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Abstract

Background

Recently, remnant lipoprotein is expected to be a new therapeutic target in the age of ‘beyond LDL-cholesterol’. The aim of this study was to clarify the clinical significance of remnant lipoprotein cholesterol (RemL-C) determination in annual health examinations with the focus on large artery atherosclerosis.

Methods and results

Subjects investigated were men (n = 528) and women (n = 318) who underwent annual health examinations at Osaka University. RemL-C was measured with a newly developed homogeneous assay. Carotid and aortic atherosclerosis was estimated by intima-media thickness (IMT) and cardio-ankle vascular index (CAVI), respectively. First, simple regression analysis revealed that the RemL-C levels positively correlated with maximum IMT, mean IMT and CAVI in the whole group (P < 0.05). Next, receiver operating characteristic curve analysis showed that the most effective levels of RemL-C for predicting carotid and aortic atherosclerosis were 0.21 mmol/L (P < 0.05) and 0.22 mmol/L (P < 0.01) or more, respectively. Odds ratios (ORs) of high RemL-C levels (0.21 mmol/L or more) for carotid and aortic atherosclerosis were significantly increased, especially in low-risk, apparently healthy women (OR: 4.20, P < 0.05 and 3.79, P < 0.01, respectively). Five out of 13 female low-risk cases (38%) with carotid atherosclerosis showed high serum RemL-C levels. It should be emphasized that conventional risk factors are still strong predictors for large artery atherosclerosis in the whole group.

Conclusions

Our results indicate that high serum RemL-C level is a predictive hallmark for large artery atherosclerosis in apparently healthy women. Determination of RemL-C should be employed as one of the parameters in annual health examinations.

Introduction

Recently, the main causes of death in developed countries have been attributed to malignant neoplasma and cardiovascular diseases associated with atherosclerosis. Statin therapy has been a central stream for the treatment of hypercholesterolemia based on ‘low-density lipoptotein theory’ but the majority of cardiovascular events are not prevented by virtue of drastic reduction of serum cholesterol levels with strong statins.^1,2 So, the age of ‘beyond LDL-cholesterol level’ has come and remnant lipoproteins (remnants) attract much attention as a candidate for one of the new therapeutic targets.

Remnants are triglyceride (TG)-rich lipoproteins, which are produced by hydrolysis of chylomicrons and VLDL in plasma.³ While healthy individuals have few remnants in their plasma even in the postprandial state, under some conditions, for example, insulin resistance leads to the accumulation of remnants in plasma.⁴ High remnant lipoprotein cholesterol (RemL-C) level is known as a risk factor of atherosclerosis⁵ but the clinical significance of determining RemL-C values in annual health examinations has not yet been completely elucidated.

A unique immunoseparation assay (remnant lipoprotein-like particle cholesterol, RLP-C) has been widely employed for RemL-C determination in many epidemiological studies.^6–8 This method, however, poses problems of reproducibility and is technically too complicated for routine examinations. One of the important aims of annual health examinations is to seek out the high-risk group of diseases in a mass group and prevent them from progressing to serious illness. A more easy and simple method for remnant determination than RLP-C has been needed to screen many subjects who undergo annual health examinations. We recently developed a convenient homogeneous assay for RemL-C,⁹ which can be run on an automated analyser. RemL-C assay is reported to reflect intermediate-density lipoprotein more closely than RLP-C.¹⁰

In the study presented here, we evaluated the clinical significance of the newly developed RemL-C assay for assessment of carotid and aortic atherosclerosis in annual health examinations. We will show the evidence that RemL-C is a predictive biomarker for large artery atherosclerosis in apparently healthy women.

Materials and methods

Study population

The subjects enrolled in the current study were individuals who underwent an annual health examination at the Osaka University Health Care Center in 2007–2009, comprising apparently healthy Japanese men (n = 528) and women (n = 318), aged 27–71 y. Low-density lipoprotein cholesterol (LDL-C) was calculated by using Friedewald's equation¹¹ and cases with serum TG levels over 4.53 mmol/L (400 mg/dL) were excluded from the analysis. After this selection, we analysed 518 men and 317 women. Each case participated in this study only once through the concerned period. Informed consent was obtained from all subjects prior to their participation in the study, and this study was approved by the Ethics Committee of Osaka University.

Laboratory measurements

Serum was collected from the subjects after overnight fasting and kept at <− 80°C until assay. We confirmed that the RemL-C assay is reliable after one freeze-and-thaw procedure (data not shown). The serum RemL-C levels were measured with a newly developed homogeneous assay, MetaboRead^® RemL-C (Kyowa Medex Co., Ltd, Tokyo, Japan), on a Cobas Mira Plus autoanalyser (Roche Diagnostics, Basle, Switzerland) according to the manufacturer's manual. Every assay was performed while referring to the high and low concentration standards for RemL-C included with the assay kit and the values measured were confirmed to be within the acceptable range. Other parameters were assayed with routine methods.

Risk factor assessment

Hypertension was defined as systolic blood pressure (SBP) ≥130 mmHg and/or diastolic blood pressure (DBP) ≥85 mmHg. The upper limit values of LDL-C, TG and non-high-density lipoprotein cholesterol (HDL-C) were set at 3.62 mmol/L (140 mg/dL), 1.70 mmol/L (150 mg/dL) and 4.39 mmol/L (170 mg/dL), respectively. Non-HDL-C was calculated by subtracting HDL-C from total cholesterol (TC) as atherogenic lipoprotein cholesterol.^12,13 Low HDL-C was defined as less than 1.03 mmol/L (40 mg/dL). Regarding glucose tolerance, the upper limits of fasting plasma glucose (FPG) and haemoglobin A1c (HbA_1c) were 6.10 mmol/L (110 mg/dL) and 5.6%, respectively. Waist circumference (WC) at the umbilical level was measured in the late exhalation phase in the standing position and visceral fat accumulation was diagnosed when WC was ≥85 cm for men and ≥90 cm for women.¹⁴ In some cases, the visceral fat area (VFA) was estimated by means of abdominal bioelectrical impedance analysis, which is a simple and non-invasive method to assess visceral fat accumulation.¹⁵ We selected ‘low-risk’ subjects whose data were within normal range in WC, blood pressure, LDL-C, HDL-C, TG, FPG and HbA_1c from non-smokers. Smoking status was considered to be positive if smoking had ever occurred. The numbers of low-risk men and women were 99 and 183, respectively.

Evaluation of carotid and aortic atherosclerosis

Ultrasound examination for intima-media thickness (IMT) was performed by a single sonographer who was blinded to all clinical information and used a LOGIQ 5 (GE Yokogawa Medical Systems Co., Tokyo, Japan) with an 8.8-MHz linear transducer. Three different longitudinal images (anterior oblique, lateral and posterior oblique) of a 1.0–1.5-cm section of the left common carotid artery (CCA) were obtained at the distal end of the CCA proximal to the carotid bulb. The investigation included transverse images to confirm the accuracy of the longitudinal images. After the examination, the best longitudinal image of each individual was analysed. Both maximum and mean IMT measurements were calculated using computer software that automatically traces the intima–media edge of the far wall. In the current study, we diagnosed a case as carotid atherosclerosis if one's maximum IMT was over 1.0 mm.¹⁶

Aortic atherosclerosis was assessed by measuring cardio-ankle vascular index (CAVI) using VaSera VS-1000 (Fukuda Denshi, Tokyo, Japan), as previously described.¹⁷ CAVI is known as a blood pressure-independent arterial wall stiffness parameter and thought to reflect the aortic stiffness.¹⁸ In the current study, we diagnosed a case as aortic atherosclerosis if one's CAVI was more than 8.0 according to the manufacturer's manual, in which the normal CAVI value is defined as less than 8.0 based on the data from the general population.¹⁸

Statistical analysis

Statistical analysis was performed with the Statcel2 (OMS Inc., Saitama, Japan) add-on package for Microsoft Excel and JMP^® (SAS Institute, Cary, NC, USA). For the logistic regression analysis, the ‘aged’ group was defined as cases 50 years or older. Pearson's correlation coefficients were calculated for skewed variables after logarithmic transformation of the variables. Statistical differences were determined by two-sided Student's t-test and values of P < 0.05 were considered statistically significant.

Results

Baseline demographics

Clinical characteristics of the study subjects are summarized in Table 1. In the whole group, the mean values of age, body mass index (BMI), WC, VFA, SBP, DBP, TG, LDL-C, non-HDL-C, RemL-C and FPG were significantly higher, and that of HDL-C was significantly lower in men than in women. There were no significant differences in the levels of TC and HbA_1c between men and women. Vascular changes, including maximum and mean IMTs and CAVI were more pronounced in men than in women as expected from the data above. The mean maximum IMT in men was over 1.0 mm, suggesting that the atherogenic profile may lead to carotid atherosclerosis in the male population of this study.

Table 1

Clinical characteristics of the study subjects

	Whole group		Low-risk group
	Men	Women	Men	Women
Number	518	317	99	183
Age (y)	49.0 ± 8.3	47.2 ± 7.5^††	46.4 ± 8.7**	44.7 ± 7.0**
Smoker	220	26	0	0
BMI (kg/m²)	23.6 ± 3.0	21.4 ± 3.0^††	21.3 ± 1.9**	20.7 ± 2.2**
WC (cm)	82.9 ± 8.4	73.5 ± 8.5^††	75.4 ± 5.3**	71.0 ± 6.5**
VFA (cm²)^§	96.3 ± 41.0	50.0 ± 25.5^††	57.7 ± 21.6**	42.0 ± 17.5**
SBP (mmHg)	121 ± 14.3	111 ± 14.4^††	112 ± 9.6**	107 ± 11.1**
DBP (mmHg)	78.4 ± 11.5	68.1 ± 11.8^††	70.5 ± 8.5**	64.7 ± 9.6**
TC (mmol/L)	5.20 ± 0.80	5.20 ± 0.84	4.80 ± 0.55**	4.89 ± 0.65**
TG (mmol/L)	1.26 ± 0.71	0.84 ± 0.48^††	0.83 ± 0.31**	0.70 ± 0.28**
LDL-C (mmol/L)	3.15 ±0.76	3.03 ± 0.76^†	2.80 ±0.45**	2.71 ± 0.51**
HDL-C (mmol/L)	1.48 ± 0.34	1.79 ± 0.35^††	1.63 ± 0.35**	1.86 ± 0.33*
Non-HDL-C (mmol/L)	3.73 ± 0.83	3.41 ± 0.82^††	3.18 ± 0.50**	3.03 ± 0.55**
RemL-C (mmol/L)	0.23 ± 0.12	0.19 ± 0.09^††	0.16 ± 0.05**	0.16 ± 0.05**
FPG (mmol/L)	5.12 ± 0.85	4.76 ± 0.72^††	4.83 ± 0.35**	4.63 ± 0.35*
HbA_1c (%)	5.15 ± 0.55	5.13 ± 0.52	4.96 ± 0.27**	4.97 ± 0.24**
Maximum IMT (mm)	1.04 ± 0.59	0.80 ± 0.39^††	0.86 ± 0.43**	0.70 ± 0.24**
Mean IMT (mm)	0.73 ± 0.24	0.63 ± 0.14^††	0.65 ± 0.15**	0.59 ± 0.09**
CAVI	7.69 ± 0.86	7.41 ± 0.75^††	7.52 ± 0.69	7.29 ± 0.64

BMI, body mass index; CAVI, cardio-ankle vascular index; DBP, diastolic blood pressure; FPG, fasting plasma glucose; HbA_1c, haemoglobin A1c; HDL-C, high-density lipoprotein cholesterol; IMT, intima–media thickness; LDL-C, low-density lipoprotein cholesterol; NS, not significant; RemL-C, remnant lipoprotein-cholesterol; SBP, systolic blood pressure; TC, total cholesterol; TG, triglyceride; VFA, visceral fat area; WC, waist circumference

^§VFA data were obtained from 414 men and 241 women. Data show mean ± SD.

^† P < 0.05, ^†† P < 0.01 versus men, *P < 0.05, **P < 0.01 versus whole group

We selected ‘low-risk’ cases as mentioned above and compared with the whole group. The values of low-risk group reasonably showed a healthy profile (Table 1).

Correlation between vascular measurements and clinical parameters

Simple correlations between vascular measurements, CAVI and clinical parameters in study subjects are shown in Table 2. Age, SBP, DBP and HbA_1c exhibited significant positive correlations with maximum IMT, mean IMT and CAVI in both gender groups. BMI unexpectedly negatively correlated with CAVI in both gender but the same tendency was reported from other group.¹⁹ Among them, age showed exceptionally strong correlation with both IMT measurements and CAVI. Age had strong positive correlation with both IMT measurements and CAVI also in the low-risk group (data not shown). Multiple stepwise regression analyses disclosed that age (β = 0.419, P < 0.0001), SBP (β = 0.098, P < 0.05) and LDL-C (β = 0.080, P < 0.05) in men and age (β = 0.308, P < 0.0001) and non-HDL-C (β = 0.158, P < 0.01) in women were independent determinants for maximum IMT. For mean IMT, the significant determinants were age (β = 0.487, P < 0.0001), BMI (β = 0.109, P < 0.01), SBP (β = 0.097, P < 0.05) and HDL-C (β = −0.086, P < 0.05) in men and age (β = 0.390, P < 0.0001), SBP (β = 0.117, P < 0.05) and non-HDL-C (β = 0.175, P < 0.001) in women, respectively. Regarding CAVI, the significant determinants were age (β = 0.565, P < 0.0001), SBP and HbA_1c (β = 0.080, P < 0.05) in men and age (β = 0.507, P < 0.0001) and TG (β = 0.104, P < 0.05) in women. Taken together, these findings confirm that conventional risk factors are strong promoters of atherosclerosis and suggest that these risk factors should be managed appropriately if the case has such risk factors.

Table 2

Simple correlations between vascular measurements and examination values in study subjects

	Maximum IMT				Mean IMT				CAVI
	Men		Women		Men		Women		Men		Women
	r	P	r	P	r	P	r	P	r	P	r	P
Age	0.449	<0.0001	0.370	<0.0001	0.509	<0.0001	0.499	<0.0001	0.630	<0.0001	0.544	<0.0001
BMI	0.087	<0.05	0.032	0.571	0.178	<0.0001	0.132	<0.05	−0.135	<0.01	−0.137	<0.05
WC	0.141	<0.01	0.098	0.080	0.225	<0.0001	0.198	<0.001	0.011	0.811	0.024	0.679
VFA	0.083	0.094	0.085	0.192	0.164	<0.001	0.177	<0.01	0.017	0.734	0.077	0.239
SBP	0.194	<0.0001	0.176	<0.01	0.236	<0.0001	0.288	<0.0001	0.325	<0.0001	0.239	<0.0001
DBP	0.117	<0.01	0.156	<0.01	0.173	<0.0001	0.242	<0.0001	0.257	<0.0001	0.175	<0.01
TC	0.093	<0.05	0.245	<0.0001	0.088	<0.05	0.313	<0.0001	0.026	0.562	0.112	<0.05
TG	0.019	0.673	0.170	<0.01	0.058	0.186	0.235	<0.0001	0.059	0.188	0.275	<0.0001
HDL-C	−0.054	0.220	−0.065	0.251	−0.102	<0.05	−0.082	0.147	0.044	0.326	−0.003	0.957
Non-HDL	0.112	<0.05	0.279	<0.0001	0.127	<0.01	0.356	<0.0001	0.007	0.880	0.117	<0.05
LDL-C	0.115	<0.01	0.261	<0.0001	0.112	<0.05	0.327	<0.0001	0.018	0.682	0.045	0.430
RemL-C	0.005	0.915	0.110	<0.05	0.050	0.257	0.168	<0.01	0.038	0.392	0.148	<0.01
FPG	0.132	<0.01	0.123	<0.05	0.197	<0.0001	0.128	<0.05	0.238	<0.0001	0.072	0.209
HbA_1c	0.153	<0.001	0.141	<0.05	0.226	<0.0001	0.137	<0.05	0.285	<0.0001	0.116	<0.05

Abbreviations and units are the same as in Table 1

Logistic regression analysis of RemL-C and factors related to large artery atherosclerosis

Next, we investigated the clinical significance of RemL-C determination in the low-risk group. We found 13 female and 22 male cases with carotid atherosclerosis in the low-risk group, who have none of the conventional risk factors. Five out of 13 female (38%) and one out of 22 male (4.5%) cases showed high serum RemL-C levels (Table 3). On the other hand, 5 (19%) and 10 (37%) out of 27 cases whose RemL-C levels were abnormally high had carotid atherosclerosis and aortic atherosclerosis, respectively, in the low-risk group. To establish the way in which to seek out atherosclerosis-prone cases, we performed logistic regression analysis as for serum RemL-C levels. To determine the optimal cut-off point of the RemL-C level, we calculated the relationship between RemL-C levels and ROC-AUC (receiver operating characteristic curve, area under the curve) in carotid atherosclerosis and aortic atherosclerosis. The result showed that the most effective criterion for high RemL-C levels in carotid atherosclerosis and aortic atherosclerosis were 0.21 mmol/L (8.2 mg/dL, P < 0.05) and 0.22 mmol/L (8.4 mg/dL, P < 0.01) or more, respectively.

Table 3

Cases with carotid atherosclerosis associated with high serum RemL-C level in the low-risk group

	Age	Gender	SBP/DBP	BMI	WC	TC	TG	HDL-C	LDL-C	FPG	HbA_1c	RemL-C	Maximum IMT	Mean IMT	CAVI
1	35	Female	97/64	21.1	69	4.73	1.10	1.42	2.82	4.44	4.9	0.25	1.12	0.59	6.5
2	42	Female	111/75	20.3	69	5.14	1.10	1.86	2.79	4.22	4.6	0.27	2.69	1.16	8.6
3	57	Female	122/83	20.9	71	6.36	1.25	2.33	3.46	4.55	5.1	0.25	1.05	0.68	7.1
4	58	Female	104/64	22.5	77	5.25	0.86	1.63	3.23	4.72	4.9	0.22	1.17	0.79	8.4
5	59	Female	118/59	21.3	73	4.44	1.67	1.40	2.30	3.88	4.6	0.27	1.14	0.95	8.8
6	58	Male	125/75	22.5	78	5.19	1.51	1.40	3.10	4.88	5.4	0.24	1.87	0.81	7.1

Abbreviations and units are the same as in Table 1

We used this finding to perform logistic regression analysis of high RemL-C for the prediction of atherosclerosis. Significant increased odds ratios (ORs) of high RemL-C (0.21 mmol/dL or more) for carotid and aortic atherosclerosis were obtained in both gender groups (Table 4). Conventional risk factors, of course, were proved to be strong determinants for large artery atherosclerosis.

Table 4

Crude odds ratio of each factor for carotid and aortic atherosclerosis by logistic regression analysis in the whole group

	Carotid atherosclerosis			Aortic atherosclerosis
	Total (n = 835)	Male (n = 518)	Female (n = 317)	Total (n = 835)	Male (n = 518)	Female (n = 317)
Aged	5.22***	5.07***	5.07***	8.58***	7.84***	9.56***
Gender (Male)	3.55***	–	–	1.87***	–	–
High RemL-C	1.71***	1.27	2.11**	1.59**	1.16	2.41**
High BMI	1.40	1.18	0.58	0.66*	0.525**	0.745
High WC	1.62*	1.18	0.45	0.74	0.525**	1.32
High TC	1.63**	1.42	2.77**	1.39	1.26	1.75
High LDL-C	1.86***	1.71**	2.29*	1.28	1.11	1.65
High TG	1.62*	1.06	3.24	1.80**	1.23	6.05**
Low HDL-C	1.38	1.01	1.38	1.2	0.785	>10**
High Non-HDL-C	1.90**	1.45	3.01**	1.44	1.23	1.72
High SBP	2.39***	1.94**	1.83	3.24***	2.84***	3.37**
High DBP	1.61*	1.25	0.88	2.91***	3.03***	1.21
High FPG	3.43***	2.52*	4.53	4.01***	3.80***	2.43
High HbA_1c	2.33***	3.08***	1.10	3.96***	5.73***	2.16
Smoking	2.24***	1.54*	1.65	1.97***	1.66**	1.78

Abbreviations are the same as in Table 1

Aged: 50 years and older

***P < 0.001, **P < 0.01, *P < 0.05

Also in the low-risk group, aging is the strongest risk factor for atherosclerosis as expected. It is noteworthy that the ORs of high RemL-C for carotid and aortic atherosclerosis were extremely high and after adjustment by age, the ORs for aortic atherosclerosis remained significant in apparently healthy women (Table 5).

Table 5

Odds ratios of each factor for carotid and aortic atherosclerosis by logistic regression analysis in the low-risk group

	Carotid atherosclerosis			Aortic atherosclerosis
	Total (n = 282)	Male (n = 99)	Female (n = 183)	Total (n = 282)	Male (n = 99)	Female (n = 183)
Gender (Male)	3.74***	–	–	1.69	–	–
Aged	5.77***	6.11***	4.34*	9.15***	6.87***	10.69***
High RemL-C	2.33	1.49	4.20*	2.10	0.76	3.79**
High RemL-C (Age adjusted)	1.75	1.12	3.29	1.52	0.48	3.02*

RemL-C, remnant lipoprotein cholesterol; Aged: 50 years and older

***P < 0.001, **P < 0.01, *P < 0.05

Further investigation needs to clarify the pathology of the increased serum RemL-C level and atherosclerosis in these cases without conventional risk factors.

Discussion

In the study presented here, we investigated the clinical significance of RemL-C determination for carotid and aortic atherosclerosis in annual health examinations by using a newly developed homogeneous assay. Based on the ROC curve analysis, we set the optimal cut-off point of RemL-C at 0.21 mmol/L (8.2 mg/dL) for carotid atherosclerosis and at 0.22 mmol/L (8.4 mg/dL) for CAVI, respectively. The ORs of high RemL-C for aortic atherosclerosis remained statistically significant after age adjustment for women without any conventional risk factors. From these results, we concluded that RemL-C determination may provide information about the concealed risk for large artery atherosclerosis in apparently health women. RemL-C determination should be added to the parameters which are investigated in annual health examination and executive check-ups. It should be emphasized that conventional risk factors are the major determinants in general population and should be managed appropriately.

Previous studies reported that RLP-C was an independent cardiovascular disease risk factor for women.⁷ Combining our data with this report indicates that accumulation of remnants should be considered as a risk factor of atherosclerosis and a therapeutic target especially for apparently healthy women. Oestrogen has the effects of increasing the number of LDL-receptors in the liver²⁰ and inhibiting hepatic triglyceride lipase activity.²¹ These effects reduce LDL-C and increase HDL-C in plasma and consequently confer an anti-atherogenic lipid profile to plasma.²² Furthermore, oestrogen is reported to inhibit LDL oxidation.^23,24 However, women do not possess a specific anti-atherogenic mechanism for remnants, especially chylomicron remnants which are mainly uptaken by remnant receptors in the liver. This might be the reason why high RemL-C is the risk factor of large artery atherosclerosis in relatively young women. Concomitant determination of serum apoB-48 will provide information in detail. Another possible cause of the sex difference may attribute to the relatively low serum levels of TG in women compared with those of men.

In the current study, RemL-C level was shown to be associated with aortic atherosclerosis estimated by CAVI. This is the supportive datum for Shoji et al. ²⁵ who reported that intermediate-density lipoprotein is an independent risk factor for aortic atherosclerosis.

Our study has a few limitations. A direct correlation between RemL-C levels and carotid atherosclerosis should have been shown in the low-risk group instead of the OR. However, there was no such correlation in this group. This may be partly due to the small number of subjects. Next, we could not eliminate the possible effects of medication for hypertension, dyslipidemia and impaired glucose tolerance on our findings because no information was available of medication given to the subjects. The last, the relationship between RemL-C levels and other atherosclerotic markers, such as high-sensitivity C-reactive protein, pentraxin 3 and adipocytokines, were not evaluated. Further investigations remain to be performed to clarify the mechanism of remnant-related atherosclerosis in the low-risk cases.

In conclusion, high RemL-C level (RemL-C ≥0.21 mmol/L) is a useful hallmark for large artery atherosclerosis in apparently healthy women. Determination of RemL-C level, therefore, should be employed as one of the parameters in annual health examinations and executive check-ups.

DECLARATIONS

Competing interests: There is no conflict of interest in this paper.

Funding: This study was in part supported by a Grant-in-Aid for Scientific Research no. 19590558 (TY) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Ethical approval: The ethics committee of Osaka University approved this study (number 13).

Guarantor: MI.

Contributorship: MT mainly performed the RemL-C assay and wrote the first draft of the manuscript. MI and TY were involved in data analysis. MN, TM and SY were involved in protocol development, gaining ethical approval and patient recruitment. All authors reviewed and edited the manuscript and approved the final version of the manuscript.

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