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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:956-962.)
© 1995 American Heart Association, Inc.

Articles

Different Effects of Subclasses of HDL Containing ApoA-I but Not ApoA-II (LpA-I) on Cholesterol Esterification in Plasma and Net Cholesterol Efflux From Foam Cells

Takao Ohta; Keijiro Saku; Kouki Takata; Rie Nakamura; Yoichiro Ikeda; Ichiro Matsuda

From the Department of Pediatrics (T.O., R.N., Y.I., I.M.), Kumamoto University School of Medicine, Kumamoto, the Department of Internal Medicine (K.S.), Fukuoka University, Fukuoka, and the Hiroshima Railway Hospital (K.T.), Hiroshima, Japan.

Correspondence to Dr Takao Ohta, Department of Pediatrics, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto-City Kumamoto 860, Japan. E-mail ohta@gpo.kumamotou.ac.jp.

	Abstract

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Abstract We investigated the effects of subclasses of plasma LpA-I (HDL containing apoA-I but not apoA-II) on cholesterol esterification in plasma and net cholesterol efflux from foam cells. LpA-I was composed of particles of three diameters: large (11.1 nm; Lg–LpA-I), medium (8.8 nm; Md–LpA-I), and small (7.7 nm; Sm–LpA-I). Plasma concentrations of LpA-I were positively correlated only with the level of Lg–LpA-I. Plasma concentrations of Lg–LpA-I were inversely correlated with the rate of cholesterol esterification in plasma and VLDL- and LDL-depleted plasma. Plasma concentrations of Md–LpA-I and Sm–LpA-I did not correlate with the rate of cholesterol esterification in plasma or VLDL- and LDL-depleted plasma. When macrophage foam cells were incubated with Md– and Sm–LpA-I, cellular cholesterol mass was reduced by approximately 70%. In contrast, the cellular cholesterol–reducing capacity of Lg–LpA-I was negligible. Lg–LpA-I inhibited net cholesterol removal from foam cells that was mediated by Md– and Sm–LpA-I and cholesteryl ester production with these particles. These results suggest that Md– and Sm–LpA-I may actively participate in cellular cholesterol removal and cholesterol esterification in plasma and HDL, while Lg–LpA-I may regulate these functions of Md– and Sm–LpA-I.

Key Words: apoA-I–containing lipoproteins • LCAT • LpA-I • LpA-I/A-II • reverse cholesterol transport

	Introduction

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HDL is present in plasma in two main forms, one containing apoA-I and apoA-II (LpA-I/A-II) and the other containing apoA-I but no apoA-II (LpA-I).^{1 2} A strong inverse relation exists between plasma LpA-I concentrations and the risk of coronary artery disease.^{3 4} In contrast, there is only a weak correlation between plasma LpA-I/A-II concentrations and coronary artery disease.^{3 4} Furthermore, recent studies in human apoA-I transgenic mice and double transgenic mice expressing human apoA-I and apoA-II clearly indicate that LpA-I may be the antiatherogenic lipoprotein fraction within HDL.^{5 6} The biochemical mechanism of the antiatherogenic nature of LpA-I is not known. However, it has been postulated that LpA-I plays a key role in the hypothetical process of reverse cholesterol transport, ie, the transport of cholesterol from peripheral cells to the liver for excretion.^{7 8} In this transport model, LpA-I is more effective in mediating net cellular cholesterol efflux than LpA-I/A-II.^{9 10}

LpA-I is itself composed of heterogeneous particles; three major subclasses (large, medium, and small LpA-I) have been characterized.^{11 12} Several studies have postulated that these subclasses are metabolically distinct and that plasma concentrations of LpA-I are mainly associated with those of large LpA-I, which suggests that large LpA-I may be a more specific marker for the risk of premature coronary heart disease and thus the more antiatherogenic subclass.^{12 13 14} We have shown that the relative amount of large LpA-I in HDL regulates the reactivity of HDL to lecithin:cholesterol acyltransferase (LCAT).¹⁵ Since the reactivity of HDL to LCAT is believed to be a key step in net cellular cholesterol efflux,^{9 16 17} as a first step in examining the underlying mechanism of the antiatherogenic nature of LpA-I we investigated the effect of large LpA-I on the rate of cholesterol esterification in HDL and plasma and on the cellular cholesterol–reducing capacity of LpA-I.

	Methods

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Subjects and Materials
Plasma for individual subject studies was obtained from 47 Japanese volunteers (17 men and 30 women; age, 18±6 years, mean±SD). Plasma for the in vitro experimental studies was obtained from five independent groups of healthy adult volunteers (each group consisted of 5 men and 5 women, 18 through 29 years of age, all of whom had fasted overnight). Plasma from the subjects in each group was combined and subjected to isolation of subclasses of LpA-I and lipoprotein-deficient plasma (LDP). None of the subjects had clinical signs or symptoms of atherosclerotic coronary heart disease. All the chemicals used were of the best grade available and were obtained from commercial sources. [7-³H]Cholesterol was purchased from Du Pont New England Nuclear.

Isolation of LpA-I and LpA-I/A-II From Plasma
LpA-I and LpA-I/A-II were isolated from the plasma samples by a combination of anti–apoA-I and anti–apoA-II immunosorbent columns.^{2 9} Briefly, plasma was applied to an anti–apoA-I column. After washing extensively with 10 mmol/L Tris, 500 mmol/L NaCl, and 1 mmol/L EDTA, pH 7.5 (buffer A), the column was eluted with 100 mmol/L acetic acid and 1 mmol/L EDTA, pH 3.0. Each effluent was immediately adjusted to pH 7.4 with 1 mol/L Tris solution and dialyzed against 150 mmol/L NaCl and 1 mmol/L EDTA, pH 7.4 (buffer B). Finally, the sample was concentrated in buffer B by using an ultrafiltration cell (Amicon Corp) equipped with a PM-10 membrane and then applied to an anti–apoA-II column. The column was washed with buffer A to obtain LpA-I. The bound fraction was eluted from the column to obtain LpA-I/A-II. Both LpA-I and LpA-I/A-II were dialyzed and concentrated in buffer B. By using this procedure, >90% of the lipids and apolipoproteins applied were recovered in both the unbound and bound fractions. When the unbound plasma fraction from the anti–apoA-I column was applied to an anti–apoA-II column, no bound fraction was obtained. Thus, we believe that all the plasma apoA-II was associated with apoA-I as LpA-I/A-II in our study subjects.

Subfractionation of LpA-I
Five sets of LpA-I isolated from five pooled plasma samples were subfractionated into HDL₂ (d=1.063 to 1.125 g/mL) and HDL₃ (d=1.125 to 1.21 g/mL) by ultracentrifugation at 150 000g for 48 hours at 4°C.¹⁸ These subfractions were dialyzed against phosphate-buffered saline (PBS) and were used in the following experiments immediately after isolation.

Isolation of LDP
LDP was isolated from fresh plasma by the method of Barter et al.¹⁹ The supernatant that was recovered after ultracentrifugation at 150 000g for 48 hours (d=1.25 g/mL) was subjected to a second 48-hour spin at d=1.25 g/mL, after which the upper third of the infranatant was recovered and added to the first 1.25-g/mL infranatant. LDP was concentrated twofold and dialyzed against PBS.

Fractional and Molar Esterification Rates in Plasma and VLDL- and LDL-Depleted Plasma
Fractional and molar esterification rates in plasma and VLDL- and LDL-depleted plasma were determined by a modification of the methods of Dobiasova et al.²⁰ VLDL- and LDL-depleted plasma was prepared by precipitating VLDL and LDL with phosphotungstate-MgCl₂.^{21 22} [³H]Free cholesterol (FC) was incorporated onto polystyrene tissue-culture wells (Corning) as follows. Absolute ethanol (100 µL) containing 0.2 µCi [³H]FC was placed in wells and then dried by flushing with nitrogen. One hundred microliters of either plasma sample in 400 µL PBS was added to each well for determining the molar rate of cholesterol esterification in plasma (MERplasma), or, for VLDL- and LDL-depleted plasma samples, for determining the fractional esterification rate in HDL (FERHDL). The [³H]FC was then equilibrated with the FC in each sample by incubation at 4°C for 16 hours. After this the [³H]FC-labeled plasma or VLDL- and LDL-depleted plasma samples were incubated at 37°C for 30 minutes. The enzyme reaction was stopped by immersing the sample tubes in an ice bath. The lipids in the incubation samples were extracted with methanol/chloroform (2:1, vol/vol), and the extract was dried by flushing with nitrogen and dissolved in 60 µL isopropanol. Aliquots (20 µL) of lipid extracts were spotted in duplicate on a thin-layer chromatography plate (Merck) and developed in n-hexane/diethyl ether/acetic acid/methanol (85:20:1:1, vol/vol/vol/vol). Spots corresponding to FC and cholesterol esters (CE) were cut from the plate, and the radioactivities were determined. The increase in [³H]CE was linear over 30 minutes of incubation. The fractional esterification rate was expressed as the difference between the percentage of radioactive cholesterol esterified before and after incubation at 37°C, and the molar rate of cholesterol esterification (micromoles per hour per liter) was calculated based on the specific activity (disintegrations per minute per nanomole FC) of each sample.

Assay of Cholesterol Esterification in Samples Containing Varying Proportions of HDL₂ and HDL₃ Subfractions of LpA-I
Subfractions of LpA-I were labeled with [³H]FC as follows. Absolute ethanol (100 µL) containing 15 µCi [³H]FC was placed in culture wells, and the ethanol was dried by flushing with nitrogen. Each lipoprotein or various mixtures of subfractions (200 to 1000 nmol FC) in 1 mL PBS was then added to the wells, and the [³H]FC was equilibrated with the FC in each sample by incubation at 4°C for 16 hours. Aliquots of [³H]FC-labeled lipoproteins were made up to a constant volume of 100 µL by adding PBS. LDP (50 µL) was then added to each incubation tube as an LCAT source, and the mixtures were incubated at 37°C for 30 minutes in a shaking water bath. The reaction was stopped by immersing the tubes in an ice bath. Lipid extraction and separation of FC and CE by thin-layer chromatography were performed as described above. The increase in [³H]CE was linear over 30 minutes of incubation. The cholesterol esterification rate was expressed as the difference between the percentage of radioactive cholesterol esterified before and after incubation at 37°C, and the esterified cholesterol mass was calculated based on the specific activity (disintegrations per minute per nanomole FC) of each sample.

Cholesterol Efflux From Macrophage Foam Cells
Experiments for determining cholesterol efflux from foam cells were performed.^{9 16} Briefly, peritoneal macrophages from Wistar rats were harvested in PBS and suspended in Dulbecco's modified minimal essential medium containing 3% bovine serum albumin (medium A). The cell suspension was placed in 22-mm plastic dishes, and the preparation was incubated for 4 hours. Adherent macrophages were converted to foam cells by a 16-hour incubation with 100 µg/mL acetyl LDL in 1.0 mL medium A. These foam cells were subjected to efflux assays by incubation with medium A containing 100 µL LDP (an LCAT source) and subclasses of LpA-I (300 µg protein each) or a combination of subclasses (HDL₃, 300 µg protein; HDL₂, 100 to 300 µg protein). Plasma concentrations of LCAT enzyme mass do not correlate with those of apoA-I and are fairly constant.²³ Thus, to create a plasma-like environment, we did not change the amount of LDP in the efflux experiments, despite different concentrations of effector protein. Parallel incubations without lipoproteins served as controls. The culture medium was removed 3, 6, 12, and 24 hours after the onset of the efflux experiments, and the cellular lipids were then extracted. Unless otherwise specified, the data derived from these efflux assays were the mean of quintuplicate runs in five separate experiments. FC and CE were extracted directly from macrophage monolayers. The mass of FC and CE was quantified by a modification of the method of Heider and Boyett.²⁴

Protein and Lipid Analysis
ApoA-I and apoA-II concentrations in plasma, LpA-I, and LpA-I/A-II were measured by radial immunodiffusion assay.^{25 26} The total cholesterol (TC), FC, triglyceride, and phospholipid concentrations of these samples were determined by enzymatic methods by using commercial kits. The concentrations of CE were calculated as (TC-FC)x1.62. HDL cholesterol (HDL-C) was measured by selective precipitation of LDL using phosphotungstate-MgCl₂.^{21 22} The protein content of each fraction from the immunosorbent columns was determined by the method of Lowry et al.²⁷ To estimate the plasma concentrations, values of lipids and apolipoproteins in LpA-I and LpA-I/A-II were corrected based on the percent recoveries during the isolation.

Electrophoretic Analysis
The apparent hydrated particle diameters of LpA-I and LpA-I/A-II were estimated by gradient polyacrylamide gel electrophoresis on Pharmacia precast PAA 4/30 gels according to the procedure specified by the manufacturer. Electrophoresis calibration kits for high-molecular-weight proteins (Pharmacia) were used. The Stokes' diameters of these high-molecular-weight proteins are (in nm) thyroglobulin, 17.0; apoferritin, 12.2; catalase, 10.4; lactate dehydrogenase, 8.2; and bovine albumin, 7.5. The stained gels were scanned with a laser scanning densitometer (model CS-9000, Shimadzu). The areas corresponding to particles were calculated after integration.

Statistical Evaluation
Linear regression analysis and paired and unpaired t tests were used to evaluate the data.

	Results

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Relation Between Plasma Concentrations of HDL-C and ApoA-I–Containing Lipoproteins
In 47 study subjects plasma concentrations of HDL-C (1.30±0.31 mmol/L, mean±SD) correlated strongly with those of LpA-I (0.57±0.12 g/L) (r=.62, P<.0001) but did not correlate with those of LpA-I/A-II (1.13±0.10 g/L) (r=.21, P>.16), which suggests that plasma concentrations of HDL-C largely depend on the plasma concentrations of LpA-I.

Relation Between Plasma Concentrations of LpA-I and Those of Its Subclasses
We have found small age- and sex-related differences in LpA-I.^{2 28} However, most of these differences were quantitative. Recently, Duverger et al¹¹ confirmed our findings and extended them to subspecies of LpA-I. Therefore, we did not consider age and sex in the present study.

On the basis of particle diameter, three subspecies of LpA-I were identified by densitometric scan of gradient gels: large (11.1 nm; Lg–LpA-I), medium (8.8 nm; Md–LpA-I), and small (7.7 nm; Sm–LpA-I) (Fig 1). These particles were distributed as follows: Lg–LpA-I, 65±9%; Md–LpA-I, 29±4%; and Sm–LpA-I, 6±5%. More than 96% of the particles in the HDL₂ fraction of LpA-I were Lg–LpA-I (Fig 1B and 1C). On the other hand, the HDL₃ fraction of LpA-I consisted mostly of Md–LpA-I and Sm–LpA-I. The chemical composition of these subspecies is summarized in the Table. The percentage of FC, the ratio of FC to CE, and the ratio of lipid to protein were all significantly higher in Lg–LpA-I (HDL₂ fraction) than in Md–LpA-I and Sm–LpA-I combined [(Md+Sm)LpA-I] (HDL₃ fraction) (P<.005).

View larger version (88K): [in this window] [in a new window]   Figure 1. Nondenaturing gradient gel electrophoresis of LpA-I (HDL containing apoA-I but not apoA-II) and subclasses of LpA-I (particles 11.1 nm [Lg–LpA-I], 8.8 nm [Md–LpA-I], and 7.7 nm [Sm–LpA-I] in diameter). A, LpA-I; B, the HDL2 fraction of LpA-I; and C, the HDL3 fraction of LpA-I. HDL2 consisted of Lg–LpA-I particles; the HDL3 fraction consisted of mostly Md–LpA-I and Sm–LpA-I, but a small amount of Lg–LpA-I was also present in this fraction. Stokes' diameters of high-molecular-weight standards are given.

View this table: [in this window] [in a new window]   Table 1. Chemical Composition of Subfractions of LpA-I

We determined the plasma concentrations of these subspecies based on their percent distribution and the plasma concentration of LpA-I (expressed as grams of protein per liter of plasma). Plasma concentrations of LpA-I were strongly correlated with those of Lg–LpA-I (Fig 2). However, there was no correlation with either Md–LpA-I or Sm–LpA-I. Plasma concentrations of HDL-C were also correlated with those of Lg–LpA-I (r=.72, P<.0001) but not with those of Md–LpA-I or Sm–LpA-I (r=.08, P>.59 and r=.04, P>.88, respectively). These results indicate that plasma concentrations of LpA-I and HDL-C apparently depend on their content of Lg–LpA-I.

View larger version (15K): [in this window] [in a new window]   Figure 2. Scatterplots showing correlations between plasma LpA-I (HDL containing apoA-I but not apoA-II) and plasma LpA-I of large (Lg–LpA-I), medium (Md–LpA-I), and small (Sm–LpA-I) diameters.

Relation Between Plasma Concentrations of Lg–LpA-I and Cholesterol Esterification in Plasma and HDL
Since most plasma CE is generated in HDL particles,²⁹ we examined the relation of LpA-I and Lg–LpA-I to the rate of cholesterol esterification in plasma and HDL. FERHDL was estimated by the cholesterol esterification rate in VLDL- and LDL-depleted plasma. Plasma concentrations of LpA-I were inversely correlated with FERHDL, and a much stronger inverse correlation was observed between Lg–LpA-I and FERHDL (Fig 3). This suggests that the reactivity of HDL to LCAT protein in plasma is determined by the concentration of LpA-I, especially that of Lg–LpA-I. Since LCAT associated with HDL can esterify cholesterol derived from VLDL and LDL,¹⁶ we studied the effect of LpA-I and Lg–LpA-I on MERplasma. Plasma concentrations of LpA-I and Lg–LpA-I were also inversely correlated with MERplasma (r=-.46, P<.0015 and r=-.41, P<.0015, respectively). In contrast, plasma concentrations of Md–LpA-I and Sm–LpA-I did not correlate with either FERHDL or MERplasma. Thus, all our data suggest that Lg–LpA-I contributes to the regulation of CE production in plasma. To clarify this regulatory mechanism, we then examined the role of subclasses of LpA-I on CE production.

View larger version (15K): [in this window] [in a new window]   Figure 3. Scatterplots showing negative correlations between the fractional esterification rate of HDL (FERHDL) and (A) plasma LpA-I (HDL containing apoA-I but not apoA-II) and (B) plasma Lg–LpA-I (large LpA-I; diameter, 11.1 nm).

Effect of Lg–LpA-I on Rate of Cholesterol Esterification in Incubations Containing (Md+Sm)LpA-I
To create a plasma-like environment, we used LDP as an LCAT source instead of purified LCAT. Lg–LpA-I suppressed the rate of cholesterol esterification in (Md+Sm)LpA-I in a dose-dependent manner (Fig 4). A similar suppression of the molar rate of cholesterol esterification by Lg–LpA-I was observed in incubations with LpA-I/A-II particles (data not shown). These results suggest that plasma CE is mainly generated in Md–LpA-I, Sm–LpA-I, and LpA-I/A-II particles, while Lg–LpA-I particles regulate the interaction between LCAT and other apoA-I–containing particles. As a result, Lg–LpA-I particles regulate CE production in plasma.

View larger version (17K): [in this window] [in a new window]   Figure 4. Plot showing effect of adding Lg–LpA-I on the rate of cholesterol esterification in incubations containing constant amounts of lecithin:cholesterol acyltransferase and (Md+Sm)LpA-I. Cholesterol esterification rates were determined in incubations containing a fixed amount of lipoprotein-deficient plasma in the presence of increasing concentrations of (Md+Sm)LpA-I (). At varying concentrations of (Md+Sm)LpA-I, the esterification rate was also determined after further increasing the concentration of free cholesterol by adding Lg–LpA-I (). (Md+Sm)LpA-I concentrations in these incubations were held constant. LpA-I indicates HDL containing apoA-I but not apoA-II; Lg–LpA-I, large (diameter, 11.1 nm) LpA-I; and (Md+Sm)LpA-I, combined medium (diameter, 8.8 nm) and small (diameter, 7.7 nm) LpA-I.

Effect of Subclasses of LpA-I on Net Cholesterol Efflux From Macrophage Foam Cells
Because LCAT activity is required for the efficient removal of cellular cholesterol by LpA-I,^{9 16} we next studied the effect of LpA-I subclasses on net cellular cholesterol efflux. Since LCAT activity in isolated subfractions is lost during ultracentrifugation, to restore LCAT activity to the subspecies of LpA-I we added LDP as an LCAT source when we incubated the subspecies of LpA-I with foam cells. The amount of LDP added was determined based on the finding that LpA-I isolated by immunoaffinity chromatography normally esterifies 25% to 30% of its own FC after incubation for 16 hours at 37°C.⁹ We reisolated immunopurified LpA-I by ultracentrifugation (d=1.21 g/mL) for 22 hours. The LCAT activity of LpA-I was completely lost during ultracentrifugation. We then added varying amounts of LDP to reisolated LpA-I, and the percent esterification of its own FC was determined. The amount of LDP used in the following experiments (100 µL) restored the rate of cholesterol esterification in reisolated LpA-I (300 µg protein) to a level similar to that in the original LpA-I (300 µg protein). The cholesterol-reducing capacity of Lg–LpA-I was much less than that of (Md+Sm)LpA-I (Fig 5). Similar results were obtained when the experiments were performed using smaller amounts of these subclasses (50 to 200 µg protein of each subclass) (data not shown). Since plasma concentrations of LpA-I are positively correlated with concentrations of Lg–LpA-I (Fig 2), we added Lg–LpA-I to constant amounts of (Md+Sm)LpA-I and found that the cholesterol-reducing capacity of (Md+Sm)LpA-I was inhibited by Lg–LpA-I (Fig 5). In our assay system, net cellular cholesterol reduction mediated by subclasses of LpA-I and combinations of such subclasses was not observed at 3 hours of incubation. Net cellular cholesterol reduction at 6 and 12 hours of incubation was less than that at 24 hours of incubation. However, the relative abilities of Lg–LpA-I and (Md+Sm)LpA-I were similar to those at 24 hours of incubation [(Md+Sm)LpA-I>Lg–LpA-I], and inhibition by Lg–LpA-I was also observed. Thus, Fig 5 shows data only at 24 hours of incubation.

View larger version (52K): [in this window] [in a new window]   Figure 5. Bar graphs showing effects of subclasses of LpA-I (HDL containing apoA-I but not apoA-II) and combinations of subclasses on (top) cholesterol ester (CE) and (bottom) free cholesterol (FC) mass in macrophage foam cells. Macrophage foam cells were incubated for 24 hours with 300 µg protein of subclasses of LpA-I and varying combinations of subclasses. As control experiments, macrophage foam cells were incubated without lipoproteins. After incubation, cellular lipids were extracted and determined for CE and FC mass. L indicates the HDL2 fraction of LpA-I (large LpA-I); (M+S), the HDL3 fraction of LpA-I (combined medium and small LpA-I); C1, 300 µg protein (M+S)+100 µg protein L; C2, 300 µg protein (M+S)+200 µg protein L; and C3, 300 µg protein (M+S)+300 µg protein L. Data are mean±SD of five experiments. Mean value of cellular CE and FC for controls (100%) was 130 and 120 nmol/mg cell protein, respectively. *P<.005 vs (M+S); **P<.005 vs (M+S) and C1.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present studies demonstrate four major points. First, plasma concentrations of HDL-C correlate with those of LpA-I but not LpA-I/A-II; second, plasma concentrations of HDL-C and LpA-I positively correlate with those of Lg–LpA-I but not Md– or Sm–LpA-I; third, the plasma concentration of Lg–LpA-I correlates inversely with the rate of cholesterol esterification in plasma and HDL; and fourth, Md– and Sm–LpA-I play an active role in the removal of cellular cholesterol and in cholesterol esterification in plasma and HDL, while Lg–LpA-I inhibits these functions of Md– and Sm–LpA-I. These results, taken together, suggest that the plasma concentration of LpA-I correlates inversely with the efficiency of reverse cholesterol transport.

Most plasma CE is generated in HDL by the action of plasma LCAT.²⁹ However, plasma LCAT enzyme mass does not correlate with the rate of CE production in plasma.^{30 31 32} Compared with the variation in the rate of plasma CE production, that of plasma LCAT enzyme mass is fairly constant.^{20 23} Thus, the substrate specificity for LCAT seems to be important.^{15 19 20} Lg–LpA-I has the lowest apparent K_m and lowest V_max to LCAT among the subspecies of apoA-I–containing lipoproteins.¹⁵ This suggests that increases in Lg–LpA-I cause a shift of LCAT from higher (Md–LpA-I, Sm–LpA-I, and LpA-I/A-II) to lower (Lg–LpA-I) V_max particles that reduces the rate of cholesterol esterification and vice versa. As a result, the relative amount of Lg–LpA-I among HDL particles regulates the reactivity of HDL to LCAT. The present results extend these findings to plasma levels. The addition of Lg–LpA-I inhibits the rate of cholesterol esterification mediated by Md– and Sm–LpA-I in the presence of a constant amount of LCAT (Fig 4). Thus, it is reasonable to consider that these data can explain the inverse correlation between the plasma concentration of Lg–LpA-I and FERHDL or MERplasma. Since plasma concentrations of LpA-I largely depend on those of Lg–LpA-I, the inverse correlation between the plasma concentrations of LpA-I and FERHDL or MERplasma can be explained similarly. Since LCAT associated with LpA-I esterifies FC from VLDL and LDL,¹⁶ MERplasma must be affected by the plasma concentrations of VLDL and LDL. Thus, the correlation between Lg–LpA-I and MERplasma (r=-.41) ought to be weaker than that between Lg–LpA-I and FERHDL (r=-.76).

Since cholesterol esterification of LpA-I particles is required for the efficient net removal of cellular cholesterol mediated by LpA-I,^{9 16} we next studied the effect of subclasses of LpA-I on net cholesterol efflux from foam cells. LpA-I can remove cellular cholesterol more efficiently than LpA-I/A-II. In the present study, we extended our previous findings⁹ to subspecies of LpA-I particles; (Md+Sm)LpA-I (the HDL₃ fraction of LpA-I) had a greater ability to reduce cellular cholesterol than did Lg–LpA-I (the HDL₂ fraction of LpA-I). This agrees with a report by Duverger et al¹¹ that Md–LpA-I is most effective in promoting cholesterol efflux from Ob 1771 cells. Furthermore, we found that Lg–LpA-I inhibits net cholesterol efflux from macrophage foam cells mediated by (Md+Sm)LpA-I. LpA-I cannot promote net cellular cholesterol efflux without LCAT activity.^{9 16} Thus, the effect of Lg–LpA-I on the rate of cholesterol esterification appears to contribute to the inhibitory effect of Lg–LpA-I on net cellular cholesterol efflux.

Since plasma concentrations of LpA-I are mainly determined by those of Lg–LpA-I (Fig 2), our data seem to indicate that LpA-I in subjects with lower plasma concentrations of LpA-I can remove more cellular cholesterol than that in subjects with higher plasma concentrations of LpA-I. If so, our data are in sharp contrast to the current concept of the antiatherogenic nature of LpA-I. In con-trast to our interpretation, one may assume that (Md+Sm)LpA-I particles are good acceptors for cellular cholesterol and that they remain so until they become Lg–LpA-I particles. A higher concentration of plasma Lg–LpA-I may represent the end product of very efficient cellular cholesterol acceptance. However, we¹³ and others¹⁴ have reported that the fractional catabolic rate of Lg–LpA-I is much less than that of (Md+Sm)LpA-I, and no precursor-product relation was found between the two fractions.¹³ Thus, our interpretation seems to be more likely. Llera Moya et al³³ report that the serum concentration of LpA-I is positively associated with the ability of whole human serum to promote unidirectional cellular cholesterol efflux but not with the cholesterol content of cells. These findings seem to be consistent with the current concept of the antiatherogenic nature of LpA-I. However, the distribution and nature of lipoproteins in plasma are quite different from those in the interstitium. In addition, it seems unlikely that unidirectional cellular cholesterol efflux with no change of cellular cholesterol mass would contribute to the antiatherogenic nature of LpA-I. Thus, the physiological significance of the ability of whole serum to promote cellular cholesterol efflux seems to be limited. According to studies of human arteries and lymph, only small HDL particles (<10 nm in diameter) can enter the interstitium.^{34 35 36 37} Since Lg–LpA-I particles (11.1 nm in diameter) appear to be excluded from the interstitium, the net cellular cholesterol efflux mediated by (Md+Sm)LpA-I may not be inhibited in this compartment. However, plasma concentrations of (Md+Sm)LpA-I did not correlate with those of LpA-I. In addition, LCAT activity in the interstitium and that required for efficient cellular cholesterol removal are lower than that in plasma,³⁸ suggesting that the cholesterol-reducing capacity of (Md+Sm)LpA-I in the interstitium is lower than that in plasma. Therefore, it is unlikely that cellular cholesterol reduction by (Md+Sm)LpA-I can account for the inverse correlation between plasma concentrations of LpA-I and the risk of coronary artery disease. Other smaller particles may be created from Lg–LpA-I by remodeling.^{39 40 41 42} The lower fractional catabolic rate of Lg–LpA-I [versus that of (Md+Sm)LpA-I] increases the likelihood that Lg–LpA-I will be remodeled,^{13 14} and as a result, the concentration of smaller particles may increase in the interstitium as a function of the plasma concentrations of Lg–LpA-I. However, we have reported that LpA-I and LpA-I/A-II particles in patients with familial LCAT deficiency (the size and shape of these particles are similar to those of remodeled HDL) possess only 30% to 40% of the cholesterol-reducing capacity of normal LpA-I and LpA-I/A-II particles.^{43 44} Therefore, the net cholesterol efflux from peripheral cells mediated by LpA-I in the interstitium may be less than that expected based on studies using plasma LpA-I. The cholesterol-reducing capacity of LpA-I in the interstitium may not depend on the plasma concentrations of LpA-I.

Dobiasova et al²⁰ report significant increases in FERHDL in subjects with coronary artery disease. We recently confirmed this finding in Japanese patients (K. Saku and T. Ohta, unpublished data, 1994). Thus, regulation of the reactivity of apoA-I–containing lipoproteins to LCAT by Lg–LpA-I can partly explain the antiatherogenic nature of plasma LpA-I.

	Acknowledgments

 
This work was supported in part by grant-in-aid No. 06670809 for Scientific Research from the Ministry of Education, Science, and Culture of Japan and by research grants from the Ono Medical Foundation and the National Milk Promotion Association of Japan.

Received January 17, 1995; accepted April 4, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Cheung MC, Albers JJ. Characterization of lipoprotein particles isolated by immunoaffinity chromatography. J Biol Chem. 1984;259:12201-12209. [Abstract/Free Full Text]
2. Ohta T, Hattori S, Nishiyama S, Matsuda I. Studies on the lipid and apolipoprotein compositions of two species of apoA-I-containing lipoproteins in normolipidemic males and females. J Lipid Res. 1988;29:721-728. [Abstract]
3. Stampfer MJ, Sacks FM, Salvini S, Willett WC, Hennekens CH. A prospective study of lipids, apolipoproteins and risk of myocardial infarction. N Engl J Med. 1991;325:373-381. [Abstract]
4. Fruchart JC, Ailhaud G. Apolipoprotein A-containing lipoprotein particles: physiological role, quantification, and clinical significance. Clin Chem. 1992;38:793-797. [Abstract/Free Full Text]
5. Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature. 1991;353:265-267. [Medline] [Order article via Infotrieve]
6. Schultz JR, Verstuyft JG, Gong EL, Nichols AV, Rubin EM. Protein composition determines the anti-atherogenic properties of HDL in transgenic mice. Nature. 1993;365:762-764. [Medline] [Order article via Infotrieve]
7. Reichl D, Miller NE. Pathophysiology of reverse cholesterol transport: insights from inherited disorders of lipoprotein metabolism. Arteriosclerosis. 1989;9:785-797. [Free Full Text]
8. Tall AR. Plasma high density lipoproteins, metabolism, and relationship to atherogenesis. J Clin Invest. 1990;86:379-384.
9. Ohta T, Nakamura R, Ikeda Y, Shinohara M, Miyazaki A, Horiuchi S, Matsuda I. Differential effect of subspecies of lipoprotein containing apolipoprotein A-I on cholesterol efflux from cholesterol-loaded macrophages: functional correlation with lecithin: cholesterol acyltransferase. Biochim Biophys Acta. 1992;1165:119-128. [Medline] [Order article via Infotrieve]
10. Barbaras R, Puchois P, Fruchart JC, Ailhaud G. Cholesterol efflux from cultured adipose cells is mediated by LpAI particles but not by LpAI:AII particles. Biochem Biophys Res Commun. 1987;142: 63-69.
11. Duverger N, Rader D, Duchateau P, Fruchart JC, Castro G, Brewer HB Jr. Biochemical characterization of the three major subclasses of lipoprotein A-I preparatively isolated from human plasma. Biochemistry. 1993;32:12372-12379. [Medline] [Order article via Infotrieve]
12. Duverger N, Rader D, Brewer HB Jr. Distribution of subclasses of HDL containing apoA-I without apoA-II (LpA-I) in normolipidemic men and women. Arterioscler Thromb. 1994;14:1594-1599. [Abstract/Free Full Text]
13. Saku K, Liu R, Ohta T, Jimi S, Matsuda I, Arakawa K. Plasma HDL levels are regulated by the catabolic rate of large particles of lipoprotein containing apoA-I. Biochem Biophys Res Commun. 1994;200:557-561. [Medline] [Order article via Infotrieve]
14. Moriguchi E, Cheung G, Colvin P, Rudel L. In vivo metabolism of subpopulations of apolipoprotein A-I–only high density lipoproteins (HDL) in primates. Circulation. 1994;90(suppl I):I-82. Abstract.
15. Ikeda Y, Ohta T, Matsuda I. Interaction between apoA-I-containing lipoproteins and lecithin:cholesterol acyltransferase. Biochim Biophys Acta. 1994;1215:307-313. [Medline] [Order article via Infotrieve]
16. Nakamura R, Ohta T, Ikeda Y, Matsuda I. LDL inhibits the mediation of cholesterol efflux from macrophage foam cells by apoA-I–containing lipoproteins, a putative mechanism for foam cell formation. Arterioscler Thromb. 1993;13:1307-1316. [Abstract/Free Full Text]
17. Fielding CJ, Fielding PE. Evidence for a lipoprotein carrier in human plasma catalyzing sterol efflux from cultured fibroblasts and its relationship to lecithin: cholesterol acyltransferase. Proc Natl Acad Sci U S A. 1981;78:3911-3914. [Abstract/Free Full Text]
18. Havel RJ, Eder HA, Brangdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.
19. Barter PJ, Hopkins GJ, Gorjatschko L, Jones ME. Competitive inhibition of plasma cholesterol esterification by human high-density lipoprotein-subfraction 2. Biochim Biophys Acta. 1984;793:260-268. [Medline] [Order article via Infotrieve]
20. Dobiasova M, Stribrna J, Sparks DL, Pritchard PH, Frohlich JJ. Cholesterol esterification rates in very-low-density lipoprotein– and low-density lipoprotein–depleted plasma: relation to high-density lipoprotein subspecies, sex, hyperlipidemia, and coronary heart disease. Arterioscler Thromb. 1991;11:64-70. [Abstract/Free Full Text]
21. Burnstein M, Scholnick HR, Morfin R. Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J Lipid Res. 1970;11:583-595. [Abstract]
22. Warnick GR, Nguyen T, Albers AA. Comparison of improved precipitation methods for quantification of high density lipoprotein cholesterol. Clin Chem. 1985;31:217-222.[Abstract/Free Full Text]
23. Miller NE, Rajput-Williams J, Nanjee MN, Samuel L, Albers JJ. Relationship of high density lipoprotein composition to plasma lecithin:cholesterol acyltransferase concentration in men. Atherosclerosis. 1988;69:123-129. [Medline] [Order article via Infotrieve]
24. Heider JG, Boyett RL. The picomole determination of free and total cholesterol in cells in culture. J Lipid Res. 1978;19:514-518. [Abstract]
25. Ohta T, Matsuda I. Lipid and apolipoprotein levels in patients with nephrotic syndrome. Clin Chim Acta. 1981;117:133-143. [Medline] [Order article via Infotrieve]
26. Goto Y, Akanuma Y, Harano Y, Hata Y, Itakura H. Determination by SRID methods of normal values of serum apolipoprotein (A-I, A-II, B, C-II, C-III, and E) in normolipidemic healthy Japanese subjects. J Clin Biochem Nutr. 1986;1:73-88.
27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]
28. Ohta T, Hattori S, Murakami M, Nishiyama S, Matsuda I. Age- and sex-related differences in lipoproteins containing apolipoprotein A-I. Arteriosclerosis. 1989;9:90-95. [Abstract/Free Full Text]
29. Glomset JA. The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res. 1968;9:155-167. [Abstract]
30. Frohlich J, Mclead R, Pritchard PH, Fesmire J, McConathy W. Plasma lipoprotein abnormalities in heterozygotes for familial lecithin: cholesterol acyltransferase deficiency. Metabolism. 1988;37:3-8. [Medline] [Order article via Infotrieve]
31. Ohta T, Nakamura R, Takata K, Saito Y, Yamashita S, Horiuchi S, Matsuda I. Structural and functional differences of subspecies of apoA-I-containing lipoprotein in patients with plasma cholesteryl ester transfer protein deficiency. J Lipid Res. 1995;36:696-704. [Abstract]
32. Ohta T, Nakamura R, Frohlich JJ, Pritchard PH, Matsuda I. Characterization of subspecies of lipoprotein containing apolipoprotein A-I in heterozygotes for familial lecithin: cholesterol acyltransferase deficiency. Atherosclerosis. 1995;114:147-155. [Medline] [Order article via Infotrieve]
33. Llera Moya M, Atger V, Paul JL, Fournier N, Moatti N, Giral P, Friday KE, Rothblat G. A cell-culture system for screening human serum for ability to promote cellular cholesterol efflux: relations between serum components and efflux, esterification, and transfer. Arterioscler Thromb. 1994;14:1056-1065. [Abstract/Free Full Text]
34. Smith EB, Ashall C. Compartmentalization of water in atherosclerotic lesions: changes in distribution and exclusion volumes for plasma macromolecules. Arteriosclerosis. 1984;4:21-27. [Abstract/Free Full Text]
35. Rudra DN, Myant NB, Pflug JJ, Reichl D. The distribution of cholesterol and apolipoprotein A-I between the lipoproteins in plasma and peripheral lymph from normal human subjects. Atherosclerosis. 1984;53:297-308. [Medline] [Order article via Infotrieve]
36. Reichl D, Rudra DN, Myant NB, Pflug JJ. Further evidence for the role of high density lipoprotein in the removal of tissue cholesterol in vivo. Atherosclerosis. 1982;44:73-84. [Medline] [Order article via Infotrieve]
37. Reichl D, Forte TM, Hong JL, Rudra DN, Pflug J. Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopy structure. J Lipid Res. 1985;26:1399-1411.[Abstract]
38. Wong L, Curtiss LK, Huang J, Mann CJ, Maldonado B, Roheim PS. Altered epitope expression of human interstitial fluid apolipoprotein A-I reduces its ability to activate lecithin cholesterol acyltransferase. J Clin Invest. 1992;90:2370-2375.
39. Tall AR, Small DS. Plasma high-density lipoproteins. N Engl J Med. 1978;299:1232-1236. [Medline] [Order article via Infotrieve]
40. Eisenberg S. High density lipoprotein metabolism. J Lipid Res. 1984;25:1017-1058. [Medline] [Order article via Infotrieve]
41. Hopkins GJ, Chang LBF, Barter PJ. Role of lipid transfers in the formation of subpopulations of small high density lipoproteins. J Lipid Res. 1985;26:218-229. [Abstract]
42. Clay MA, Newnham HH, Forte TM, Barter PJ. Cholesteryl ester transfer protein and hepatic lipase activity promote shedding of apoA-I from HDL and subsequent formation of discoidal HDL. Biochim Biophys Acta. 1992;1124:52-58. [Medline] [Order article via Infotrieve]
43. Ohta T, Hattori S, Nakamura R, Horiuchi S, Frohlich J, Takata K, Ikeda Y, Saito Y, Matsuda I. Characterization of subspecies of apolipoprotein A-I–containing lipoprotein in homozygotes for familial lecithin:cholesterol acyltransferase deficiency. Arterioscler Thromb. 1994;14:1137-1145. [Abstract/Free Full Text]
44. Ohta T, Nakamura R, Ikeda Y, Frohlich JJ, Takata K, Saito Y, Horiuchi S, Matsuda I. Evidence for impaired cellular cholesterol removal mediated by apoA-I-containing lipoproteins in patients with familial lecithin: cholesterol acyltransferase deficiency. Biochim Biophys Acta. 1994;1213:295-301.[Medline] [Order article via Infotrieve]

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