22)

57 (21)

VLDL-cholesterol (mg/dL)

13 (4)

14 (8)

LDL-cholesterol (mg/dL)

92 (12)

96 (23)

ACAT (pmol/5 min/mg cell protein)

45 (28)

Cell total cholesterol (kglmg cell protein)

49 (13)

Cell free cholesterol (bg/mg cell protein)

45 (12)

Cholesterol (mg/dL) Triglycerides

(mg/dL)

I

Treatment

Type IV (n = 8)

Type III (n = 6) After

Before

After

238 (53)

216 (40)*

384 (145)

276 (61)*

478 (147)

178 (58)t

675 (660)

291 (131,

95 (46)

36 (13)t

231 (170)

112 (62)*

111 (26)

145 (31)t

123 (46)

133 (25)

45 (28)

84 (52)

60 (36)”

72 (46)

73 (36)

45111)

49 (II)

49 (4)

43 (7.4)

42 (8)

42 (8)

45 (13)

47 (6)

40 (IO)

36 (6)

Before

Statistically different from before treatment (“P < .05, tP < .Ol) by the Wilcoxon matched-pairs test.

158

DALLONGEVILLE,

activity in freshly isolated MNC.” This suggests that in vivo, sufficient cholesterol enters the cells despite low LDLreceptor numbers and regulates the activity of ACAT and HMG-CoA R.“’ Type IV hypertriglyceridemic subjects had a mean ACAT activity which was higher than that of controls. VLDL from hypertriglyceridemic subjects are known to inhibit HMG-CoA R in vitro,” indicating that this lipoprotein regulates intracellular enzyme activity. Our observation suggests that ACAT might be regulated in vivo by a similar mechanism. Finally, ACAT activity was not significantly different between type III patients and controls. (5VLDL, the characteristic lipoprotein that accumulates in type III hyperlipidemia, is heterogeneous.’ In vitro studies have shown that this lipoprotein stimulates cholesterol esterification with a variable intensity,‘.’ depending on the relative amount of pre+ to P-VLDL in the d < 1.006 g/mL plasma fraction.’ This could explain the observed differences between the type III and type IV groups. Supplementation of the diet with o-3 fatty acids was shown to stimulate cholesterol esterification and ACAT activity in rat and rabbit liver.30,3’Conversely, when hepatocytes and intestinal cells were incubated in vitro with w-3 fatty acids, ACAT activity was depressed.32.‘”We found that the reduction in plasma lipid and lipoprotein concentrations in type IV hyperlipidemic subjects was associated with a significant reduction in ACAT activity. All subjects but one had a lower rate of cholesterol esterification after treatment, consistent with the hypothesis that elevated levels of hypertriglyceridemic VLDL regulate ACAT activity and that it is reduced by normalization of lipid levels. In

DAVIGNON,

AND LUSSIER-CACAN

type III hypertriglyceridemic subjects, the effect was less clear. As with type IV subjects, ACAT activity decreased in three dysbetalipoproteinemic subjects after fish oil supplementation, but increased in the three others. It is possible that treatment with fish oil in the latter subjects altered their VLDL structure in such a way that it was taken up by the cell more efficiently and thereby produced ACAT activation. In summary, we assessed ACAT activity in freshly isolated, human MNC from normolipidemic and hyperlipidemic subjects. The rate of cholesterol esterification in these cells depends on multiple factors, including diet, lipoprotein concentrations, and hormone concentrations. This combination resulted in a higher activity in MNC from hypercholesterolemic and hypertriglyceridemic subjects that could be partially normalized with treatment. Since cholesterol ester accumulation in MNC is one of the initial stages of atherosclerosis, it is tempting to speculate that subjects with higher levels of ACAT are more prone to atherosclerosis. Further investigations should clarify this relationship in humans. ACKNOWLEDGMENT

The authors thank Denise Dubreuil, Helene Mailloux, Suzanne Quidoz, Rina Riberdy, and Angele Richard for their clinical assistance; Mireille Amyot, Lucie Boulet, Louis-Jacques Fortin, Helene Jacques, Chantal Lefebvre, and Michel Tremblay for their technical help; and Lyne Lacoste for technical and clerical assistance. We also thank Parke-Davis of Warner-Lambert, Canada, for generously providing Promega capsules for this study.

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