High Level of Estimated Small Dense Low-Density Lipoprotein Cholesterol as an Independent Risk Factor for the Development of Ischemic Heart Disease Regardless of Low-Density Lipoprotein Cholesterol Level　― A 10-Year Cohort Study ―

Abstract

Background: We previously reported that a high level of small dense low-density lipoprotein cholesterol (sdLDL-C) calculated by the Sampson equation was independently associated with the development of ischemic heart disease (IHD), but it remains unclear whether the effect depends on the level of low-density lipoprotein cholesterol (LDL-C).

Methods and Results: We investigated the associations of new onset of IHD with categorized groups of high (H-) and low (L-) levels of estimated sdLDL-C and LDL-C using 25^th percentile levels of sdLDL-C level (25.2 mg/dL) and LDL-C (100 mg/dL) as cutoff values in 17,963 Japanese individuals (men/women: 11,508/6,455, mean age: 48 years) who underwent annual health checkups. During a 10-year follow-up period, 570 subjects (men/women: 449/121) had new development of IHD. Multivariable Cox proportional hazard analyses after adjustment of age, sex, smoking habit, hypertension and diabetes mellitus at baseline showed that the hazard ratio (HR) [95% confidence interval (CI)] for new onset of IHD was significantly higher in subjects with H-sdLDL-C/H-LDL-C (1.49 [1.06–2.08]) and subjects with H-sdLDL-C/L-LDL-C (1.49 [1.00–2.22]) than in subjects with L-sdLDL-C/L-LDL-C as the reference.

Conclusions: A high level of sdLDL-C estimated by the Sampson equation was a predominant predictor for the development of IHD, regardless of the level of LDL-C, in a general Japanese population.

Cardiovascular disease, primarily ischemic heart disease (IHD), is the leading cause of death and disability worldwide,¹ and identifying risk factors for the development of IHD is an important public health issue.² Recent epidemiological studies have revealed several, including smoking, obesity, hypertension, diabetes mellitus and dyslipidemia.³ In the treatment of dyslipidemia, lowering the levels of low-density lipoprotein cholesterol (LDL-C) is essential for the prevention of atherosclerotic cardiovascular disease including IHD.^4–6 When levels of triglycerides are <400 mg/dL, the Friedewald equation for estimating LDL-C is conventionally used.⁷ LDL contains lipoproteins that are composed of multiple subfractions with different particle sizes and specific gravities ranging from 1.019 to 1.063 g/mL, and LDL consists of particles for small dense LDL (sdLDL) and large buoyant LDL (lbLDL).⁸ sdLDL is defined as LDL particles ≤25.5 nm and a specific gravity of 1.044–1.063 g/mL.⁹ It has been reported that sdLDL particles are a more potent risk factor than lbLDL particles for coronary artery disease.^9–11

A fully automated assay for direct measurement of sdLDL cholesterol (sdLDL-C) has been developed, but its measurement is time-consuming and labor-intensive.^12,13 Recently, Sampson et al. developed a calculation method to estimate sdLDL-C using levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), non-HDL-C, triglycerides, and LDL-C, known as the Sampson equation.^14,15 It has been reported that there were strong correlations between estimated and measured levels of sdLDL-C in 673 healthy Japanese subjects (R²=0.736), and in 1,542 Japanese patients with diabetes mellitus (R²=0.614).¹⁶ We also showed a strong correlation (R²=0.701) between directly measured sdLDL-C level and estimated sdLDL-C level calculated by the Sampson equation in 605 subjects who had undergone annual health examinations.¹⁷ Furthermore, we recently showed that a high level of sdLDL-C calculated by the Sampson equation was independently associated with new onset of IHD,¹⁸ as well as hypertension,¹⁹ during a 10-year period in a Japanese general population. However, it remains unclear whether the effect depends on the level of LDL-C. In the present study, we investigated the associations of new-onset IHD with categorized groups of high (H-) and low (L-) levels of sdLDL-C calculated by the Sampson equation and LDL-C calculated by the Friedewald equation⁷ in a large number of Japanese individuals who had undergone annual health checkups.

Methods

Study Subjects and Clinical Endpoint

This study was a retrospective cohort study using prospectively obtained data for Japanese individuals who had an annual health checkup in Keijinkai Maruyama Clinic, a major health checkup institute in Sapporo, Japan.²⁰ All individuals who had undergone their annual health checkups in 2006 were initially enrolled in the present study (n=28,990) (Figure 1). Subjects without data for TC, triglycerides and HDL-C levels, those with levels of triglycerides ≥400 mg/dL and those with IHD at baseline were excluded, leaving a total of 17,963 subjects (men/women: 11,508/6,455) who had undergone a health checkup at least once between 2007 and 2016 participating in the present analyses. The clinical endpoint was new onset of IHD determined by a self-reported questionnaire at annual health checkups during a 10-year follow-up period. This study conformed to the principles outlined in the Declaration of Helsinki and was approved by the Ethics Committee of Sapporo Medical University (Number: 30-2-32). Written informed consent was given by all subjects.

Figure 1.

Flowchart of the study. Among 28,990 individuals who underwent a health examination in 2006, a total of 17,963 subjects (men/women: 11,508/6,455) were finally recruited for analyses in the present study.

Measurements

Medical examinations and sampling of urine and blood were performed after an overnight fast. Blood pressure was measured twice consecutively by a nurse using a sphygmomanometer (#601, Kenzmedico, Saitama, Japan) on the subject’s upper arm while seated, and the average blood pressure was used for analysis. Body mass index (BMI) was calculated as body weight in kilograms divided by height in meters squared. Estimated glomerular filtration rate (eGFR, mL/min/1.73 m²) was calculated for Japanese people as: 194 × serum creatinine^−1.094 × age^−0.287 × 0.782 (if female).²¹ The level of non-HDL-C was calculated by subtracting the HDL-C level from the TC level. LDL-C was calculated by the Friedewald equation: LDL-C = TC − HDL-C − triglycerides / 5. sdLDL-C was calculated by the 3-step Sampson equation: LDL-C¹⁴ = TC / 0.948 − HDL-C / 0.971 − (triglycerides / 8.56 + [triglycerides × non-HDL-C] / 2,140 − triglycerides² / 16,100) − 9.44; lbLDL-C¹² = 1.43 × LDL-C − (0.14 × (In [triglycerides] × LDL-C) − 8.99; sdLDL-C¹⁵ = LDL-C − lbLDL-C.

A self-administered questionnaire survey was performed to obtain information on habits of current smoking and alcohol drinking, and medical histories of hypertension, diabetes mellitus, dyslipidemia and IHD at baseline. Obesity was defined as BMI ≥25 in accordance with the Japan Society for the Study of Obesity.²² Hypertension was diagnosed in accordance with the guidelines of the Japanese Hypertension Society:²³ systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg or self-reported use of antihypertensive drugs. Diabetes mellitus was diagnosed in accordance with the guidelines of the American Diabetes Association:²⁴ fasting plasma glucose ≥126 mg/dL, HbA1c ≥6.5% or self-reported use of antidiabetic drugs. Dyslipidemia was diagnosed as LDL-C ≥140 mg/dL, HDL-C <40 mg/dL, non-HDL-C ≥170 mg/dL, triglycerides ≥150 mg/dL or self-reported use of antidyslipidemic drugs.

Statistical Analysis

Numeric variables are expressed as mean±SD for normal distributions or median (interquartile range) for skewed distributions. The distribution of each parameter was tested for its normality using the Shapiro-Wilk test. Comparisons between groups for parametric and nonparametric factors were performed with Student’s t-test and the Mann-Whitney U test, respectively. Intergroup differences in percentages of demographic parameters were examined by the chi-square test. Subjects were divided into 4 groups by using the 25^th percentile levels of sdLDL-C calculated by the Sampson equation and LDL-C calculated by the Friedewald equation at baseline as cutoff values: H-sdLDL-C/H-LDL-C, H-sdLDL-C/L-LDL-C, L-sdLDL-C/H-LDL-C, and L-sdLDL-C/L-LDL-C groups. One-way analysis of variance was used for detecting significant differences between data in multiple groups. The cumulative incidence of new onset of IHD was analyzed by the log-rank test of Kaplan-Meier curves. The associations of the development of IHD with the 4 subgroups were investigated by multivariable Cox proportional hazard models after adjustment of confounders including age, sex, smoking habit, hypertension and diabetes mellitus at baseline. Interaction between sex and the subgroups for the development of IHD was also investigated, and hazard ratios (HRs), 95% confidence intervals (CIs) and Akaike’s information criterion were calculated. A P value <0.05 was considered statistically significant. All data were analyzed by using EZR²⁵ and JMP pro17.1.0 (SAS Institute, Cary, NC).

Results

Characteristics of the Study Subjects

Baseline characteristics of the enrolled and excluded subjects are shown in Supplementary Table 1. Compared with the enrolled subjects, the excluded subjects were significantly younger, included a lower percentage of men, had lower levels of TC, non-HDL-C, LDL-C, triglycerides and sdLDL-C, and had a higher level of HDL-C. The proportion of subjects with dyslipidemia was significantly lower in the excluded subjects than in the enrolled subjects. Baseline characteristics of the recruited subjects are shown in Table 1. Men had higher systolic and diastolic blood pressures, higher levels of TC, non-HDL-C, LDL-C, triglycerides and sdLDL-C and a lower level of HDL-C than did women. The percentages of subjects with dyslipidemia and subjects receiving treatment for dyslipidemia were higher in men than in women.

Table 1.

Baseline Characteristics of the Subjects

	All (n=17,963)	Men (n=11,508)	Women (n=6,455)	P value
Age (years)	48±10	48±10	47±11	<0.001
Body mass index	23±3	24±3	22±3	<0.001
Obesity	5,623 (31.3)	4,534 (39.4)	1,089 (16.9)	<0.001
SBP (mmHg)	117±17	120±16	110±17	<0.001
DBP (mmHg)	74±11	77±11	69±11	<0.001
Current smoking habit	6,269 (34.9)	5,078 (44.1)	1,191 (18.5)	<0.001
Alcohol drinking habit	8,113 (45.2)	6,454 (56.1)	1,659 (25.7)	<0.001
Comorbidities
Hypertension	3,058 (17.0)	2,376 (20.6)	682 (10.6)	<0.001
Diabetes mellitus	1,051 (5.7)	875 (7.6)	147 (2.3)	<0.001
Dyslipidemia	8,265 (46.0)	6,158 (53.5)	2,107 (32.6)	<0.001
Treatment of dyslipidemia	2,873 (16.0)	1,921 (16.7)	952 (14.7)	<0.001
Biochemical data
Hemoglobin (g/dL)	14.3±1.5	15.1±1.1	12.9±1.2	<0.001
Albumin (g/dL)	4.4±0.2	4.4±0.2	4.3±0.2	<0.001
Creatinine (mg/dL)	0.74±0.28	0.81±0.30	0.60±0.17	<0.001
eGFR (mL/min/1.73 m2)	85±15	84±15	87±16	<0.001
Uric acid (mg/dL)	5.5±1.4	6.1±1.2	4.4±0.9	<0.001
FPG (mg/dL)	93±19	96±21	87±14	<0.001
Hemoglobin A1c (%)	5.3±0.7	5.4±0.8	5.2±0.5	<0.001
TC (mg/dL)	204±33	204±33	203±34	<0.001
HDL-C (mg/dL)	61±16	56±14	69±15	<0.001
Non-HDL-C (mg/dL)	143±35	149±35	133±35	<0.001
LDL-C (mg/dL)	121±31	123±31	118±31	<0.001
Triglycerides (mg/dL)	91 [63–135]	109 [77–156]	67 [49–93]	<0.001
sdLDL-C (mg/dL)	33.5 [25.2–43.0]	37.1 [29.0–46.0]	27.0 [20.7–35.4]	<0.001
Subgroups
L-sdLDL-C/L-LDL-C	2,588 (14.4)	1,062 (9.2)	1,526 (23.6)	<0.001
L-sdLDL-C/H-LDL-C	1,874 (10.4)	636 (5.5)	1,238 (19.2)	<0.001
H-sdLDL-C/L-LDL-C	1,874 (10.4)	1,480 (12.9)	394 (6.1)	<0.001
H-sdLDL-C/H-LDL-C	11,627 (64.7)	8,330 (72.4)	3,297 (51.1)	<0.001

Variables are expressed as number (%), mean±SD or median [interquartile range]. DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; FPG, fasting plasma glucose; H-, high-; HDL-C, high-density lipoprotein cholesterol; L-, low-; LDL-C, low-density lipoprotein cholesterol; sdLDL-C, small dense LDL-C; SBP, systolic blood pressure; TC, total cholesterol.

Baseline characteristics of subjects divided into the 4 subgroups according to 25^th percentile levels of estimated sdLDL-C (25.2 mg/dL) and estimated LDL-C (100 mg/dL) at baseline are shown in Table 2. Subjects in the H-sdLDL-C/H-LDL-C and H-sdLDL-C/L-LDL-C groups were older than subjects in the L-sdLDL-C/H-LDL-C and L-sdLDL-C/L-LDL-C groups and they included higher percentages of men, had higher frequencies of diabetes mellitus and hypertension, and had higher levels of TC, non-HDL-C and triglycerides and lower levels of eGFR.

Table 2.

Characteristics of the Subjects According to Levels of sdLDL-C and LDL-C at Baseline

	L-sdLDL-C/L-LDL-C (n=2,588)	L-sdLDL-C/H-LDL-C (n=1,874)	H-sdLDL-C/L-LDL-C (n=1,874)	H-sdLDL-C/H-LDL-C (n=11,627)	P value
Age (years)	42±10	45±10	47±11	49±10	<0.001
Body mass index	21±3	21±3	24±3	24±3	<0.001
Obesity	233 (13.2)	211 (15.4)	682 (42.7)	4,497 (42.1)	<0.001
Sex (male)	1,062 (47.1)	636 (40.2)	1,480 (83.9)	8,330 (74.4)	<0.001
SBP (mmHg)	109±16	111±16	119±16	119±17	<0.001
DBP (mmHg)	69±11	70±11	76±11	76±11	<0.001
Current smoking habit	785 (32.0)	382 (22.3)	901 (47.0)	4,201 (36.9)	<0.001
Alcohol drinking habit	1,184 (46.2)	599 (32.3)	1,111 (59.1)	5,219 (44.8)	0.034
Comorbidities					<0.001
Hypertension	196 (10.0)	163 (11.0)	421 (24.1)	2,278 (20.6)	<0.001
Diabetes mellitus	60 (3.2)	41 (2.6)	171 (9.4)	750 (6.9)	<0.001
Dyslipidemia	105 (4.1)	234 (12.5)	892 (47.6)	7,034 (60.5)	<0.001
Treatment of dyslipidemia	71 (2.7)	104 (5.5)	273 (14.6)	2,425 (20.9)	<0.001
Biochemical data
Hemoglobin (g/dL)	13.5±1.6	13.4±1.5	14.7±1.4	14.6±1.4	<0.001
Albumin (g/dL)	4.3±0.2	4.3±0.2	4.4±0.2	4.4±0.2	<0.001
Creatinine (mg/dL)	0.68±0.34	0.66±0.13	0.76±0.37	0.76±0.26	<0.001
eGFR (mL/min/1.73 m2)	90±16	87±14	86±16	83±15	<0.001
Uric acid (mg/dL)	4.8±1.3	4.7±1.2	5.8±1.4	5.7±1.4	<0.001
FPG (mg/dL)	87±15	87±13	96±23	95±20	<0.001
Hemoglobin A1c (%)	5.1±0.5	5.2±0.5	5.4±0.8	5.4±0.7	<0.001
TC (mg/dL)	163±20	198±21	175±18	219±28	<0.001
HDL-C (mg/dL)	70±16	72±15	57±15	58±14	<0.001
Non-HDL-C (mg/dL)	93±12	126±14	118±15	161±28	<0.001
LDL-C (mg/dL)	81±13	117±15	87±11	136±25	<0.001
Triglycerides (mg/dL)	54 [43–67]	47 [40–53]	127 [97–185]	106 [79–148]	<0.001
sdLDL-C (mg/dL)	19.5 [16.5–22.2]	21.7 [19.2–23.6]	30.9 [27.7–36.4]	39.3 [32.5–47.6]	<0.001
sdLDL-C/LDL-C	0.24 [0.20–0.27]	0.19 [0.16–0.21]	0.36 [0.31–0.43]	0.29 [0.25–0.34]	<0.001

Variables are expressed as number (%), mean±SD or median [interquartile range]. Abbreviations as in Table 1.

Incidence Rate for New Onset of IHD During Follow-up

Among the 17,963 recruited subjects (men/women: 11,508/6,455), 570 subjects (men/women: 449/121) developed new onset of IHD during the 10-year follow-up period (Table 3). The follow-up summation was 117,916 (men/women: 75,558/42,358) person-years, and the incidence rate of IHD was 4.8 (men/women: 5.9/2.9) per 1,000 person-years. The incidence rates of IHD were approximately 3-fold higher in the H-sdLDL-C/H-LDL-C and H-sdLDL-C/L-LDL-C groups than in the L-sdLDL-C/L-LDL-C and L-sdLDL-C/H-LDL-C groups.

Table 3.

Incidence Rate for the Development of IHD During Follow-up

	L-sdLDL-C/L-LDL-C	L-sdLDL-C/H-LDL-C	H-sdLDL-C/L-LDL-C	H-sdLDL-C/H-LDL-C
N
All	2,588	1,874	1,874	11,627
Men	1,062	636	1,480	8,330
Women	1,526	1,238	394	3,297
Follow-up summation (person-years)
All	17,371	12,905	11,914	75,726
Men	6,970	4,276	9,491	54,821
Women	10,401	8,629	2,423	20,905
No. of cases of new onset of IHD
All	38	26	70	436
Men	29	11	57	352
Women	9	15	13	84
Incidence rate of IHD, value per 1,000 person-years
All	2.2	2.0	5.9	5.8
Men	4.2	2.6	6.0	6.4
Women	0.9	1.7	5.4	4.0

Variables are expressed as number or incidence rate. IHD, ischemic heart disease. Other abbreviations as in Table 1.

Kaplan-Meier survival curves showed that there was a significant difference in the cumulative incidence for new onset of IHD among the 4 subgroups (log-rank test, P<0.001) and that cumulative incidences were high in subjects with high levels of sdLDL-C regardless of levels of LDL-C (Figure 2).

Figure 2.

Cumulative incidence of ischemic heart disease (IHD) in the sdLDL-C/LDL-C group. Kaplan-Meier survival curve analysis of cumulative incidence of IHD in 4 subgroups according to high (H-) and low (L-) levels of LDL-C and sdLDL-C. LDL-C, low-density lipoprotein cholesterol; sdLDL-C, small dense LDL-C.

HRs for New Onset of IHD in the 4 Subgroups

Cox proportional hazard analysis showed that the unadjusted HRs for new onset of IHD were significantly higher in the H-sdLDL-C/L-LDL-C and H-sdLDL-C/H-LDL-C groups than in the L-sdLDL-C/L-LDL-C group as a reference (Model 1) (Figure 3A). After adjustment for age and sex (Model 2), the HRs in the H-sdLDL-C/H-LDL-C and H-sdLDL-C/L-LDL-C groups were significantly higher than the HR in the L-sdLDL-C/L-LDL-C group (Figure 3B). With further additional incorporation of hypertension, diabetes, and current smoking habit into Model 2 (Model 3), the HRs in the H-sdLDL-C/H-LDL-C and H-sdLDL-C/L-LDL-C groups were still significantly higher than the HR in the L-sdLDL-C/L-LDL-C group (Figure 3C). There were no significant differences in HRs between the L-sdLDL-C/L-LDL-C and L-sdLDL-C/H-LDL-C groups in models 1–3 (Supplementary Table 2). With further additional incorporation of treatment of dyslipidemia into Model 3 (Model 4), HRs of the 4 subgroups of sdLDL-C and LDL-C were comparable, although treatment of dyslipidemia was a significant risk factor for the development of IHD (Supplementary Table 3). There were no significant interactions of sex with the subgroups for the adjusted HRs of development of IHD (models 2–4).

Figure 3.

Hazard ratios (HRs) and 95% confidence intervals (CIs) for new onset of IHD in 4 subgroups according to high (H-) and low (L-) levels of low-density lipoprotein cholesterol (LDL-C) and small dense LDL-C (sdLDL-C) analyzed by multivariable Cox proportional hazard models with unadjusted analysis (Model 1) (A), adjusted analysis for age and sex (Model 2) (B) and adjusted analysis for age, sex, hypertension, diabetes mellites, and current smoking habit (Model 3) (C).

As additional analyses, subjects were also divided into 4 subgroups according to 25^th percentile levels of estimated sdLDL-C (25.2 mg/dL) and HDL-C (49 mg/dL) or TG (63 mg/dL) at baseline. Similar to the results of the 4 subgroups divided by levels of sdLDL-C and LDL-C, the HRs for new onset of IHD in the H-sdLDL-C/H-HDL-C and H-sdLDL-C/L-HDL-C groups were significantly higher than the HR in the L-sdLDL-C/L-LDL-C group (models 1–3, Supplementary Table 4). On the other hand, the unadjusted HRs for new onset of IHD were significantly higher in the L-sdLDL-C/H-TG, H-sdLDL-C/L-TG and H-sdLDL-C/H-TG groups than in the L-sdLDL-C/L-TG group as a reference (Model 1) (Supplementary Table 5). In models 2 and 3, the HRs in the H-sdLDL-C/H-TG group were significantly higher than the HR in the L-sdLDL-C/L-TG group.

Discussion

In the present study, we showed that a high level of sdLDL-C estimated by the Sampson equation was significantly associated with new onset of IHD during a 10-year period, regardless of LDL-C level calculated by the Friedewald equation, in a Japanese general population. Cumulative incidences in the H-sdLDL-C/H-LDL-C and H-sdLDL-C/L-LDL-C groups increased with a similar tendency. After adjustment for age, sex, smoking habit, hypertension and diabetes mellitus, HRs for the development of IHD in the H-sdLDL-C/H-LDL-C and H-sdLDL-C/L-LDL-C groups were comparable and significantly higher than the HR in the L-sdLDL-C/L-LDL-C group. On the other hand, the HR in the L-sdLDL-C/H-LDL-C group was almost the same as that in the L-sdLDL-C/L-LDL-C group. These results suggested that a high level of estimated sdLDL-C, as well as directly measured sdLDL-C, is an independent risk factor for the development of IHD regardless of LDL-C level.

To the best of our knowledge, there is no previous study focusing on the development of IHD in categorized groups according to estimated levels of sdLDL-C and LDL-C, although there have been 3 longitudinal studies in which the association of the development of atherosclerotic cardiovascular disease with directly measured sdLDL-C was investigated.^26–28 The Atherosclerosis Risk in Communities (ARIC) study of 11,419 USA subjects for a mean follow-up of 11 years showed that high levels of measured sdLDL-C were associated with risk of the development of coronary artery disease regardless of the levels of LDL-C calculated by the Friedewald equation.²⁶ The Multi Ethnic Study of Atherosclerosis (MESA) comprising 4,387 USA subjects for a mean follow-up of 8.5 years also showed that levels of measured sdLDL-C were independently associated with the risk of coronary artery disease regardless of directly measured LDL-C in patients without diabetes mellitus.²⁷ The Hisayama study of 3,080 Japanese subjects for a mean follow-up of 8.3 years showed that the adjusted HR for IHD almost doubled in subjects with measured sdLDL-C ≥32.9 mg/dL regardless of measured LDL-C levels compared with subjects with sdLDL-C <32.9 mg/dL and LDL-C <120.1 mg/dL as a reference.²⁸ Those results are consistent with the results of the present study using estimated sdLDL-C levels, although the mean age of subjects was older in the previous studies (mean age: ARIC study/MESA/Hisayama study, respectively: 63±6/62±10/>60 years) than in the present study (mean age: 48±10 years).^26–28 Taken together, the results of the previous and present studies support the notion that an elevation of measured or estimated sdLDL-C regardless of the level of LDL-C is an important risk factor for the development of IHD. Furthermore, the present study allowed us to assess the risk of IHD in subjects with a low level of LDL-C by calculating sdLDL-C using clinically assessed levels of TC, triglycerides and HDL-C.

In a recent review of the present study, Koba et al. investigated the effects of various lipid markers, including direct sdLDL-C and sdLDL-C calculated by the Sampson equation, on secondary, but not primary, prevention of IHD and showed that direct sdLDL-C, but not sdLDL-C estimated by the Sampson equation, could predict subsequent recurrence of IHD.²⁹ Further studies are needed to investigate the association between sdLDL-C and primary prevention of IHD including a comparison of directly measured and estimated levels of sdLDL-C in multicenter trials using a large number of subjects.

Several mechanisms for the greater effect of sdLDL-C than that of lbLDL-C on atherosclerotic cardiovascular disease have been proposed.^8,30,31 It has been reported that sdLDL particles have lower affinity than larger LDL particles for the LDL receptor, resulting in longer residence time in the blood,^8,30 and that sdLDL particles easily infiltrate the vessel wall than do larger LDL particles.⁸ There has been reported differential susceptibility of LDL subfractions to oxidative stress, an important factor in atherogenesis.³⁰ It has been reported that lbLDL is less oxidized and that sdLDL is more oxidized.³¹ In addition, we previously showed that estimated sdLDL-C, unlike LDL-C, is associated with the development of hypertension as an important risk factor for IHD.¹⁹ It has also been reported that insulin has a direct inhibitory effect on the production of very low-density lipoprotein (VLDL) in the liver and that acute hyperinsulinemia reduced VLDL concentrations and changed the LDL subfraction profile to lower density LDL in insulin-sensitive subjects but not in insulin-resistant subjects, indicating that insulin resistance increases the proportion of sdLDL.⁸ Furthermore, the sdLDL-C level has been reported to be increased in subjects with metabolic syndrome.³² Therefore, sdLDL-C levels might be a surrogate marker of insulin resistance, and because insulin resistance has been reported as a risk factor for the development of IHD,³³ it is possible that insulin resistance contributes to further coronary atherosclerosis.

In the present study, the adjusted HRs between the H-sdLDL-C/L-TG and L-sdLDL-C/L-TG groups were not statistically different, although the adjusted HR in the H-sdLDL-C/H-TG group was significantly higher than the HR in the L-sdLDL-C/L-TG group. The reasons are unclear, but one possibility is the difference in statistical power. The number of patients in the H-sdLDL-C/H-TG group was 12,603, whereas that of the H-sdLDL-C/L-TG was 898. Another passible reason is the difference in biochemical factors that affect LDL particles. It has been reported that the number of LDL particles positively correlates with the non-HDL-C level and that LDL size negatively correlates with TG level.³⁴

Because the HRs of the 4 subgroups divided by levels of sdLDL-C and LDL-C were comparable after adjustment of drug treatment for dyslipidemia (Model 4, Supplementary Table 3), lipid-lowering drugs may reduce risk developing IHD in subjects with high levels of sdLDL-C. Several lipid-lowering drugs can reduce the levels of sdLDL-C,³⁵ so optimal interventions would be important to reduce the risk of IHD in subjects with high levels of sdLDL-C. Taken together, it appears to be important to routinely assess sdLDL-C for prevention of IHD, even in subjects with low levels of LDL-C but who may have high levels of sdLDL-C.

Study Limitations

First, the possibility of selection bias cannot be ruled out because the study subjects were only those who underwent annual physical examinations at a single clinic in an urban area. The reduced number of subjects during the follow-up period may also have caused bias. Second, information on precise dietary habits and exercise that might influence the development of cardiovascular disease was not available. Third, we were unable to examine the effect of extreme hypertriglyceridemia on IHD, because subjects with triglycerides >400 mg/dL (n=512) were excluded in order to calculate LDL-C using the Friedewald equation.⁷ Fourth, although risk factors for IHD at baseline were adjusted in the multivariate Cox proportional hazards model, those risk factors during the follow-up period were not included in the adjustment. Finally, directly measured sdLDL-C levels were not used in the present study.

Conclusions

A high level of sdLDL-C estimated by the Sampson equation was a predominant predictor for the development of IHD, regardless of LDL-C level calculated by the Friedewald equation, in a general Japanese population. Estimation of sdLDL-C level by the Sampson equation may be useful for determining the risk for IHD even in individuals whose LDL-C levels are within the normal range. Further elucidation of the mechanisms by which estimated sdLDL-C levels affect IHD may allow for the development of new therapeutic strategies for prevention of IHD.

Acknowledgments

M.T. and M.F. were supported by grants from swJapan Society for the Promotion of Science (22K08313, 23K07993). The authors are grateful to Keita Numata and Takashi Hisasue for data management. We appreciate the invaluable discussion with members of Sapporo Medical University Adiposcience Research Group (SMARG) and KA8 (Tue. PM8) Research Group.

Disclosures

The authors declare that they have no competing interests.

IRB Information

The study was performed with the approval of the Ethics Committee of Sapporo Medical University (Number: 30-2-32).

Data Availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Supplementary Files

Please find supplementary file(s);

https://doi.org/10.1253/circj.CJ-24-0770

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