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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:446-451.)
© 1995 American Heart Association, Inc.

Articles

Bimodal Distribution of Cholesteryl Ester Transfer Protein Activities in Normotriglyceridemic Men With Low HDL Cholesterol Concentrations

Federico Tato; Gloria Lena Vega; Scott M. Grundy

From the Center for Human Nutrition (F.T., G.L.V., S.M.G.) and the 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.

Correspondence to Gloria Lena Vega, PhD, Center for Human Nutrition (Y3.206), University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9052.

	Abstract

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Abstract Increased plasma activities of cholesteryl ester transfer protein (CETP) theoretically could lower HDL cholesterol levels due to enhanced transfer of cholesteryl esters from HDL to apo B–containing lipoproteins. To determine whether high CETP activities are associated with isolated hypoalphalipoproteinemia, CETP activities were measured in 109 adult men with HDL cholesterol <35 mg/dL, plasma triglycerides <200 mg/dL, and LDL cholesterol <160 mg/dL; the results were compared with those of 50 normolipidemic (HDL cholesterol >40 mg/dL) male subjects. CETP activities were assayed in vitro and expressed as the percent of [³H]cholesteryl ester transferred from HDL₃ to LDL during a 16-hour incubation. In addition, postheparin plasma activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were determined in 71 patients with a low HDL cholesterol level. Distributions of CETP activities were unimodal in control subjects (mean±SD, 23.1±5.0%), but they were bimodal in the low-HDL patients. Among the latter, 27 patients had elevated CETP activities (40.8±4.6%), whereas 82 patients had CETP activities that overlapped the normal range (26.14±7.6%). Low-HDL patients with normal CETP activities had 20% lower LPL activities (P=.01), 25% higher HTGL activities (P=.03), and 63% lower LPL/HTGL ratios (P<.001) than those of low-HDL patients with increased CETP activity. Furthermore, mean LPL and HTGL activities in the low-HDL patients with elevated CETP activities were in the normal range. Another important distinction between the two subgroups with low HDL was that the subgroup with high CETP activity had only a 30% prevalence of coronary heart disease compared with a 70% prevalence in the subgroup with normal CETP activity (P<.01). These findings suggest that elevated CETP activity may be a significant factor in causing low HDL cholesterol levels in a distinct subgroup of normolipidemic patients with low HDL cholesterol levels.

Key Words: cholesteryl ester transfer protein • HDL • normotriglyceridemia • postheparin plasma lipase activities

	Introduction

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The inverse relation between plasma levels of HDL cholesterol and the risk for coronary heart disease (CHD) is well established.^{1 2 3} This association is strong enough to designate a low HDL cholesterol level as a major risk factor for CHD.⁴ Although reduced levels of HDL cholesterol occur in hypertriglyceridemia^{5 6} and other forms of hyperlipidemia,⁷ a low HDL cholesterol concentration in the presence of normal plasma lipids (normolipidemia) is also a common lipoprotein pattern in patients with CHD. In fact, recent studies indicate that in the United States as many as one third of patients who develop CHD have a total cholesterol level <200 mg/dL^{8 9} ; moreover, in a large number of these patients, triglyceride concentrations also are normal, but HDL cholesterol levels are low.^{8 9 10}

The causes of low HDL cholesterol levels in patients with normal triglycerides are not fully understood. These patients, however, usually have an increased fractional catabolic rate for apo A-I, indicative of an abnormality in HDL metabolism.^{11 12 13 14} Although a low production rate of apo A-I may contribute to low HDL levels in some patients,¹² tracer kinetic studies indicate that increased catabolism of apo A-I is a more consistent abnormality.^{11 12 13 14} The mechanisms responsible for the accelerated catabolism of HDL are probably heterogeneous. A low activity of lipoprotein lipase (LPL) and a high activity of hepatic triglyceride lipase (HTGL) have both been linked to low levels of HDL cholesterol^{15 16 17 18} as well as to increased catabolism of apo A-I.^{11 13 15 19} These abnormal lipase activities are the most common metabolic changes so far identified in normotriglyceridemic patients with low HDL levels. However, as reported recently from this laboratory,¹⁸ abnormal lipase activities cannot explain low HDL levels in all patients. Therefore, other factors, yet to be identified, must be involved in the etiology of this condition.

In recent years, cholesteryl ester transfer protein (CETP) has emerged as another plasma component of importance in the metabolism of HDL cholesterol. CETP facilitates the exchange of neutral lipids among plasma lipoproteins.^{20 21 22 23} CETP also induces a net movement of cholesteryl esters from HDL to triglyceride-rich lipoproteins in exchange for triglyceride.^{24 25 26 27} This redistribution process theoretically could affect HDL cholesterol concentrations. Indeed, the absence of CETP in hereditary CETP deficiency results in greatly increased levels of HDL cholesterol; furthermore, in this condition, there are low levels of LDL and VLDL cholesterol.^{28 29 30}

These observations raise the possibility that high CETP activity is one cause of low HDL cholesterol levels. In humans, increased CETP mass and/or activity have been reported in several conditions associated with low HDL cholesterol; these include probucol treatment,^{31 32 33 34 35 36} diabetes mellitus,^{34 36} and dyslipidemia of the nephrotic syndrome.^{37 38} However, evidence that elevated CETP levels may cause low HDL cholesterol concentrations in normolipidemic humans is lacking. Thus, the present study was designed to determine whether normolipidemic men who have reduced HDL cholesterol levels also have abnormalities in CETP activity. In addition, the relative frequencies of abnormal CETP activities were compared with the frequencies of abnormal LPL and HTGL activities in these same patients.

	Methods

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

The primary aim of the study was to measure CETP activity in patients with normolipidemia and a low HDL cholesterol level and to compare the findings with those of normolipidemic subjects with normal HDL levels. Selection of plasma samples according to the criteria described below was made from among a large number of samples obtained from patients who were being evaluated for dyslipidemia at the time of the study or from samples of patients who were seen before the study was initiated. The latter samples had been stored at -70°C. In the present study, the term "normolipidemia with low HDL" was defined as an HDL cholesterol level <35 mg/dL, an LDL cholesterol level <160 mg/dL, and triglycerides <200 mg/dL.⁴ A total of 109 men were identified as having this lipoprotein phenotype. Fifty normolipidemic men with normal HDL cholesterol levels served as control subjects. These same men also were control subjects for another recent study,³⁹ but their measurements were made simultaneously with those of the present study. These normolipidemic control subjects had LDL cholesterol <160 mg/dL, HDL cholesterol >40 mg/dL, and triglycerides <200 mg/dL. All CETP and lipase measurements reported in this study were done on the same plasma samples that were used for plasma lipid and lipoprotein levels.

The clinical characteristics and lipid profiles of the patients in both groups are summarized in Table 1. All subjects were middle-aged men. On average, patients in the low-HDL group had a higher body mass index (BMI) and a higher prevalence of CHD than did the normolipidemic group. The two groups did not differ in the frequency of hypertension, smoking, or treatment with ß-adrenergic–blocking agents (ß-blockers). The levels of fasting plasma triglycerides and non-HDL cholesterol (VLDL+ IDL+LDL) were higher in the low-HDL group. The two groups did not differ in LDL cholesterol levels. As a result of selection criteria, the patients in the low-HDL group had about 41% lower HDL cholesterol levels than the control subjects.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics and Levels of Plasma Lipids and Lipoprotein Cholesterol

General Procedures
Blood samples were collected into tubes containing Na₂-EDTA at a concentration of 1 mg/mL of blood. All blood samples were collected after a 12-hour fast. Aliquots of plasma samples were set aside for measurement of CETP activity, and the remainder was used for measurement of plasma lipids and lipoprotein cholesterol. CETP activity was measured in 57% of plasma samples soon after collection, and the remaining 43% was frozen at -70°C for a maximum of 3 years. Postheparin activities of LPL and HTGL were measured in 71 low-HDL patients who were compared with 51 control men with normal HDL cholesterol levels (normal-HDL control subjects) reported previously.¹⁸

Plasma CETP Activities
Plasma CETP activities were determined according to the method of Albers et al,²² as recently modified in our laboratory.³⁹ Briefly, plasma was obtained from fasting normolipidemic volunteers and pooled to isolate the acceptor lipoprotein, LDL (density=1.019 g/mL to 1.063 g/mL) and the plasma fraction of density >1.125 g/mL. Both fractions were isolated by preparative ultracentrifugation. Subsequently, they were dialyzed against a Tris buffer (10 mmol/L Tris base, 150 mmol/L NaCl, 2 mmol/L EDTA, and 0.01% NaN₃, pH 7.4). LDL was diluted to a final cholesterol concentration of 200 mg/dL and stored in aliquots at -20°C. The plasma fraction of density >1.125 g/mL was incubated at 37°C for 18 hours in the presence of tritiated unesterified cholesterol. This procedure allowed for the esterification of radiolabeled cholesterol by the enzyme lecithin:cholesterol acyltransferase. Subsequently, the density of the plasma fraction was adjusted to 1.21 g/mL and subjected to ultracentrifugation. The donor lipoprotein, HDL₃, containing tritiated cholesterol esters ([³H]HDL₃), was isolated, dialyzed against the Tris buffer, and diluted to a final cholesterol concentration of 40 mg/dL. Aliquots of the lipoprotein were stored at 4°C for use in the assays. For the assay, 50 µL [³H]HDL₃ was mixed with 200 µL LDL and 20 µL of test plasma that had been diluted (1:3, vol/vol) with Tris buffer. Blanks and quality controls also were included in each assay. All assay samples were incubated for 16 hours at 37°C, and the reaction was stopped by placing the tubes in ice for 15 minutes. Donor and acceptor lipoproteins were separated by heparin/MnCl₂ precipitation of LDL.⁴⁰ An aliquot of the supernatant was counted for 20 minutes in a liquid scintillation counter. CETP activity was expressed as the percentage of radioactivity transferred from donor to acceptor lipoprotein during the 16-hour incubation. Values for blanks were subtracted from the sample radioactivity values. As recently demonstrated,³⁹ measurements of CETP activity are highly correlated with CETP mass (r=.85, P<.0001), which was measured immunologically in the laboratory of Dr Alan Tall, Columbia University, New York, NY. CETP specific activities (activity divided by mass) were independent of lipoprotein levels. To demonstrate this, specific CETP activities of 31 plasma samples with a mean HDL cholesterol level of 29±3 mg/dL were compared with those of 19 samples with a mean HDL cholesterol level of 43±5 mg/dL. The former group had a mean CETP specific activity of 11.9±2.8 (percent transferred per nanogram), and the latter, a mean of 11.5±2.0 (percent transferred per nanogram). Mean specific activities were not significantly different. Thus, the assay for CETP activity is highly correlated with CETP mass, regardless of HDL cholesterol levels. The intra-assay coefficient of variation for the samples with high activity was 2.62% and for the low-activity samples, 4.42%. The physiological coefficient of variation was 5.76±2.52%.³⁹

Plasma Lipids and Lipoprotein Cholesterol
Levels of plasma total cholesterol, HDL cholesterol, and triglycerides were measured enzymatically.^{41 42} Concentration of HDL cholesterol was measured in whole plasma after precipitation of apo B–containing lipoproteins with 0.55 mmol/L phosphotungstic acid and 25 mmol/L MgCl₂.⁴³ This procedure gives values for HDL cholesterol that are similar to those obtained by precipitation with heparin-manganese, as detailed in the Lipid Research Clinics procedures.^{44 45} LDL cholesterol was calculated using the formula of Friedewald et al.⁴⁶

Postheparin LPL Activities
LPL and HTGL activities were measured in postheparin plasma samples of 71 normolipidemic patients with low HDL. Their results were compared with those of 51 men with normal HDL cholesterol levels. Results for this group have been reported previously.¹⁸ Among the 71 low-HDL patients of the present study, some were included among the previously presented normolipidemic, low-HDL patients.³⁹ The method employed was a modification of the method of Baginsky and Brown⁴⁷ as detailed recently.¹⁸ Briefly, patients received an intravenous injection of 75 IU heparin, and blood was drawn by venipuncture before and 15 minutes after injection. The blood was spun at 4°C to obtain plasma, and the latter was frozen at -70°C until measurements of LPL and HTGL activities were made. The substrate for the assay consisted of an emulsion containing 15 mmol/L triolein mixed with tritiated triolein (glycerol tri[9,10,[³H]oleate) and stabilized with gum arabic. The assay for hepatic LPL consisted of substrate, 25 µL postheparin plasma from the patient, 0.2 mmol/L Tris chloride buffer (pH 8.2), and 0.75 mol/L NaCl to inhibit LPL activity. The assay mixture was incubated at 28°C. The LPL assay consisted of 25 µL of the patient's postheparin plasma (which was incubated with heat-inactivated serum as a source of apo C-II), substrate, and 25 µL of 50 mmol/L SDS to inhibit HTGL. In both assays, the final concentration of fatty acid–free bovine serum albumin (fraction V) was 50 g/L; the final assay volume, 0.5 mL; and the reaction time, 1 hour. Free, tritiated oleic acid that was released from tritiated triolein by lipase activity was extracted by the method of Belfrage and Vaughan as detailed previously.¹⁸ [1-¹⁴C]oleic acid was used routinely⁴⁸ as an internal standard. The interassay and intra-assay coefficients of variation were <5% for each assay.

Statistical Analysis of the Data
Results are given as mean±SD. Statistical significance of differences between means was assessed using two-sample t tests. Differences in the frequency of categorical variables (CHD, hypertension, smoking, and intake of ß-blockers) were tested with the ² test. ANOVA models were used to determine the effect of potentially confounding variables on the difference in CETP activity between the low-HDL group and the control subjects. Specifically, ANCOVA was employed to adjust CETP activity for age, BMI, LDL cholesterol, and triglycerides. Interaction of the categorical variables with CETP activity was determined by two-way ANOVA. All analyses were carried out in CLINFO using BMDP software.

Normal mixture analysis was performed using the program PC-NORMIX 2.0.^{48 49} Bimodality was tested by comparing the log-likelihood statistic for a mixture of two normal distributions with the log-likelihood statistic for a single normal distribution. The likelihood ratio was then evaluated by ² distribution with two degrees of freedom. A value of P<.05 was considered significant.^{49 50}

	Results

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The distributions of CETP activities are presented for normolipidemic control subjects and low-HDL patients in the upper and lower panels, respectively, of the Figure. The distribution of CETP activities in normolipidemic control subjects was unimodal, as reported previously.³⁹ In contrast, in low-HDL patients, the distribution appeared to be bimodal. The statistical significance of bimodality was assessed by mixture analysis and was found to be a mixture of two normal distributions; the probability of true bimodality was estimated at .016. The cut point between the two curves was a CETP activity of 35.5% of transfer. Thus, values for 82 patients, designated as group A, fell into the lower distribution curve, whereas values for the other 27 patients (group B) fell within the higher curve. Group A had a range of CETP activities similar to that in normolipidemic control subjects.

View larger version (19K): [in this window] [in a new window]   Figure 1. A, Distribution of cholesteryl ester transfer protein (CETP) activities in 50 normolipidemic men. The histograms are shown along with the corresponding normal distribution curve as determined by normal mixture analysis. Data are consistent with a unimodal distribution. B, Distribution of CETP activities in 109 normotriglyceridemic men with hypoalphalipoproteinemia. The histograms are shown along with the corresponding normal distributions determined by normal mixture analysis. The data best fit a mixture of two normal distributions (P=.016).

Table 2 compares levels and distributions of CETP activities among normolipidemic control subjects and members of the low-HDL group. When all low-HDL patients were compared with control subjects, average CETP activities were significantly higher in the former. However, when all low-HDL patients were subdivided into groups A and B, the significance of difference compared with control was much less for group A (P<.05) than for group B (P<.0001); the latter showed minimal overlap with the range of activities observed in the control group (the Figure and Table 2).

View this table: [in this window] [in a new window]   Table 2. Levels and Distribution of CETP Activity1

The characteristics for groups A and B are compared in more detail in Table 3. The two groups were of similar age, and there were no significant differences between the groups in terms of percentages of those who were hypertensive, were smokers, or were taking ß-blockers. However, group B had a lower percentage of patients with CHD. Average BMI was somewhat higher in group B, but no significant differences were noted for plasma lipid or lipoprotein cholesterol levels.

View this table: [in this window] [in a new window]   Table 3. Clinical Characteristics of the Two Groups of Normotriglyceridemic Subjects With Low HDL Cholesterol

The available measurements of postheparin LPL and HTGL activities for patients in groups A and B were compared with corresponding activities in 51 normal-HDL subjects (Table 4). Group A had significantly lower activities of postheparin LPL, higher activities of postheparin HTGL, and lower LPL/HTGL ratios than did the normal-HDL group. In contrast, no significant differences were found for any of these parameters between low-HDL group B and the normal-HDL group.

View this table: [in this window] [in a new window]   Table 4. Activities of Lipoprotein Lipase and Hepatic Triglyceride Lipase

Pearson's correlation coefficients for CETP activity and other parameters were determined for each group of subjects (data points not shown). For the normolipidemic control group, linear regression analysis revealed significant positive correlations between CETP activity and LDL cholesterol levels (r=.38, P<.01) and between CETP activity and non-HDL/HDL-cholesterol ratios (r=.44, P<.001). For the two low-HDL groups, none of these correlations were significant. Two-way ANOVA revealed no interaction between CETP activity and the presence of hypertension, smoking, or treatment with ß-blockers (for all, P>.3). However, a reciprocal interaction was noted between CETP activity and CHD (P<.05).

	Discussion

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CETP is a potentially important plasma protein for regulating HDL metabolism. This protein transfers cholesteryl esters from HDL to triglyceride-rich lipoproteins in exchange for triglycerides. The important role that CETP plays in this process in humans is illustrated by the disorder of inherited CETP deficiency.^{28 29 30} Patients with this condition have marked increases in plasma concentrations of HDL cholesterol, which presumably reflect an impediment in the transfer of cholesteryl esters from HDL to triglyceride-rich lipoproteins. In contrast, treatment with the cholesterol-lowering drug probucol raises plasma CETP levels,^{31 32 33} and this response may partly account for the HDL cholesterol–lowering action of this agent. Other metabolic disorders, such as diabetes mellitus^{34 35 36} and the nephrotic syndrome,^{37 38} have likewise been reported to be accompanied by both elevated CETP activities and low HDL levels. The present study was thus designed to determine whether normolipidemic patients with low HDL cholesterol levels have concomitant elevations in CETP activities.

The results of the study reveal that CETP activities in low-HDL patients are bimodally distributed. In contrast, normolipidemic subjects with normal HDL levels manifest a unimodal distribution of CETP activities. The majority of current patients with low HDL levels fell within a distribution of activities that overlapped the normal distribution. These patients were designated group A patients. However, a minority of patients (about one fourth) clearly had elevated CETP activities (group B). For this group, it is possible that the high CETP activities were a contributing factor to lower HDL concentrations. The converse possibility, namely, that low HDL levels are a cause of high CETP activities, seems unlikely, because the majority of patients with low HDL levels had normal CETP activities. As indicated in "Methods," average specific CETP activities were essentially identical for low-HDL and normal-HDL subjects; this finding seemingly rules out the possibility that the observed CETP activities do not reflect CETP masses in patients with low HDL levels.

When the two low-HDL groups were compared, certain differences were noted that may help to explain the mechanisms underlying the low HDL cholesterol concentrations in each group. These comparisons are secondary to the primary question under investigation, but the findings are provocative and could serve as the basis for further investigation. Of particular interest are the comparisons of postheparin lipase activities that were measured in a subgroup of patients from groups A and B.

Postheparin LPL and HTGL activities have both been reported to be altered in many patients with low HDL cholesterol levels. Recently, Blades et al¹⁸ reported that average postheparin LPL activities were significantly reduced in low-HDL patients, whether they had hypertriglyceridemia or not. Furthermore, HTGL activities, on average, were elevated in both normotriglyceridemic and hypertriglyceridemic patients with low HDL levels.¹⁸ In this study, average LPL/HTGL ratios were particularly reduced in low-HDL patients. Despite these differences in mean activities, only about one third of low-HDL patients had definitely reduced LPL, and only another third had elevated HTGL, although about half of all of these patients had reduced LPL/HTGL ratios. Of interest in the current study, only group A patients, ie, those with normal CETP activities, had significantly reduced LPL activities, elevated HTGL activities, and decreased LPL/HTGL ratios. In contrast, group B patients had normal postheparin LPL and HTGL activities and LPL/HTGL ratios. These observations increase the likelihood that an elevated CETP was the only detectable abnormality responsible for reduced HDL cholesterol levels in group B.

Another interesting difference between the two low-HDL groups was the lower prevalence of CHD among group B patients. Only 30% of group B patients had CHD, whereas 70% of group A patients had CHD. Although this difference raises the possibility that high CETP activities are a less atherogenic risk factor than are other causes of low HDL, larger prospective studies obviously would be required to verify the reality of this difference.

In a recent study from this laboratory,³⁹ CETP activities were investigated in a group of patients with primary hypercholesterolemia (elevated LDL cholesterol). In this other study,³⁹ the distribution curve for CETP activities for hypercholesterolemic patients was shifted to higher values than that of control subjects; even so and in contrast to the current low-HDL group, no bimodal distribution was observed in hypercholesterolemic patients. About 60% of hypercholesterolemic patients had elevated CETP activities, and HDL cholesterol levels likewise were significantly reduced. These results are compatible with the possibility that high CETP activities contribute to both elevated LDL cholesterol and reduced HDL cholesterol in hypercholesterolemic patients. In the current investigation, patients in group B did not have higher LDL cholesterol levels than those of group A. It must be noted, however, that all low-HDL patients in the current study were selected for their "normal" LDL cholesterol levels. Thus, even if elevated CETP activities in group B patients had contributed to some increase in LDL cholesterol levels, the increase was not sufficient to elevate LDL concentrations to the hypercholesterolemic range, ie, >160 mg/dL. Undoubtedly, many other factors contribute to high LDL cholesterol levels besides an elevated CETP.

In summary, the current data are consistent with the concept that high levels of CETP contribute to low HDL cholesterol levels in some patients. About one fourth of our low-HDL patients had abnormally high CETP activities. If this association can be extended to a causal connection by future investigation, elevated CETP activities could prove to be a significant cause of low HDL cholesterol. Because CETP activities in low-HDL patients were bimodally distributed, a monogenic hereditary disorder must be considered; of course, this possibility will require family and genetic studies for proof. In any case, on the basis of the current data and the known physiological functions of CETP, we suggest that high levels of circulating CETP are a strong candidate as one cause of low HDL levels.

	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; and The Southwestern Medical Foundation, Dallas, Tex. The authors appreciate the excellent technical assistance of Biman Pramanik, Hahn Nguyen Tran, Han Tran, Champa Vitalla, and Long Nguyen. 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 November 14, 1994; accepted February 2, 1995.

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