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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:112-120.)
© 1995 American Heart Association, Inc.

Articles

Relation Between Cholesterol Ester Transfer Protein Activities and Lipoprotein Cholesterol in Patients With Hypercholesterolemia and Combined Hyperlipidemia

Federico Tatò; Gloria Lena Vega; Alan R. Tall; Scott M. Grundy

From the Center for Human Nutrition (F.T., G.L.V., S.M.G.) and Departments of Clinical Nutrition (G.L.V., S.M.G.), Internal Medicine (S.M.G.), and Biochemistry (S.M.G.), University of Texas Southwestern Medical Center, Dallas, and the Department of Internal Medicine (A.R.T.), College of Physicians and Surgeons, Columbia University Medical Center, New York, NY.

Correspondence to Gloria Lena Vega, PhD, Center for Human Nutrition, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Room Y3.206, Dallas, TX 75235-9052.

	Abstract

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Abstract Cholesterol ester transfer protein (CETP) promotes the transfer of cholesterol esters among different lipoprotein classes–high-density lipoproteins (HDL), very-low-density lipoproteins, intermediate-density lipoproteins, and low-density lipoproteins (LDL). The current study was carried out to determine whether CETP activities are correlated with lipoprotein cholesterol levels in a large number of patients having elevated LDL cholesterol and normal triglycerides (hypercholesterolemia) and elevated LDL cholesterol and high triglycerides (combined hyperlipidemia). Compared with 50 normolipidemic male patients, 113 hypercholesterolemic patients had a 42% higher mean activity of CETP, and approximately 60% of these patients had CETP activities outside the normal range. A similar elevation of CETP was observed in 47 patients with combined hyperlipidemia. Furthermore, in those with combined hyperlipidemia, CETP activities were highly correlated with LDL cholesterol, non-HDL cholesterol, and non-HDL/HDL ratios. Similar high correlations were obtained by combining normotriglyceridemic patients with and without elevated LDL cholesterol. Since patients with elevated LDL cholesterol had a significantly lower mean level of HDL cholesterol, a high CETP activity also was related to a reduced HDL cholesterol level. Our results are consistent with this concept, although they do not constitute final proof that high CETP activities contribute to elevated cholesterol concentrations and reduced HDL cholesterol levels in patients with hypercholesterolemia and in those with combined hyperlipidemia.

Key Words: hypercholesterolemia • combined hyperlipidemia • cholesterol ester transfer protein

	Introduction

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Cholesterol ester transfer protein (CETP) is recognized as an important factor regulating the distribution of cholesterol esters (CE) among the plasma lipoproteins.¹ According to current concepts, CETP facilitates the transfer of CE from high-density lipoproteins (HDL) to very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), and low-density lipoproteins (LDL) in exchange for triglyceride.^{2 3 4 5} In patients with a genetic deficiency of CETP, HDL cholesterol levels are markedly elevated,^{6 7 8} suggesting a block in the transfer of CE from HDL to other lipoproteins. However, whether CETP levels significantly influence cholesterol concentrations in different lipoprotein fractions, or the distribution of cholesterol among lipoproteins, in the absence of a genetic deficiency of CETP has not been studied extensively.

Nonetheless, several reports^{9 10 11 12 13 14} indicate that patients with various forms of hyperlipidemia often have relatively high activities or levels of CETP. This observation raises the possibility that an increased activity of CETP contributes to elevated cholesterol levels in lipoproteins containing apolipoprotein B (apo B). The present study therefore was carried out to examine the strength of the association between plasma CETP activities and cholesterol concentrations in patients with hypercholesterolemia (elevated LDL cholesterol and normal triglyceride concentrations) and in those with combined hyperlipidemia (elevated LDL cholesterol and high triglycerides). Furthermore, the relation between CETP activities and the distribution of cholesterol between apo B–containing lipoproteins and HDL was examined. Normolipidemic patients served as a reference group to evaluate these relations. A sizable number of patients were selected for each group to obtain more detailed information on the strength of the association between CETP activities and lipoprotein cholesterol concentrations.

	Methods

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Patients
This study was performed on samples obtained from 210 patients from the Veterans Affairs Medical Center at Dallas, Tex. Those who were taking lipid-lowering drugs were excluded from the study. Other exclusion criteria were treatment with steroids, a history of excessive alcohol intake, diabetes mellitus, other endocrine disorders, renal disease, clinically severe cardiopulmonary disease, or diseases of the gastrointestinal tract and liver. The presence of stable coronary heart disease (CHD) did not exclude patients from the study.

Experimental Design
The primary purpose of the study was to determine CETP activities in patients with primary hypercholesterolemia and with combined hyperlipidemia. Therefore, the population under investigation was enriched with patients of this type. This selection was aided by studying patients who were referred to the lipid clinic of the medical center. Another source included frozen plasma from patients who had been seen previously in the clinic and in whom lipid and lipoprotein values had already been obtained. In this study, the term hypercholesterolemia was defined as a plasma LDL cholesterol level 160 mg/dL and a triglyceride level <200 mg/dL. Combined hyperlipidemia was defined as an LDL cholesterol 160 mg/dL and a triglyceride level between 200 and 500 mg/dL. Normolipidemia was defined as an LDL cholesterol <160 mg/dL, triglycerides <200 mg/dL, and HDL cholesterol 40 mg/dL. The LDL cholesterol and triglyceride levels selected for classification generally conform to those of the National Cholesterol Education Program guidelines.¹⁵ Although most of the patients had previous measurements of lipids and lipoproteins, the values used in the current analysis represented the lipid and lipoprotein levels obtained on the same plasma samples in which CETP activity was measured.

Measurement of Lipids and Lipoprotein Cholesterol
Blood samples were drawn by venipuncture and collected into tubes containing disodium ethylenediamine tetraacetate (EDTA) after a 12-hour fast. On these samples, levels of plasma triglyceride, cholesterol, and lipoprotein cholesterol were measured. Aliquots of plasma also were frozen at -70°C. Levels of plasma total cholesterol and triglycerides were measured enzymatically.^{16 17} Concentrations of HDL cholesterol were measured in plasma after precipitation of apo B–containing lipoproteins with 0.55 mmol/L phosphotungstic acid and 25 mmol/L magnesium chloride.¹⁸ This method has been reported to give results for HDL cholesterol similar to levels obtained by heparin-manganese precipitation of apo B–containing lipoproteins^{19 20} ; this close similarity was confirmed in our laboratory. LDL cholesterol was calculated using the Friedewald formula²¹ when triglycerides were <300 mg/dL. When triglycerides were 300 mg/dL, the plasma was subjected to ultracentrifugation at d<1.0063 g/mL, and LDL cholesterol was quantified after precipitation of HDL cholesterol from the plasma infranatant of d>1.0063 g/mL.¹⁹

Measurement of CETP Activity
General Approach
In this study, a method was used to measure CETP activity in a way that approximates CETP mass concentration. The method was a modification of the procedure of Tollefson and Albers,²² and a recent report¹⁰ indicates that there is a close correspondence between measured activities of CETP and mass determined by radioimmunoassay. To confirm the correspondence, a comparison was made between estimated concentrations and activities in 50 plasma samples. Activities were measured on frozen samples in the Dallas laboratory and mass concentrations were determined at Columbia University, New York City, by one of the authors (A.R.T.). A solid-phase competitive radioimmunoassay²³ was used for mass measurements.

The assay for plasma CETP activity was standardized for linearity as a function of incubation time and volume of plasma used in the assay. Coefficients of interassay, intra-assay, and physiological variation were determined. The latter was measured in 25 subjects during their admission to the metabolic ward for evaluation for dyslipidemia. These measurements were done on plasma obtained during three consecutive days. In addition, the physiological variability in CETP activity over a longer period was studied in 19 patients. In these patients, CETP activity was assayed in a fresh plasma sample and in a sample that had been drawn 2 to 3 years previously and had been frozen at -70°C.

Procedure
CETP activity was measured as the percent of the total tritiated CE (³H-CE) transferred from HDL₃ (donor lipoprotein) to LDL (acceptor lipoprotein) in the presence of a small volume of the patient's plasma. Donor and acceptor lipoproteins were prepared from 300 mL of plasma obtained from six healthy, normolipidemic volunteers. LDL (d=1.02 to 1.063 g/mL) and the plasma fraction of d>1.125 g/mL were isolated by preparative ultracentrifugation.²⁴ LDL was dialyzed against Tris buffer (10 mol/L Tris base, 2 mmol/L EDTA, 150 mmol/L NaCl, and 0.01% NaN₃, pH 7.4). In addition, the LDL was diluted with the Tris buffer to a final concentration of 200 mg/dL and stored frozen in aliquots at -20°C until used in the assay. The plasma fraction of d>1.125 g/mL was also dialyzed against Tris buffer and used for the preparation of radiolabeled HDL₃. Briefly, 1.5 µCi of tritiated cholesterol ([7(n)-³H]cholesterol, 6.1 Ci/mmol, Amersham Searle) per milligram of cholesterol was injected below the surface of the d>1.125 g/mL plasma fraction while stirring. This was followed by incubation at 37°C for 18 hours to allow for the formation of ³H-CE, a reaction catalyzed by lecithin cholesteryl acyl transferase. HDL₃ (d=1.125 to 1.21 g/mL) was isolated by preparative ultracentrifugation. The lipoprotein was dialyzed against Tris buffer and diluted to a final cholesterol concentration of 40 mg/dL. The specific activity of HDL CE averaged 3333 dpm/mg. The radiolabeled HDL₃ was stored at 4°C until used in the assay. The distribution of radiolabeled unesterified and esterified cholesterol was determined in HDL₃ after separation of the lipids by thin-layer chromatography. More than 90% of the radiolabeled cholesterol was esterified. Donor and acceptor lipoproteins were stable for a period of 6 months.

The CETP assay consisted of 50 µL of the radiolabeled HDL₃ preparation, 200 µL of the LDL preparation, and 20 µL of plasma diluted 1:3 (vol/vol) in Tris buffer or 20 µL of Tris buffer used as blank. The total assay volume was 270 µL. Two quality controls and blanks were included in each assay. The quality controls consisted of a plasma sample with a low CETP activity (<20% transfer/16 hours) and another with high CETP activity (>35% transfer/16 hours). The blanks and quality controls were measured in triplicate for each assay. Plasma samples of control subjects and patients were assayed in duplicate.

Samples, blanks, and quality controls were incubated at 37°C for 16 hours, and the reaction was stopped by transferring the assay tubes to ice for 15 minutes. Donor and acceptor lipoproteins were separated by precipitation of LDL with heparin/MnCl₂. The precipitation was carried out using 92 mmol/L MnCl₂²⁵ and 0.22 mg/dL of heparin. This modification in the heparin concentration was found to minimize the coprecipitation of HDL without interfering with the complete precipitation of LDL. One hundred fifty microliters of the supernatant was counted for 20 minutes in a liquid scintillation counter. CETP activity was expressed as the percentage of radioactivity transferred from ³H-HDL₃ to LDL during 16 hours of incubation. Values were corrected for blanks and variation of quality controls.

Standardization of CETP Activity
The linearity of the assay as a function of plasma volume used in the assay and of incubation time is shown in Fig 1. The assay was linear for a sample volume ranging from 1.25 to 7.5 µL. Samples with high activity or low activity exhibited similar degrees of linearity. The assay was also linear for each sample as a function of incubation time between 3 and 18 hours. Therefore, all subsequent assays were performed using 5 µL of plasma sample and 16-hour incubations.

View larger version (20K): [in this window] [in a new window]   Figure 1. Plots show effect of time of incubation (A) and volume of plasma sample (B) on the percentage of 3H-cholesterol esters (CE) transferred from high-density lipoprotein (subfraction) (HDL3) to low-density lipoprotein (LDL) during incubation at 37°C. The incubation mixture contained 200 µL of unlabeled LDL (200 mg cholesterol/dL), 50 µL HDL3 labeled with 3H-CE (40 mg cholesterol/dL), and whole plasma as the source of cholesterol ester transfer protein (CETP) in a final volume of 270 µL. The effect of incubation time was tested at a constant sample volume of 5 µL; the effect of the sample volume was tested at a constant incubation time of 16 hours. Triangles represent a sample from a patient with hypercholesterolemia (LDL cholesterol, 165 mg/dL) and high CETP activity; circles represent a sample with low LDL cholesterol (98 mg/dL), high HDL cholesterol (68 mg/dL), and normal CETP activity. Values shown are means of four measurements ±SD.

The intra-assay coefficient of variation for the sample with high activity was 2.62%, and for the low-activity sample it was 4.42%. To determine the coefficients of variation, each sample was measured 18 times. The interassay coefficients of variation were 4.55% and 6.71% for the high- and low-activity samples, respectively. These coefficients were estimated from 12 individual measurements of the sample with high and low activities. The physiological coefficient of variation for 25 individuals averaged 5.76±2.52%. To estimate this variation, each subject had a measurement of CETP activity during each of 3 consecutive days during their evaluation for dyslipidemia.

The activity of CETP was compared between plasma samples that had been frozen and stored for a maximum time of 3 years and freshly isolated samples from the same individual (Fig 2A). These measurements were carried out in plasma of 19 different subjects. The coefficient of correlation for the two measurements was 0.96 (P<.0001). Moreover, the largest difference between the two measurements did not exceed 10.9%.

View larger version (22K): [in this window] [in a new window]   Figure 2. Plot A shows the correlation between two measure-ments of CETP activity, one in a fresh plasma sample and one in a plasma sample from the same subject that had been stored frozen at -70°C for a period of 2 to 3 years (n=19). Plot B presents the correlation between CETP activity (% transfer) and CETP mass (micrograms per milliliter) in 50 plasma samples. CETP activity was assayed in vitro using 200 µL of unlabeled LDL (200 mg cholesterol/dL) as acceptor, 50 µL HDL3 labeled with 3H-CE (40 mg cholesterol/dL) as donor, and 5 µL of whole plasma as the source of CETP. Incubation time was 16 hours. All samples had been stored frozen at -70°C until assayed. See Fig 1 for abbreviations.

CETP activity and mass measurements were comparable (Fig 2B). The coefficient of correlation for the two methods was 0.85 (P<.0001). This agreement extended to dyslipidemic and normolipidemic samples. The dyslipidemic samples included 18 subjects with normotriglyceridemia and low HDL cholesterol, 16 subjects with hypercholesterolemia, and 12 subjects with combined hyperlipidemia. The phenotype of dyslipidemia had no effect on the relation of CETP mass and activity.

Statistics
Results are summarized as mean±SD. Comparison of means was made by using one-way analysis of variance followed by Bonferroni-adjusted t tests for multiple comparisons. Logarithmic transformations of CETP activity were performed to correct for differences in variance between the 3 groups of patients. Differences in the frequency of categorical variables (CHD, hypertension, smoking, intake of ß-blockers) were tested with the ² test. The distribution of CETP activity was tested for normality using Wild-Shapiro tests for n50 and Anderson-Darling tests for n>50. Levene's test was performed to test for homogeneity of variances. Modeling of the distributions of CETP activity was carried out using the normal probability density function. Correlations were calculated by linear regression and stepwise regression analysis. A value of P=.05 was considered significant. Data management and statistical analyses were performed using CLINFO and BMDP software.

	Results

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Clinical Characteristics and Lipids and Lipoprotein Levels
The 210 patients of this study fell into three groups: 50 patients with normal levels of triglyceride and LDL cholesterol (normolipidemia), 113 patients with normal triglycerides and elevated LDL cholesterol (hypercholesterolemia), and the remaining 47 patients, who had increased levels of both triglycerides and LDL cholesterol (combined hyperlipidemia). The clinical characteristics of the three groups are shown in Table 1. The group with combined hyperlipidemia had a higher level of mean body mass index (BMI). Otherwise, the three groups had similar prevalence rates of CHD, hypertension, treatment with ß-blockers, and cigarette smoking. Mean levels of plasma lipids, lipoprotein cholesterol, and non-HDL cholesterol/HDL cholesterol (non-HDL/HDL) ratios are presented in Table 2. Differences were largely defined by the recruitment design. However, the group with combined hyperlipidemia had the highest levels of non-HDL cholesterol, the lowest HDL cholesterol levels, and the highest non-HDL/HDL ratios.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Three Groups of Patients

View this table: [in this window] [in a new window]   Table 2. Plasma Lipids and Lipoprotein Cholesterol Levels in Three Groups of Patients

CETP Activities
Mean activities and distribution characteristics of CETP in the three groups are detailed in Table 3. The frequency distributions of CETP activities in the three groups are presented in Figs 3 through 5. Best-fit distribution curves are shown for each group. Both hypercholesterolemic and combined hyperlipidemic groups had significantly higher mean activities of CETP than the normolipidemic group (Table 3). Ranges and variances were greater in the two hyperlipidemic cohorts, but in both, distributions were shifted to higher values (Figs 3 through 5).

View this table: [in this window] [in a new window]   Table 3. Mean Values and Distributional Characteristics of Plasma Cholesterol Ester Transfer Protein Activity in Three Groups of Patients

View larger version (18K): [in this window] [in a new window]   Figure 3. Frequency distribution of cholesterol ester transfer protein (CETP) activity in normolipidemic patients. The histogram is superimposed on the corresponding normal distribution calculated by the normal probability density function (dashed line).

View larger version (19K): [in this window] [in a new window]   Figure 4. Frequency distribution of cholesterol ester transfer protein (CETP) activity in patients with hypercholesterolemia and normal triglycerides. The histogram is superimposed on the corresponding distribution curve calculated by the normal probability density function (solid line). Dashed line shows the distribution curve for the normolipidemic patients.

View larger version (21K): [in this window] [in a new window]   Figure 5. Frequency distribution of cholesterol ester transfer protein (CETP) activity in patients with combined hyperlipidemia. The histogram is superimposed on the corresponding distribution curve calculated by the normal probability density function (solid line). Dashed line shows the distribution curve for the normolipidemic patients.

Correlations between CETP activities and various parameters are presented in Table 4. Data are given for correlations only within each group. The correlation coefficients (r²) presented represent the appropriate statistical contribution of CETP activities to each parameter. There were no significant relations between CETP activities and age, BMI, or total triglycerides. Total cholesterol levels were significantly correlated with CETP activity only within the hypercholesterolemic group, but LDL cholesterol levels were positively and significantly correlated with CETP activities for all three groups. The same was true for non-HDL cholesterol levels and for non-HDL/HDL ratios in normolipidemic and combined hyperlipidemic patients. CETP activities showed a trend toward a negative correlation with HDL cholesterol levels, but the correlation was significant only for the group with combined hyperlipidemia.

View this table: [in this window] [in a new window]   Table 4. Pearson Correlation Coefficients (r2) With Plasma Activity of Cholesterol Ester Transfer Protein in Three Groups of Patients

The failure to detect a significant correlation between CETP activities and non-HDL cholesterol levels in the hypercholesterolemic group may have been due to an insufficiently wide range of cholesterol levels in this fraction. The marked difference in mean CETP activities between normolipidemic and hypercholesterolemic groups, both having normal triglyceride levels, suggested that CETP activities may be significantly correlated with cholesterol levels over a broader range of levels in normotriglyceridemic subjects. To examine this possibility, a number of patients equal to those with normolipidemia were selected from the hypercholesterolemic group, and the two sets were combined and analyzed together. The 50 patients from the hypercholesterolemic group were those with the 50 lowest levels of triglycerides, which made their triglyceride concentrations similar to the normolipidemic group. The data showed a high correlation between CETP activities and non-HDL cholesterol (r²=.42; P<.0001), LDL cholesterol (r²=.43; P<.0001), and non-HDL/HDL ratios (r²=.28; P<.0001) (Fig 6). Also, there was a significant inverse correlation (r²=.12; P<.001) between CETP activities and HDL cholesterol concentrations.

View larger version (21K): [in this window] [in a new window]   Figure 6. Scatterplots show comparison of CETP activities to LDL cholesterol (A), non-HDL cholesterol (B), and non-HDL/HDL ratio (C) in 100 normotriglyceridemic patients. The figure includes the 50 normolipidemic subjects described in the "Results" section and 50 hypercholesterolemic subjects (LDL cholesterol, 188±30 mg/dL) with plasma triglyceride levels <200 mg/dL. See Fig 1 for abbreviations.

	Discussion

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This study demonstrates that patients with elevated LDL cholesterol levels, with or without increased triglyceride concentrations, have higher average activities of CETP than do comparable normolipidemic patients. Since a high correlation was found between estimates of CETP activity and CETP mass concentrations (Fig 2B), it is presumed that the observed differences extend to CETP concentrations. Another study¹⁰ using similar methodology also found a close correspondence between activities and concentrations of CETP. Our data further demonstrate that CETP activities are positively and significantly correlated with LDL cholesterol and non-HDL cholesterol concentrations over a broad range of cholesterol levels in patients with normal or elevated triglycerides. This finding raises the issue of the nature of the relation between elevated CETP activities and high concentrations of cholesterol in apo B–containing lipoproteins. Of particular interest is whether the latter are causally related to the former.

Several studies^{9 10 11 12 13 14} have demonstrated that patients with elevated cholesterol levels tend to have increased CETP activity, but recent opinion seems to be that plasma CETP levels actually have little influence on concentrations of lipoprotein cholesterol. Rather, the prevalent view holds that CETP plays an important role in the shuttling of CE between lipoproteins in their passage through the plasma, but beyond this, it has little effect on cholesterol concentrations. In accord, the predominant hypothesis is that high CETP activities are secondary to increased cholesterol levels; that is, hypercholesterolemia causes high CETP activity. An important question is whether this hypothesis is correct. For example, in the presence of a genetic deficiency of CETP, concentrations of lipoproteins are markedly altered. While this may be an extreme case, it raises the question of whether lesser variations in CETP activity could affect lipoprotein concentrations. In the discussion to follow, we examine the current findings to see whether they shed any light on the nature of the relation between CETP activities and lipoprotein levels.

Normotriglyceridemic Patients
Normolipidemic Patients
Even though our patients in this category had a relatively narrow range of CETP activities, within the group there nevertheless were positive, significant correlations between CETP activities and LDL cholesterol levels, non-HDL cholesterol levels, and non-HDL/HDL ratios. If these correlations were to represent a causal connection, then in normolipidemic subjects, differences in CETP activities could account for 13% to 14% of the variability in levels of cholesterol in apo B–containing lipoproteins.

Hypercholesterolemic Patients
This group of patients had significantly higher CETP levels than normolipidemic subjects. The increment averaged 42% (Table 3). Approximately 60% of hypercholesterolemic patients had CETP activities outside the normal distribution curve (Fig 4). Thus, high CETP activities appear to be characteristic of the majority of normotriglyceridemic patients with hypercholesterolemia. Although CETP values for the group were shifted to higher values, there was a relatively small but significant correlation between CETP activities and LDL cholesterol levels within this group; variability in activities of CETP appeared to account for only about 4% of the variation in LDL cholesterol concentrations within the group. This small effect, however, should not obscure the marked difference in CETP activities between normolipidemic and hypercholesterolemic groups.

Summed Normotriglyceridemic Patients
Although the two normotriglyceridemic groups were selected separately, we have attempted to determine the correlation between CETP activities and lipoprotein levels across the entire range of cholesterol concentrations. This could not be done simply by combining the two groups. Therefore, an effort was made to obtain "comparable" samples by selecting equal numbers from both groups having similar triglyceride levels. The findings shown in Fig 6 (A and B) indicate that a strong, positive correlation was obtained between CETP activities and concentrations of both LDL cholesterol and non-HDL cholesterol. Variation in CETP activities appeared to account for about 45% of the variance in cholesterol concentrations in these two fractions. While it can be questioned whether CETP variation could explain such a high proportion of the variation in cholesterol levels, the results do not appear to be much out of line with the marked difference in CETP activities between normolipidemic and hypercholesterolemic groups. Thus, there is a strong link between CETP activities and cholesterol concentrations in patients with normotriglyceridemia. In this summed analysis, there also was a high correlation between CETP activities and non-HDL/HDL ratios (Fig 6C).

Combined Hyperlipidemia
In this group, mean CETP activities were strikingly elevated compared with the normolipidemic group. Furthermore, within this group, CETP activities were significantly correlated with LDL cholesterol, non-HDL cholesterol, and particularly with non-HDL/HDL ratios (Table 4). These relatively high correlations are conspicuous considering how relatively narrow was the range of cholesterol levels. This finding suggests that CETP may be particularly active in the presence of high triglyceride concentrations, as previously suggested by Mann et al.²⁶

Interpretation
The above observations are at least compatible with the concept that CETP may be one of several factors affecting concentrations of cholesterol in apo B–containing lipoproteins. The mechanisms whereby a high CETP activity could elevate cholesterol concentrations in these lipoproteins theoretically is related to the basic action of the protein. CETP is known to transfer CE from HDL to VLDL, IDL, and LDL.^{2 3 4 5} The resulting enrichment with CE could increase the cholesterol concentrations in these lipoprotein fractions in two ways. First, the enrichment process itself could raise the cholesterol concentration even without an increase in the number of circulating lipoprotein particles. For example, a recent report from our laboratory²⁷ revealed that a sizable proportion of patients with primary hypercholesterolemia exhibit high cholesterol levels solely on the basis of enrichment of LDL particles with cholesterol; the number of LDL particles, as reflected by LDL–apo B concentrations, was not increased. The hypothesis that high CETP activities may enhance rates of redistribution of CE is supported by the relatively strong correlations between CETP activities and non-HDL/HDL ratios (Table 4 and Fig 6). Moreover, it is well known that patients with hypercholesterolemia and combined hyperlipidemia frequently have low HDL cholesterol concentrations, as observed in the present study. This association seemingly adds support to the concept that high levels of CETP in hypercholesterolemic states are physiologically active in the redistribution of CE among the different lipoprotein fractions.

A second but more tenuous possibility is that an augmentation of the cholesterol content of lipoprotein particles can slow their catabolism, which would further increase cholesterol concentrations. Although this mechanism has not been validated, it is consistent with the presence of elevated CETP activities in patients having increased concentrations of cholesterol in apo B–containing lipoproteins. If high CETP activities actually raise cholesterol concentrations beyond that caused by enrichment of lipoprotein particles with CE, a mechanism of this second type must be at play.

The findings of the present study do not exclude the alternate postulate for the relation between CETP activities and cholesterol concentrations, namely, that high cholesterol levels cause high CETP levels. If this proposal actually pertains, increased CETP levels presumably would have very little effect on cholesterol distribution among lipoproteins in a manner that would affect lipoprotein cholesterol levels. This hypothesis is based on two observations. First, high CETP activities have been reported in several different forms of hypercholesterolemia,^{9 10 11 12 13 14} which might imply a common secondary response; second, feeding of excess cholesterol raises CETP levels, both in rabbits²⁸ and humans.²⁹ Cholesterol feeding also increases mRNA for CETP in transgenic mice.³⁰ These latter observations^{28 29 30} appear to be solid, but they may not be related to the first set of findings.^{9 10 11 12 13 14} The feeding of excess cholesterol undoubtedly raises tissue concentrations of cholesterol, and this could stimulate the synthesis of CETP independent of any action of dietary cholesterol to raise serum cholesterol levels. In other words, the rise in serum cholesterol per se might not be the cause of increased CETP activity.

If moderately high plasma concentrations of cholesterol indeed do directly induce an enhanced CETP synthesis and secretion, then regulation of CETP synthesis in tissues must be exquisitely sensitive to plasma cholesterol levels. It is questionable whether such a mechanism could be explained by increased receptor-mediated uptake of lipoproteins because this process tightly controls rate of entrance of cholesterol into cells. In other words, high plasma cholesterol concentrations per se do not of necessity indicate increased delivery of cholesterol into cells via LDL receptors. A nonreceptor pathway for lipoprotein cholesterol uptake more likely would have to be invoked, yet there is little evidence that nonreceptor uptake of cholesterol is significantly enhanced in the presence of moderate increases in plasma cholesterol concentrations. If such a process could elicit a striking increase in CETP synthesis, as would be required to explain the higher CETP levels observed in our patients, this would indeed be a remarkable response. If this explanation pertains, the critical question becomes: How can tissue regulatory enzymes for CETP synthesis sense the presence of elevated cholesterol concentrations in lipoproteins containing apo B-100? In the absence of a satisfactory answer to this question, we suggest that it is less likely that high cholesterol levels cause increased CETP activities than vice versa. Nonetheless, the former possibility deserves further investigation.

The finding that high CETP levels attend several different hypercholesterolemic states^{9 10 11 12 13 14} could actually support the concept that they raise cholesterol concentrations. For example, in patients who have modest defects in lipoprotein metabolism, the addition of a high CETP level might enhance the cholesterol level sufficiently to bring them to clinical notice. Of particular notice is the finding that patients with familial dysbetalipoproteinemia frequently have high CETP levels.¹² The incidence of clinical dysbetalipoproteinemia in the patients with the apo E2/E2 genotype is relatively low, and it is conceivable that in E2/E2 patients, the presence of a high CETP level augments ß-VLDL levels by packing VLDL remnants with CE and retarding their catabolism. Thus, a high CETP level may be one factor that elicits the clinical picture of dysbetalipoproteinemia in patients with the E2/E2 genotype. A similar example occurs in patients with the nephrotic syndrome; those nephrotic patients who have cholesterol-enriched VLDL also have elevated CETP levels.^{13 14}

Summary
The current study provides strong additional support for the existence of a significant positive relation between the activity of CETP and concentrations of LDL cholesterol and non-HDL cholesterol. This relation has been documented in a large number of patients with different forms of elevated cholesterol levels. The results are in accord with the concept that high CETP levels are a definite factor augmenting cholesterol levels. This seemingly is true for both primary hypercholesterolemia and combined hyperlipidemia. At the same time, high CETP levels may help to confer the low HDL cholesterol levels found in hypercholesterolemic patients, and this in turn could increase the non-HDL/HDL ratio. The current observation that CETP activities are positively correlated with cholesterol concentrations in apo B–containing lipoproteins and inversely correlated with HDL cholesterol levels lends support to the concept that CETP activities do influence the concentrations of lipoprotein cholesterol. If future studies can demonstrate with certainty a causal connection between high CETP levels and these lipoprotein abnormalities, then high concentrations of CETP could prove to be an important atherogenic factor, as suggested by animal models,^{28 31 32} and potentially could be a target for therapy.

	Acknowledgments

 
This work was supported by the Department of Veterans Affairs grants HL-29252 and HL-22682; National Institutes of Health grant MO-IRR00663, Bethesda, Md; Merck & Co, Inc, West Point, Pa; an unrestricted grant from Bristol-Myers Squibb, New Brunswick, NJ; the Southwestern Medical Foundation, Dallas, Tex; and the Moss Heart Foundation, Dallas, Tex. The authors appreciate the excellent technical assistance of Biman Pramanik, Hahn Nguyen Tron, Hahn Tran, and Champa Vitalla. The assistance of Kathleen Gray, RN, Terri Shamway, RN, and the clinical staff of the metabolic unit at the Veterans Affairs Medical Center also is appreciated. Beverly Adams, MS, Program Analyst of the General Clinical Research Center, assisted in the data management and analysis.

Received July 25, 1994; accepted October 18, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Tall AR. Plasma cholesterol ester transfer protein. J Lipid Res. 1993;34:1255-1274. [Medline] [Order article via Infotrieve]
2. Rehnborg CS, Nicols AV. The fate of cholesteryl esters in human serum incubated in vitro at 38°C. Biochim Biophys Acta. 1964;84:596-603. [Medline] [Order article via Infotrieve]
3. Morton RE, Zilversmit DB. Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma. J Biol Chem. 1983;258:11751-11757. [Abstract/Free Full Text]
4. Hesler CB, Swenson TL, Tall AR. Purification and characterization of human plasma cholesteryl ester transfer protein. J Biol Chem. 1987;262:2275-2282. [Abstract/Free Full Text]
5. Yen FY, Deckelbaum RJ, Mann CJ, Marcel YL, Milne RW, Tall AR. Inhibition of cholesteryl ester transfer protein activity by monoclonal antibody: effects on cholesteryl ester formation and neutral lipid mass transfer in human plasma. J Clin Invest. 1989;83:2018-2024.
6. Koizumi J, Mabuchi H, Yoshimura A, Michishita I, Takeda M, Itoh H, Sakai Y, Sakai T, Ueda K, Takeda R. Deficiency of serum cholesteryl ester transfer activity in patients with familial hyperalphalipoproteinemia. Atherosclerosis. 1985;58:175-186. [Medline] [Order article via Infotrieve]
7. Inazu A, Brown ML, Hesler CB, Agellon LB, Koizumi J, Takata K, Maruhama Y, Mabuchi H, Tall AR. Increased high density lipoprotein caused by a common cholesteryl ester transfer protein gene mutation. N Engl J Med. 1990;323:1234-1238. [Abstract]
8. Koizumi J, Inazu A, Kunimas Y, Ichiro K, Uno Y, Kajinami K, Miyamoto S, Moulin P, Tall AR, Mabuchi H, et al. Serum lipoprotein lipids concentrations and composition in homozygous and heterozygous patients with cholesteryl ester transfer protein deficiency. Atherosclerosis. 1991;90:189-196. [Medline] [Order article via Infotrieve]
9. Bagdade JD, Ritter MC, Subbaiah PV. Accelerated cholesteryl ester transfer in plasma of patients with hypercholesterolemia. J Clin Invest. 1991;87:1259-1265.
10. McPherson R, Mann CJ, Tall AR, Hogue M, Martin L, Milne RW, Marcel YL. Plasma concentration of cholesteryl ester transfer protein activity and other lipoprotein variables. Arterioscler Thromb. 1991;11:797-804. [Abstract/Free Full Text]
11. Inazu A, Koizumi J, Mabuchi H, Kajinami K, Takeda R. Enhanced cholesteryl ester transfer protein activities and abnormalities of high density lipoproteins in familial hypercholesterolemia. Hormone Metabol Res. 1992;24:284-288. [Medline] [Order article via Infotrieve]
12. Tall AR, Granot E, Brocia R, Tabas I, Hesler C, Williams K, Denke MA. Accelerated transfer of cholesteryl esters in dyslipidemic plasma: role of cholesteryl ester transfer protein. J Clin Invest. 1987;79:1217-1225.
13. Moulin P, Appel GB, Ginsberg HN, Tall AR. Increased concentration of plasma cholesteryl ester transfer protein in nephrotic syndrome: role in dyslipidemia. J Lipid Res. 1992;33:1817-1822. [Abstract]
14. Dullaart RPF, Gansevoort RT, Dikkeschei BD, De Zeeuw D, De Jong PE, van Tol A. Role of elevated lecithin: cholesterol acyltransferase and cholesteryl ester transfer protein activities in abnormal lipoproteins from proteinuric patients. Kidney Int. 1993;44:91-97. [Medline] [Order article via Infotrieve]
15. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. National Cholesterol Education Program: second report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). Circulation. 1994;89:1329-1445.
16. Roeschlau P, Bernt E, Gruber W. Enzymatic determination of total cholesterol in serum. Z Clin Chem Clin Biochem. 1974;12:226-227.
17. McGowan MW, Artis JD, Strandbergh DR, Zack B. A peroxidase coupled method for the colorimetric determination of serum triglycerides. Clin Chem. 1983;29:538-542. [Abstract/Free Full Text]
18. Warnick GR, Cheung MC, Albers JJ. Comparison of current methods for high-density lipoprotein cholesterol quantitation. Clin Chem. 1979;25:596-604. [Abstract/Free Full Text]
19. Lipid Research Clinics Program. Manual of Laboratory Operations, Lipid and Lipoprotein Analysis. Bethesda, Md: US Government Printing Office; 1984.
20. Assmann G, Schriewer H, Schmitz G, Hagele E-O. Quantification of high-density-lipoprotein cholesterol by precipitation with phosphotungstic acid MgCl₂. Clin Chem. 1983;29:2026-2030. [Abstract/Free Full Text]
21. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18:499-502. [Abstract]
22. Tollefson JH, Albers JJ. Isolation, characterization, and assay of plasma lipid transfer proteins. In: Albers, JJ, Segrest, JP, eds. Methods of Enzymology. New York: Academic Press, Inc; 1994:797-812.
23. Marcel YL, McPherson R, Hogue M, Czarnecka H, Zawadzki Z, Weech PK, Whitlock ME, Tall AR, Milne RW. Distribution and concentration of cholesteryl ester transfer protein in plasma of normolipidemic subjects. J Clin Invest. 1990;85:10-17.
24. Lindgren FJ, Jensen LC, Hatch FT. The isolation and quantitative analysis of serum lipoproteins. In: Nelson, GS, ed. Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism. New York: Wiley Interscience; 1972:181-274.
25. Warnick GR, Albers JJ. A comprehensive evaluation of the heparin-manganese precipitation procedure for estimating high density lipoprotein cholesterol. J Lipid Res. 1978;19:65-76. [Abstract]
26. Mann CJ, Yen FT, Grant AM, Bihain BE. Mechanism of plasma cholesteryl ester transfer in hypertriglyceridemia. J Clin Invest. 1991;88:2059-2066.
27. Vega GL, Denke MA, Grundy SM. Metabolic basis of primary hypercholesterolemia. Circulation. 1991;84:118-128. [Abstract/Free Full Text]
28. Quinet EM, Agellon LB, Kroon PA, Marcel YL, Lee YC, Whitlock ME, Tall AR. Atherogenic diet increases cholesteryl ester transfer protein (CETP) mRNA levels in rabbit liver. J Clin Invest. 1990;85:357-363.
29. Martin LJ, Connelly PW, Nancoo D, Wood N, Zhang ZJ, Maguire G, Quinet E, Tall AR, Marcel YL, McPherson R. Cholesteryl ester transfer protein and high density lipoprotein responses to cholesterol feeding in men: relationship to apolipoprotein E genotype. J Lipid Res. 1993;34:437-446. [Abstract]
30. Jiang XC, Agellon LB, Walsh A, Breslow JL, Tall AR. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice: dependence on natural flanking sequences. J Clin Invest. 1992;90:1290-1295.
31. Agellon LB, Walsh A, Hayek T, Moulin P, Jiang XC, Shelanski SA, Breslow JL, Tall AR. Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer protein transgenic mice. J Biol Chem. 1990;266:10796-10801. [Abstract/Free Full Text]
32. Marotti KR, Castle CK, Boyle TP, Lin AH, Murray RW, Melchior GW. Severe atherosclerosis in transgenic mice expressing simian cholesteryl ester transfer protein. Nature. 1993;364:73-75.[Medline] [Order article via Infotrieve]

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