Changes in ultracentrifugally separated plasma lipoprotein subfractions in patients with polygenic hypercholesterolemia, familial combined hyperlipoproteinemia, and familial hypercholesterolemia after treatment with atorvastatin

Accepted Manuscript Changes in ultracentrifugally separated plasma lipoprotein subfractions in patients with polygenic hypercholesterolemia, familial combined hyperlipoproteinemia, and familial hypercholesterolemia following treatment with atorvastatin Koichiro Homma, Yasuhiko Homma, Tadashi Yoshida, Hideki Ozawa, Yutaka Shiina, Shu Wakino, Koichi Hayashi, Hiroshi Itoh, Shingo Hori PII:

S1933-2874(14)00419-X

DOI:

10.1016/j.jacl.2014.12.007

Reference:

JACL 718

To appear in:

Journal of Clinical Lipidology

Received Date: 7 June 2014 Revised Date:

6 December 2014

Accepted Date: 9 December 2014

Please cite this article as: Homma K, Homma Y, Yoshida T, Ozawa H, Shiina Y, Wakino S, Hayashi K, Itoh H, Hori S, Changes in ultracentrifugally separated plasma lipoprotein subfractions in patients with polygenic hypercholesterolemia, familial combined hyperlipoproteinemia, and familial hypercholesterolemia following treatment with atorvastatin, Journal of Clinical Lipidology (2015), doi: 10.1016/j.jacl.2014.12.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Changes in ultracentrifugally separated plasma lipoprotein subfractions in patients with polygenic hypercholesterolemia, familial combined hyperlipoproteinemia, and familial

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hypercholesterolemia following treatment with atorvastatin

Koichiro Hommaa,b, Yasuhiko Hommad, Tadashi Yoshidac, Hideki Ozawae, Yutaka Shiinad, Shu

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Wakinoa, Koichi Hayashia, Hiroshi Itoha, Shingo Horib

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Departments of aInternal Medicine, bEmergency Medicine, and cApheresis and Dialysis Center, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan Departments of dClinical Health Science and eInternal Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara 259-1193, Japan

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Short title: Atorvastatin and lipoprotein subfractions

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Correspondence: Koichiro Homma, M.D., Ph.D. Department of Internal Medicine, School of Medicine, Keio University

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35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Tel.: 81-3-5363-3796 Fax: 81-3-3359-2745

E-mail: [email protected]

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Highlights

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Statins reduce plasma LDL by stimulating hepatic LDL uptake via

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LDL-receptors. It is unclear whether statins equally stimulate uptake of all subfractions via LDL-receptors.

LDL-receptor activity and the decrease in plasma LDL subfractions was studied.

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Atorvastatin treatment reduced plasma levels of the 3 LDL subfractions.

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LDL-receptor activity was negatively correlated only with md-LDL decreases.

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Abstract

Background

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Plasma levels of low-density lipoproteins (LDLs) are decreased through stimulation of their hepatic uptake by statins via an LDL-receptor. However, it is unclear whether statins equally stimulate the hepatic uptake of all LDL subfractions.

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Objective

We compared the effects of atorvastatin on 3 LDL subfractions, and their associations with

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LDL-receptor activities, in Japanese patients with polygenic hypercholesterolemia (PHC), familial combined hyperlipoproteinemia (FCHL), and familial hypercholesterolemia (FH). Materials and Methods

Atorvastatin was administered to patients with PHC (n = 11), FCHL (n = 16), and FH (n = 13). We measured plasma levels of lipids, remnant-like particle cholesterol, apoproteins, and

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cholesterol in lipoprotein fractions. Sequential ultracentrifugation was performed to subfractionate the plasma lipoproteins, and lymphocyte LDL-receptor activities were estimated using flow cytometry.

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Results

The average daily dosage of atorvastatin was 10 mg, 27 mg, and 40 mg in patients with PHC,

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FCHL, and FH, respectively; after 12 months of atorvastatin treatment, LDL-cholesterol (LDL-C) plasma levels decreased by 44%, 50%, and 53%, respectively (all, p < 0.0001). Atorvastatin reduced low-density LDL-C plasma levels in patients with PHC (48% reduction), FCHL (53%), and FH (46%) (all, p < 0.0001). Plasma levels of medium-density (md)- and high-density LDL-C were also significantly reduced in the 3 patient groups (all, p ≤ 0.0147). LDL-receptor activity was negatively correlated with baseline levels of md-LDL-C and with the decreases in plasma md-LDL-C levels. Conclusion 3

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Atorvastatin decreased the levels of the 3 LDL fractions. The md-LDL decrease appeared to be mainly due to stimulation of LDL-receptor activity.

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Key words: Atorvastatin, Lipoprotein subfractions, LDL-receptor activity, polygenic hypercholesterolemia, hypercholesterolemia, Familial combined hyperlipoproteinemia, Familial

Abbreviations:

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LDL: low-density lipoprotein

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hypercholesterolemia

FCHL: familial combined hyperlipoproteinemia FH: familial hypercholesterolemia

C: cholesterol

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ld: low density

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PHC: polygenic hypercholesterolemia

md: medium density hd: high density

RPL-C: remnant-like particle cholesterol VLDL: very low-density lipoprotein IDL: intermediate density lipoprotein NMR: nuclear magnetic resonance TG: triglyceride 4

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d: density HDL: high density lipoprotein SD: standard deviation

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DM; diabetes mellitus

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Introduction

Numerous large-scale, double-blind, placebo-controlled studies have shown that statins are

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effective for the primary and secondary prevention of coronary artery and cerebrovascular diseases, primarily by decreasing plasma low-density lipoprotein (LDL) cholesterol (LDL-C) levels. LDL consists of several subfractions (1-7); the levels of the large, less-dense LDLs are elevated in patients with familial hypercholesterolemia (FH), and the small, more-dense LDL

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levels are elevated in patients with familial combined hyperlipoproteinemia (FCHL) (8, 9).

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Small, dense LDLs have also been reported to be more susceptible to oxidation as compared to large, less-dense LDLs (10, 11). The main mechanism of statin-mediated lowering of plasma LDL-C levels involves the stimulation of hepatic LDL uptake by increasing LDL-receptor activity (12, 13). Large, less-dense LDL molecules are reported to have a higher affinity for the

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LDL-receptors as compared to the small, dense LDL molecules (14, 15).

The statin-induced decreases in plasma levels of cholesterol in LDL subfractions are controversial (16-20), and the effects of atorvastatin are not always consistent (16, 19, 20). In

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particular, atorvastatin has been reported to decrease the plasma levels of cholesterol in less-dense LDLs (20); cholesterol in the small, dense LDLs (19); and cholesterol in all LDL

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subfractions (16). Ultracentrifugation was the first reported method for separating plasma lipoproteins (21), and in the present study, we used sequential ultracentrifugation to separate the 3 plasma LDL subfractions. In this study, we aimed to compare the effect of atorvastatin on each LDL subfraction in Japanese patients with polygenic hypercholesterolemia (PHC), FCHL, and FH. In addition, we examined the correlation between atorvastatin and lymphocyte LDL-receptor activity.

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Materials and Methods This study was approved by the Ethics Committee of Tokai University Hospital and was started

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after informed consent was obtained from all the participants.

Patients

Forty Japanese patients were divided into 3 groups, according to their type of dyslipidemia: PHC (n = 11), FCHL (n = 16), and FH (n = 13). Those with PHC had plasma LDL-cholesterol

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(C) levels of ≥160 mg/dL and triglyceride (TG) levels of <150 mg/dL, but did not have blood

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relatives with hyperlipoproteinemia. FH was diagnosed in patients with plasma LDL-C levels of ≥200 mg/dL and TG levels of <150 mg/dL, radiographically determined Achilles tendon thicknesses of ≥10 mm, and blood relatives with a history of hypercholesterolemia. FCHL was diagnosed in patients with LDL-C levels of ≥200 mg/dL, TG levels of ≥200 mg/dL, apo(lipoprotein) B levels of ≥150 mg/dL, and Achilles tendon thicknesses of <10 mm. Among

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the FCHL patients, combined hyperlipoproteinemia was also found in some blood relatives; patients with plasma TG levels of <200 mg/dL and those who had >1 blood relative with combined hyperlipoproteinemia were also included in the FCHL group. Information regarding

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Study protocol

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family members was obtained from the patients.

The administration of atorvastatin began after 2 months of observation. Dietary treatment, started at the beginning of the observation period, included a daily energy intake of 1800 kcal, daily fat intake equivalent to 25% of the total energy intake, a polyunsaturated/saturated fat ratio of 1.0, and daily cholesterol intake of 300–400 mg. Patients with FCHL were also requested to reduce their intake of sweets, fruits, and alcohol. The initial daily dose of atorvastatin was 10 mg, and was increased, as necessary, to 20 mg/day or 40 mg/day (the maximum dose allowed in Japan) to reduce the LDL-C concentration to the target level of <130 mg/dL within the first 3 7

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months. Thereafter, the dose was maintained constant throughout the following 12 months of treatment. Patients visited the outpatient lipid clinic of Tokai University Hospital, once a month,

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for plasma lipid level and drug safety assessments.

Lymphocyte LDL-receptor activity was assayed during the observation period. Plasma lipids, apo(lipo)proteins, and cholesterol in the lipoprotein subfractions were compared between the

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start and end of treatment.

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Laboratory procedures

Plasma lipid levels were measured enzymatically using an autoanalyzer. Apo(lipo)protein levels were estimated using a turbidimetric immunoassay (22). Remnant-like particle cholesterol (RLP-C) levels, equivalent to the TG-rich lipoprotein remnant, were determined using immunoprecipitation involving monoclonal antibodies against apo(protein) AI and B-100 (23).

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Plasma lipoproteins were subfractionated, using sequential ultracentrifugation, into very low-density lipoproteins (VLDL, density <1.006), intermediate-density lipoproteins (IDL, 1.006 < density < 1.019), low-density (ld)-LDL (1.019 < density < 1.035), medium density (md)-LDL

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(1.035 < density < 1.045), high density (hd)-LDL (1.045 < density < 1.063), HDL2 (1.063 < density < 1.125), and HDL3 (density > 1.125), according to the method of Havel et al. (21). The

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duration of ultracentrifugation was 20 h for VLDL, IDL, and LDL separation, and 24 h for HDL separation at 1 × 105 × g.

Lymphocyte LDL-receptor activity was assayed using flow cytometry; peripheral blood lymphocytes were cultured for 72 h in a lipoprotein-free medium, as described elsewhere (24). The reference LDL-receptor activity (100%) was determined in healthy controls for each receptor assay.

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Statistical analysis All values, except for the numbers of males and females and TG, RLP-C, VLDL-C, and IDL-C levels are reported as means (standard deviation); the TG, RLP-C, VLDL-C, and IDL-C levels

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are expressed as medians (25th percentile, 75th percentile) because their distributions were not normal. A software program, SPSS (IBM, Armonk, NY, USA), was used to perform the statistical analyses. The comparisons among the 3 patient groups were made using Scheffe’s multiple comparison analysis on items with F values of p < 0.05, using analysis of variance

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calculations. Comparisons between the start and end of treatment were made using paired t-tests,

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except for the comparisons of TG, RLP-C, VLDL-C, and IDL-C levels. Comparisons for these variables (TG, RLP-C, VLDL-C, and IDL-C levels) were made using Wilcoxon signed-rank test. Comparisons of coronary artery disease, cerebrovascular disease, and diabetes mellitus (DM) complication rates were made using the exact test. Correlation coefficients were calculated for LDL-receptor activity and plasma cholesterol levels of each LDL subfraction. A p-value <

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0.05/3 for Scheffe’s analysis and p-values < 0.05 for the paired t-test, Wilcoxon signed-rank test,

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and exact test and correlation coefficients were considered statistically significant.

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Results

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The patient characteristics are provided in Table 1. None of the patients were diagnosed with DM, and none received hypotensive drugs that would influence their plasma lipid levels, such as β-blockers or diuretics. The plasma total cholesterol and LDL-C levels were highest in the FH group and lowest in the PHC group. The plasma TG and RLP-C levels were highest in the

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FCHL group. The plasma HDL-C levels are usually low in hypertriglyceridemic individuals, but

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the plasma levels of HDL-C were similar among all groups. The plasma levels of apo B were highest in the FH group and lowest in the PHC group. The plasma apo E levels were high in the FCHL and FH groups, whereas the plasma levels of IDL-C were highest in the FCHL group. There were no differences in the plasma levels of ld-LDL-C among the groups. However, the plasma levels of md-LDL-C were high in the FCHL and FH groups, and the plasma levels of

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hd-LDL-C were higher in the FCHL group than in the PHC group. The plasma levels of hd-LDL-C in the FCHL and FH groups were not significantly different, but the md-LDL-C/hd-LDL-C ratio was significantly higher in the FH group than in the FCHL group.

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This ratio was not significantly different between the PHC and FCHL groups or between the PHC and FH groups. The plasma levels of HDL2-C and HDL3-C were similar among the 3

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groups.

The lymphocyte LDL-receptor activity was significantly lower in the FH group than in the other 2 groups.

At the conclusion of the 12 months of treatment, the daily dosages (mean ± SD) of atorvastatin for each group of patients were 10 ± 0 mg for PHC patients, 26.9 ± 14.0 mg for FCHL patients, and 40 ± 0 mg for FH patients. 10

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The plasma levels of lipids and apoproteins at the start and end of the 12-month atorvastatin treatment, and the percent changes in the plasma levels throughout the treatment period, are

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shown in Table 2. The plasma levels of total cholesterol and LDL-C were markedly decreased at 12 months in all groups (all, p < 0.0001), whereas the absolute decrease in the LDL-C levels was largest in the FH group and smallest in the PHC group (PHC vs. FH, p < 0.0001; PHC vs. FCHL, p = 0.0014; FCHL vs. FH, p = 0.0002). However, the percent change in LDL-C levels

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was not significantly different between the groups. The decreases in plasma TG levels were

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significant only in the FCHL group. The plasma levels of RLP-C, apo B, and apo E were also significantly decreased in all 3 groups. However, the absolute decrease in plasma apo B levels was significantly greater in the FH group than in the PHC group (p = 0.0014), although this value did not differ between the PHC and FCHL groups or between the FCHC and FH groups; the percent change was similar among the 3 groups. The plasma levels of HDL-C and apo AI

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were unchanged during the 12-month treatment period in all 3 groups.

The effects of the 12-month atorvastatin treatment on plasma cholesterol levels in the

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lipoprotein subfractions are shown in Table 3. The decreases in VLDL-C levels were significant only in the FCHL group. The plasma IDL-C levels decreased in the 3 groups (PHC, 0.0076;

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FCHL, 0.0012; FH, 0.0009). The plasma levels of ld-LDL-C, md-LDL-C, and hd-LDL-C were also significantly decreased in the 3 groups (ld-LDL: PHC and FCHL, p < 0.0001; FH, p = 0.0006; md-LDL: all p < 0.0001; hd-LDL: PHC, p = 0.0087; FCHL, p = 0.0004; FH, p = 0.0047). The absolute and percent changes in ld-LDL-C did not differ among the 3 groups. However, the absolute decrease in md-LDL-C levels was largest in the FH group, as compared to the other 2 groups (PHC vs. FH, p = 0.0019; PHC vs. FCHL, not significant; FCHL vs. FH: p = 0.0048). The absolute decreases in hd-LDL-C levels were not significantly different among the 3 groups, but the percent changes were larger in the FCHL and FH groups than in the PHC 11

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group (PHC vs. FH, p < 0.0001; PHC vs. FCHL, p < 0.0001; FCHL vs. FH, not significant). No changes in the plasma levels of HDL2-C and HDL3-C were observed in any group, except for a

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small, but significant increase in the HDL2-C level in FH patients.

Table 4 shows the correlation between lymphocyte LDL-receptor activity at baseline and the baseline plasma levels of lipids, apolipoproteins, and cholesterol in each lipoprotein subfraction. Lymphocyte LDL-receptor activity was negatively correlated with plasma levels of total

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cholesterol, LDL-C, apo B, and md-LDL-C. Table 4 also shows the correlation between

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lymphocyte LDL-receptor activity and absolute changes in plasma levels of lipids, apolipoproteins, and cholesterol in the 3 LDL subfractions, after 12 months of atorvastatin treatment. LDL-receptor activity was negatively correlated with the absolute decrease in the levels of total cholesterol, LDL-C, and md-LDL-C, but did not correlate with the percent

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decreases.

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No subjective or objective side-effects were observed throughout the study period.

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Discussion A daily dose of 10 mg was sufficient for the treatment of PHC, and a 10–40 mg/day dose was sufficient for the treatment of FCHL. A daily dose of 40 mg, which is the maximum dose

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permitted in Japan, was insufficient for the treatment of FH.

Compared with PHC patients, the plasma levels of md-LDL-C were higher in patients with FCHL and FH. Md-LDL has been reported to have a higher affinity for LDL-receptors than

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ld-LDL and hd-LDL (14, 15), and the negative correlation between lymphocyte LDL-receptor

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activity and md-LDL-C levels in the present study confirmed this finding. The high plasma levels of md-LDL-C in FCHL patients do not seem to be induced by the same mechanism in patients with FH because LDL-receptor activity in most patients with FCHL was within the normal range. In this study, the high plasma levels of hd-LDL-C, which is a small, dense LDL (25), could not be clearly demonstrated in FCHL patients, as previously reported (9, 19). The

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md-LDL-C/hd-LDL-C ratio should be lower in FCHL patients, thus resulting in a skewing of the LDL subfractions towards the smaller and denser LDL subfraction. However, in this study, this ratio was clearly not higher in the FCHL patients as compared to the other 2 groups,

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possibly because of dietary adjustments prior to the start of atorvastatin treatment. The levels of the remnants of the TG-rich lipoproteins, corresponding to RLP and IDL, were high in patients

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with FCHL. This finding is reasonable because plasma TG-rich lipoprotein levels are high in patients with FCHL.

The decrease in plasma LDL-C levels, in the 3 patient groups during atorvastatin treatment, was dependent on the decrease in the levels of cholesterol in all 3 subfractions, and not just a specific one. The main LDL subfraction, md-LDL, seemed to be most actively removed via the LDL-receptor-based mechanism (26, 27). The negative correlation between lymphocyte LDL-receptor activity and the decrease in plasma md-LDL-C levels confirmed this finding. 13

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Atorvastatin treatment also reduced plasma LDL-C and md-LDL-C levels more effectively in patients with lower baseline LDL-receptor activities. Our data suggest that the measurement of baseline LDL-receptor activities could be used to estimate the efficacy of statin treatment before

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starting treatment. The decrease in the cholesterol levels associated with ld-LDL and hd-LDL could not be explained using the LDL-receptor mechanism alone because the decreases in ld-LDL and hd-LDL levels were not correlated with LDL-receptor activity. Thus, the decrease

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in ld-LDL-C and hd-LDL-C levels by atorvastatin is likely mediated by another mechanism(s).

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The plasma levels of HDL-C have been reported to increase with statin treatment (2-7). In the present study, the plasma levels of HDL-C, HDL2-C, and HDL3-C did not change during atorvastatin treatment, and the plasma levels of HDL-C were relatively high. This finding makes

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the effect of atorvastatin treatment on plasma HDL-C levels unclear.

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Conclusion

In the present study, atorvastatin reduced the plasma levels of 3 LDL subfractions in Japanese

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patients with PHC, FCHL, and FH. Md-LDL seemed to be removed mainly by the LDL-receptor mechanism, although the decrease in ld-LDL and hd-LDL levels could not be

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Acknowledgement

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explained by this mechanism, alone.

Statistical analyses were performed by an expert statistician, Dr. Takeo Shibata, Department of

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Molecular Life Science, Tokai University School of Medicine.

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Table 1. Patient demographics

FH

11 (4/7)

16 (5/11)

13 (7/6)

63 (6)

61 (9)

24 (2)

22 (2)

TC (mg/dL)

281 (17)

356 (42)

TG (mg/dL)

122 (112, 149) 4 184 (82, 220)

LDL-C (mg/dL)

196 (24)

267 (49)

HDL-C (mg/dL)

60 (21)

61 (11)

RLP-C (mg/dL)

6 (5, 7) 4 141 (38)

Apo B (mg/dL)

154 (23)

Apo E (mg/dL)

5 (1)

22 (3)

417 (53) ad, bd, cd 3

124 (82, 135) af, bf 336 (41) ad, bd, cd 53 (9)

10 (7, 12)

7 (6, 8) af, be

131 (20)

119 (15)

199 (29)

227 (32) ad, bf, cd

7 (1)

7 (1) ad, cf

13 (9, 16) 4

20 (13, 28)

11 (7, 14) bf

IDL-C (mg/dL)

15 (10, 18) 4

20 (15, 27)

14 (12, 15) af, be

56 (11)

73 (37)

76 (31)

77 (15)

91 (30)

143 (35) ad, cd

28 (12)

46 (19)

39 (20) af

3.1 (1.3)

2.7 (1.6)

5.2 (3.5) bd

HDL2-C (mg/dL)

28 (18)

19 (5)

19 (7)

HDL3-C (mg/dL)

23 (3)

24 (5)

27 (8)

109 (32)

100 (23)

64 (13) bd, cd

CAD, n (%)

2 (18.1)

3 (19.8)

2 (15.3)

CVD, n (%)

0 (0)

0 (0)

0 (0)

DM, n (%)

0 (0)

0 (0)

0 (0)

ld-LDL-C (mg/dL) md-LDL-C (mg/dL) hd-LDL-C (mg/dL)

AC C

md-LDL-C/hd-LDL

TE D

VLDL-C (mg/dL)

EP

Apo AI (mg/dL)

57 (10)

M AN U

BMI (kg/m2)

RI PT

Age (years)

FCHL

SC

Males/Females

PHC

LDL-R activity (%) Complications

1) Statistical significance using Scheffe’s multiple comparison test, after calculating F values using analysis of variance for comparisons among 3 groups; P < 0.05/3 was considered statistically significant. Comparisons of complication rates for CAD, CVD, and DM were

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made using the exact test; P < 0.05 was considered significant. 2) Data (except the numbers of males and females and TG, RLP-C, VLDL-C, and IDL-C levels) are reported as mean (standard deviation). 3) P values (a, PHL vs. FCHL; b, FCHL vs. FH; c, PHC vs. FH; d, p < 0.001; e, p < 0.01; f, p

RI PT

< 0.05/3)

4) TG, RLP-C, VLDL-C, and IDL-C levels are expressed as median (25th percentile, 75th percentile).

5) Abbreviations: PHC, polygenic hypercholesterolemia; FCHL, familial combined

SC

hyperlipoproteinemia, FH, familial hypercholesterolemia; BMI, body mass index; TC, total cholesterol; TG, triglyceride; C, cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein; RLP, remnant-like particle; Apo, apolipoprotein; VLDL, very

M AN U

low-density lipoprotein; IDL, intermediate-density lipoprotein; ld, low-density; md, medium-density; hd, high-density; R, receptor; CAD, coronary artery disease; CVD,

AC C

EP

TE D

cerebrovascular disease; DM, diabetes mellitus.

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Table 2. Changes in plasma lipid and apoprotein levels after 12 months of atorvastatin treatment

HDL-C (mg/dL)

RLP-C (mg/dL)

Apo A I (mg/dL)

PHC

281 (17)

FCHL

356 (42)

214 (31)

<0.0001

-40 (6)

FH

417 (53)

225 (21)

<0.0001

-45 (7)

122 (112, 149)

95 (61, 135)

FCHL

184 (82, 222)

95 (73, 152)

FH

124 (83, 135)

83 (67, 122)

PHC

196 (24)

110 (31)

FCHL

267 (49)

132 (31)

FH

336 (41)

<0.0001

NS

-34 (6)

0.0105

-28 (16)

-18 (41)

NS

-36 (24)

<0.0001

-44 (8)

<0.0001

-50 (8)

156 (23)

<0.0001

-53 (9)

64 (22)

NS

12 (43)

55 (12)

NS

8 (17)

55 (11)

NS

5 (16)

4 (3, 5)

0.0044

-43 (25)

PHC

60 (21)

FCHL

51 (11)

FH

53 (9)

PHC

6 (5, 7)

FCHL

10 (7, 12)

4 (3, 5)

0.0006

-47 (32)

FH

7 (6, 8)

4 (3, 5)

0.0156

-45 (28)

144 (38)

132 (48)

PHC

131 (20)

EP FH

PHC

119 (15)

154 (23)

NS

-8 (15)

141 (22)

NS

9 (15)

129 (20)

NS

15 (14)

<0.0001

-43 (20)

87 (35)

FCHL

199 (29)

113 (22)

<0.0001

-43 (9)

FH

227 (32)

115 (15)

<0.0001

-49 (9)

PHC

5 (1)

AC C

Apo E (mg/dL)

% change

RI PT

PHC

FCHL

Apo B (mg/dL)

187 (25)

p2

SC

LDL-C (mg/dL)

12 months1

M AN U

TG (mg/dL)

Start1

TE D

TC (mg/dL)

Group

3 (1)

<0.0001

-34 (13)

FCHL

7 (1)

4 (1)

<0.0001

-37 (14)

FH

7 (1)

4 (1)

<0.0001

-38 (7)

1) All values, except for TG and RLP-C levels, are presented as means (standard deviation); the levels of TG and RLP-C are expressed as medians (25th percentile, 75th percentile) 2) Statistical significance (except TG and RLP-C levels) was determined using paired t-tests; for TG and RLP-C levels, Wilcoxon’s signed-rank test was used (start vs. 12

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months of treatment). P < 0.05was considered statistically significant. 3) Abbreviations: PHC, polygenic hypercholesterolemia; FCHL, familial combined hyperlipoproteinemia; FH, familial hypercholesterolemia; TC, total cholesterol; TG,

AC C

EP

TE D

M AN U

SC

lipoprotein; RLP, remnant-like particle; Apo, apolipoprotein

RI PT

triglyceride; C, cholesterol; LDL, low-density lipoprotein; HDL, high-density

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Table 3. Changes in plasma cholesterol levels in the lipoprotein subfractions, after 12 months of treatment

13 (9,16)

8 (5)

FCHL

20 (13, 28)

11 (7)

0.0084

-34 (47)

FH

11 (7, 14)

7 (6)

NS

-51 (31)

PHC

15 (10, 18)

6 (5,9)

-33 (53)

0.0076

-53 (16)

20 (15, 17)

8 (5, 9)

0.0012

-55 (23)

FH

14 (12, 15)

6 (4, 10)

0.0009

-51 (22)

<0.0001

-48 (16) -53 (19)

56 (11)

29 (8)

M AN U

PHC FCHL

75 (37)

31 (14)

<0.0001

FH

76 (31)

37 (19)

0.0006

-46 (34) -45 (10)

PHC

77 (15)

42 (10)

<0.0001

FCHL

94 (29)

52 (18)

<0.0001

-39 (23)

FH

134 (39)

60 (14)

<0.0001

-53 (21)

PHC

28 (12)

18 (7)

0.0087

-28 (31)

FCHL

44 (17)

18 (5)

0.0004

-42 (31)

FH

38 (23)

18 (5)

0.0047

-45 (27)

PHC

29 (12)

27 (15)

NS

4 (20) 3 (28)

EP

TE D

md-LDL-C (md/dL)

FCHL

19 (5)

20 (8)

NS

FH

18 (6)

23 (6)

0.0366

21 (32)

PHC

22 (4)

23 (3)

NS

3 (18)

FCHL

23 (5)

23 (3)

NS

2 (17)

FH

26 (8)

26 (4)

NS

4 (21)

AC C

HDL3-C (mg/dL)

NS

FCHL

ld-LDL-C (mg/dL)

HDL2-C (mg/dL)

% change

PHC

IDL-C (mg/dL)

hd-LDL-C (mg/dL)

p2

SC

VLDL-C (mg/dL)

12 months1

RI PT

Start1

Group

1) All values are presented as means (standard deviation), except for VLDL-C and IDL-C levels; the VLDL-C and IDL-C levels are expressed as medians (25th percentile, 75th percentile). 2) Statistical significance (except for VLDL-C and IDL-C levels) was determined using paired t-tests; for VLDL-C and IDL-C levels, Wilcoxon’s signed-rank test was used (start vs. 12 months of treatment). P < 0.05 was considered statistically significant

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3) Abbreviations: PHC, polygenic hypercholesterolemia; FCHL, familial combined hyperlipoproteinemia; FH, familial hypercholesterolemia; VLDL, very low-density lipoprotein; C, cholesterol; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; ld, low-density; md, medium-density; hd, high-density; HDL, high-density

AC C

EP

TE D

M AN U

SC

RI PT

lipoprotein

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Table 4. Correlations between LDL-receptor activities and plasma levels of lipids and apolipoproteins

r1

p2

vs. Absolute changes after 12 months of

levels (md/dL)

TG

-0.563 0.0004 0.021

NS

LDL-C

-0.568 0.0002

HDL-C

0.207

Apo AI

0.291

∆TC

NS NS

∆TG

∆LDL-C

SC

TC

treatment (mg/dL)

-0.449 0.0042 -0080

NS

-0.403 0.0115 -0.292

NS

∆Apo AI

0.182

NS

∆Apo B

-0.296

NS

∆HDL

Apo B

-0.559 0.0002

Apo E

-0.125

NS

∆Apo E

-0.014

NS

RLP-C

-0.115

NS

∆RLP-C

-0.281

NS

0.061

NS

∆VLDL-C

0.039

NS

IDL-C

-0.047

NS

∆IDL-C

0.039

NS

ld-LDL-C

-0.253

NS

∆ld-LDL-C

-0.073

NS

md-LDL-C -0.508 0.0009

∆md-LDL-C

-0.402 0.0118

hd-LDL-C

TE D

VLDL-C

M AN U

activities (%)

p

RI PT

LDL-receptor vs. Baseline

r

NS

∆hd-LDL-C

-0.099

NS

HDL2--C

0.177

NS

∆HDL2-C

-0.018

NS

HDL3-C

-0.187

NS

∆HDL3-C

0.044

NS

EP

-0.088

1) Correlation coefficients (r) were calculated. P < 0.05 was considered statistically significant. 2) Abbreviations: LDL, low-density lipoprotein; TC, total cholesterol; ∆, absolute change; TG,

AC C

triglyceride; C, cholesterol; HDL, high-density lipoprotein; Apo, apolipoprotein; RLP, remnant-particle; VLDL, very low-density lipoprotein; IDL, intermediate density lipoprotein， ld；low-density; md, medium-density; hd, high-density