Association of Serum Levels of Cholesterol Absorption and Synthesis Markers with the Presence of Cardiovascular Disease: The CACHE Study CVD Analysis

Abstract

Aim: Serum levels of cholesterol absorption and synthesis markers have been associated with cardiovascular risk in the United States and European countries. In this study, we examined the relevance of these biomarkers and the presence of cardiovascular disease (CVD) in Japanese individuals.

Methods: The CACHE consortium, comprising of 13 research groups in Japan possessing data on campesterol, an absorption marker, and lathosterol, a synthesis marker measured by gas chromatography, compiled the clinical data using the REDCap system.

Results: Among the 2,944 individuals in the CACHE population, those with missing campesterol or lathosterol data were excluded. This cross-sectional study was able to analyze data from 2,895 individuals, including 339 coronary artery disease (CAD) patients, 108 cerebrovascular disease (CeVD) patients, and 88 peripheral artery disease (PAD) patients. The median age was 57 years, 43% were female, and the median low-density lipoprotein cholesterol and triglyceride levels were 118 mg/dL and 98 mg/dL, respectively. We assessed the associations of campesterol, lathosterol, and the ratio of campesterol to lathosterol (Campe/Latho ratio) with the odds of CVD using multivariable-adjusted nonlinear regression models. The prevalence of CVD, especially CAD, showed positive, inverse, and positive associations with campesterol, lathosterol, and the Campe/Latho ratio, respectively. These associations remained significant even after excluding individuals using statins and/or ezetimibe. The associations of the cholesterol biomarkers with PAD were determined weaker than those with CAD. Contrarily, no significant association was noted between cholesterol metabolism biomarkers and CeVD.

Conclusion: This study showed that both high cholesterol absorption and low cholesterol synthesis biomarker levels were associated with high odds of CVD, especially CAD.

See editorial vol. 30: 1759-1760

Introduction

Cardiovascular disease (CVD), including coronary artery disease (CAD), cerebrovascular disease (CeVD) and peripheral artery disease (PAD), has been identified as the leading cause of death and economic burdens worldwide¹⁾. Alterations in cholesterol metabolism may affect the risk of CVD apart from serum lipids and lipoprotein levels.

Dietary cholesterol is absorbed from the intestine through Niemann-Pick C1-like protein 1 (NPC1L1), which is an intestinal cholesterol transporter²⁾. Cholesterol absorption can be assessed by the serum levels of plant sterols such as campesterol and sitosterol³⁾, which are not synthesized in humans but exogenously absorbed through NPC1L1. Alternatively, cholesterol synthesis can be assessed by the serum levels of cholesterol precursors such as lathosterol³⁾. As per previous studies, higher cholesterol absorption and lower synthesis are independent predictors of CVD relative to established coronary risk factors^{4, 5)}. However, cohort studies examining the association between CVD and cholesterol metabolism markers are mainly reported in the United States and European countries. Therefore, it is unknown whether this can be applied to Asians, including the Japanese, who have a lower incidence of CVD than Americans and Europeans. Moreover, to the best of our knowledge, no studies have reported the associations between cholesterol metabolism biomarkers and the individual diseases such as CAD, CeVD and PAD in patients with CVD.

Therefore, in this study, we aim to address the following research questions in the Japanese population. First, are cholesterol metabolism biomarkers associated with the presence of CVD, a composite of CAD, CeVD, and PAD? Second, are cholesterol metabolism biomarkers associated with the presence of these individual diseases separately? Third, how are these associations in analyses in which those treated with cholesterol-lowering drugs such as statins and/or ezetimibe that affect cholesterol metabolism are excluded?

Materials and Methods

Ethical Consideration

This study adhered to the latest version of the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects by the Ministry of Health, Labor, and Welfare and the Ministry of Education, Japan (the original version in 2016 was modified in 2017). The study protocol was reviewed and approved by the Ethics Committee, Osaka City University Graduate School of Medicine, Osaka, Japan (Approval No. 3871) and registered at UMIN-CTR (UMIN000030635)⁶⁾. Additionally, the protocol of this study was approved by the review board of each participating institution prior to the study⁶⁾.

Clinical Data Collection

In total, there are 13 research groups in Japan that make up the CACHE consortium; they possess data on serum markers of cholesterol metabolism. CACHE stands for Cholesterol Absorption and Cholesterol synthesis in High-risk patiEnts. Clinical data, including serum biomarkers of cholesterol metabolism, were collected and compiled using the web-based system called Research Electronic Data Capture (REDCap)^{7, 8)} (https://projectredcap.org/about/) at Osaka City University (http://www.hosp.med.osaka-cu.ac.jp/self/hyokac/redcap/index.shtml).

The CACHE study collected clinical data from medical records or datasets for research purposes regarding the following items: 1) clinical background including age, sex, smoking status, weight, height, high-risk conditions (prior CAD, prior stroke, prior PAD, diabetes mellitus, chronic kidney disease (CKD) including dialysis, and familial hypercholesterolemia), and comorbidity such as hypertension and hyperuricemia; 2) blood tests including total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting plasma glucose, hemoglobin A1c (HbA1c), serum creatinine, estimated glomerular filtration rate (eGFR), uric acid, serum albumin, AST, ALT, C-reactive protein, red blood cell, hemoglobin, mean corpuscular volume, white blood cell, and platelet counts; 3) physical examination and vital signs including height, body weight, body mass index (BMI), systolic blood pressure, diastolic blood pressure, and pulse rate; 4) medication use including drugs for dyslipidemia, hypertension, diabetes mellitus, and hyperuricemia; and 5) specific treatments including hemodialysis and LDL apheresis.

CAD, CeVD, and PAD were classified based on medical records or history taking. CAD was defined as a history of stable ischemic heart disease, acute coronary syndrome, or coronary revascularization with percutaneous coronary intervention and/or coronary artery bypass graft surgery, as confirmed by electrocardiography and cardiac imaging. Atypical chest pain was not included in the definition of CAD. CeVD was defined as symptomatic stroke, including hemorrhagic stroke, confirmed by magnetic resonance imaging or computed tomography. Asymptomatic cerebrovascular disease was not included in the definition of CeVD. PAD was diagnosed by an ankle-brachial index of 0.9 or lower, stenosis or occlusion confirmed by vascular imaging, or lower limb amputation due to ischemia. Leg symptoms that could not be confirmed by any of the tests were not included in the definition of PAD.

Diabetes mellitus was defined by either previous diagnosis of diabetes mellitus, use of any antidiabetic medication, fasting plasma glucose of 126 mg/dL or higher, or HbA1c by the National Glycohemoglobin Standardization Program (NGSP) value of 6.5% or higher according to the diagnostic criteria of the American Diabetes Association and the Japan Diabetes Society^{9, 10)}. If the HbA1c value previously used by the Japan Diabetes Society was entered, it was converted to the NGSP value using a conversion formula provided by the JDS¹¹⁾.

Hypertension was defined either by the use of any antihypertensive medication, systolic blood pressure of 140 mmHg or higher, or diastolic blood pressure of 90 mmHg or higher according to the criteria by the Japanese Society of Hypertension¹²⁾.

In this study, CKD was defined by eGFR lower than 60 mL/min/1.73 m² using the equation for the Japanese¹³⁾. Because the CACHE study did not collect data on proteinuria; therefore, it was not used in the definition of CKD in this study. Patients with kidney failure treated with hemodialysis were included in patients with CKD.

Familial hypercholesterolemia was diagnosed using the criteria from the Japan Atherosclerosis Society¹⁴⁾.

With regard to lipid parameters, we used the following rules: 1) triglyceride (TG) and HDL-C values were used as entered. 2) Using total cholesterol (TC), TG, and HDL-C, non-HDL-C was calculated by subtracting HDL-C from TC, and LDL-C was calculated using the Friedewald formula¹⁵⁾. 3) If LDL-C measured by a homogenous assay was entered but TC was unavailable, the LDL-C by a homogenous assay was used for analysis, and non-HDL-C was thereafter calculated as LDL-C plus TG/5. 4) If LDL-C or non-HDL-C cannot be calculated because of missing values for TG or HDL-C, it was handled as missing. In the CACHE-CKD analysis⁶⁾, we presented data for TG, HDL-C, non-HDL-C, and LDL-C.

Selection of the CACHE Population and Individuals for this Analysis

The inclusion criteria for the CACHE study were as follows: 1) patients at high risk of CVD (CAD, CeVD, PAD, diabetes mellitus, CKD including those treated with dialysis, and FH), or individuals who were examined for the screening of these conditions and 2) individuals whose cholesterol metabolism markers were already measured (serum lathosterol, campesterol, and sitosterol levels). From the total 3,597 records accumulated in the REDCap system, we selected the CACHE population for analysis by excluding 1) duplicate records from the same individual and 2) individuals with missing values for age, sex, or both height and weight. For this “CACHE study CVD analysis,” individuals were further excluded if serum campesterol or lathosterol data were missing.

Assays for Campesterol and Lathosterol Concentrations

Serum concentrations of campesterol and lathosterol were measured as biomarkers for cholesterol synthesis and absorption, respectively, using gas chromatography at SRL Inc., Tokyo, Japan. The gas chromatography procedure has been described elsewhere in detail¹⁶⁾. In addition to the concentrations of campesterol and lathosterol, we calculated the campesterol to lathosterol ratio (Campe/Latho ratio) in order to assess the relative status of cholesterol absorption to cholesterol synthesis¹⁷⁾.

Statistical Analysis

The clinical characteristics of the individuals selected for this analysis were presented. Continuous and categorical variables were summarized by medians (interquartile ranges) and numbers (percentages), respectively, and were compared using the Kruskal–Wallis test or Fisher’s exact test.

The associations of the serum biomarkers for cholesterol metabolism (exposure) with the presence of CVD (outcome) were examined using multivariable-adjusted nonlinear regression models that considered the restricted cubic spline term for the exposure variables with three knots (10th, 50th, and 90th percentile levels). The rms function was implemented in the rms and Hmisc packages in R. The presence of CVD was handled as a composite of CAD, CeVD, and PAD, or the individual vascular disease. Adjustment was made for age, sex, the presence of diabetes mellitus, hypertension, current smoking, CKD, BMI, non-HDL-C, HDL-C, and TG. To avoid the possible influence of the use of statins and ezetimibe, the above analyses in the total individuals were repeated in the subgroup of individuals excluding those with statin and/or ezetimibe use.

All statistical inferences were conducted with a two-sided 5% significance level using R software version 4.0.3 (https://cran.r-project.org/).

Results

Selection of Individuals for this Analysis

Fig.1 shows the selection of individuals for this CACHE study CVD analysis. In this study, we were able to collect 3,597 records for 2,989 individuals, and the repeated records were not used. By excluding 45 individuals with missing data on age, sex, or both height and weight, the CACHE population (N=2,944) was determined. For the purpose of this CACHE study CVD analysis, 49 individuals were further excluded because of missing values of serum campesterol or lathosterol. Finally, 2,895 individuals were selected for this analysis.

Fig.1. Selection of individuals for this CACHE study CVD analysis

The initial dataset contained 3,597 records from 2,989 independent individuals. We eliminated duplicate records from the same individuals. We further excluded 45 individuals who had missing values for age, sex, and/or height and body weight. The remaining 2,944 were defined as the CACHE population for further analyses, including this CACHE study CVD analysis. We excluded 49 individuals with missing values for serum campesterol or lathosterol, and the remaining 2,895 individuals were analyzed in this CACHE study CVD analysis.

Table 1 provides the characteristics of the study subjects. It was noted that the number of patients with CAD was highest among those with CVD (N=339, 11.7% of the total for CAD, N=108, 3.7% of the total for CeVD and N=88, 3.0% of the total for CeVD). The median serum concentrations of campesterol and lathosterol were 4.70 µg/mL and 1.90 µg/mL, respectively. The median Campe/Latho ratio was determined to be 2.31.

Table 1. Characteristics of the individuals in this analysis

Variable	Unit	Total individuals	Subgroup excluding patients using statins and/or ezetimibe
Number of individuals	---	2895	2490
Age	years	57 [46, 65]	56 [44, 63]
Sex (M)	N (%)	1651 (57.0)	1405 (56.4)
Smoking	N (%)	474 (19.6)	407 (20.2)
Body mass index (BMI)	kg/m2	22.57 [20.52, 24.83]	22.34 [20.41, 24.54]
Total cholesterol	mg/dL	198 [169, 228]	201 [172, 230]
Triglyceride	mg/dL	98 [70, 142]	96 [68, 139]
HDL-C	mg/dL	55.1 [44, 69]	56.75 [45, 70]
Non-HDL-C	mg/dL	141.9 [113, 170]	143 [114.45, 171.00]
LDL-C	mg/dL	118.6 [93.2, 143.6]	120.4 [95, 144.8]
Campesterol	µg/mL	4.70 [3.30, 6.40]	4.60 [3.30, 6.40]
Lathosterol	µg/mL	1.90 [1.20, 3.00]	2.10 [1.40, 3.20]
Campesterol/Lathosterol ratio	no unit	2.31 [1.36, 4.00]	2.13 [1.29, 3.60]
Sitosterol	µg/mL	2.50 [1.80, 3.40]	2.50 [1.80, 3.40]
Comorbidities
Coronary artery disease (CAD)	N (%)	339 (11.7)	119 (4.8)
Cerebrovascular disease (CeVD)	N (%)	108 (3.7)	85 (3.4)
Peripheral artery disease (PAD)	N (%)	88 (3.0)	55 (2.2)
Familial hypercholesterolemia	N (%)	158 (5.5)	58 (2.3)
Diabetes	N (%)	782 (36.6)	623 (34.4)
Chronic kidney disease (CKD)	N (%)	821 (37.3)	686 (37.5)
CKD treated with hemodialysis	N (%)	522 (18.0)	495 (19.9)
Hypertension	N (%)	1300 (45.5)	1003 (40.7)
Obesity	N (%)	686 (23.8)	538 (21.7)
Dyslipidemia	N (%)	1475 (51.0)	1258 (50.6)
Lipid-lowering medications	N (%)	431 (14.9)	26 (1.0)
Statins	N (%)	397 (13.7)	0 (0)
Ezetimibe	N (%)	63 (2.2)	0 (0)
PCSK9 inhibitors	N (%)	4 (0.1)	0 (0)
Fibrate	N (%)	15 (0.5)	12 (0.5)
n3-PUFA	N (%)	30 (1.0)	7 (0.3)

The table gives medians (interquartile ranges) for continuous variables and numbers (percentages) for categorical variables. Abbreviations: BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; CKD, chronic kidney disease; PCSK9, Proprotein convertase subtilisin/kexin type 9; PUFA, polyunsaturated fatty acid.

Associations between Biomarkers of Cholesterol Metabolism and CVD

Fig.2A shows the multivariable-adjusted association of the biomarkers of cholesterol metabolism with the odds of having a composite of CVD among the total individuals (N=2,895). The odds of CVD had a positive association with the campesterol concentration. Moreover, the odds of CVD exhibited a U-shaped association with lathosterol, and the association was inverse in individuals with lathosterol concentrations of 3 µg/mL or lower, which represents approximately 75% of the individuals. The odds of CVD had a positive association with the Campe/Latho ratio.

Fig.2. Association between biomarkers of cholesterol metabolism and composite CVD

The associations of serum levels of campesterol and lathosterol and the Campe/Latho ratio with CVD were analyzed in the total individuals (A) and individuals excluding those using statins and/or ezetimibe (B) via a multivariable-adjusted nonlinear regression model. The curves and shaded areas indicate the means and 95% confidence intervals, respectively. Adjustment was made for age, sex, diabetes mellitus, hypertension, smoking status, body mass index, nonhigh-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides.

Abbreviations: Campe, campesterol; Latho, lathosterol.

We then performed additional analyses in 2,490 individuals, excluding those treated with statins and/or ezetimibe (Fig.2B). As per analysis results, the odds of CVD showed a U-shaped association with the campesterol concentration, namely, a positive association in the range of 6–7 mg/mL or lower and an inverse trend in the range of 6–7 mg/mL or higher. Lathosterol and the Campe/Latho ratio showed the same patterns as in the total individuals.

Adjusted odds ratios for the presence of CVD in the 75th versus the 25th percentile levels of campesterol concentration, lathosterol concentration, and Campe/Latho ratio were 2.07 [1.66, 2.59], 0.21 [0.15, 0.27], and 3.24 [2.41, 4.35] in the total individuals, respectively, and 1.53 [1.15, 2.02], 0.55 [0.39, 0.77], and 1.80 [1.26, 2.56] in the individuals excluding those using statins and/or ezetimibe, respectively (Table 2).

Table 2. Odds ratios for the presence of cardiovascular disease in the 75th versus 25th percentile levels of serum biomarkers for cholesterol metabolism

Population	Exposure	25th and 75th percentile levels	Outcome	Odds ratio to have outcome
Total individuals (N = 2,895)	Campe	3.3 and 6.4 mg/mL	Any CVD	2.074 [1.662, 2.589]
			CAD	2.748 [2.121, 3.562]
			CeVD	0.683 [0.469, 0.994]
			PAD	1.771 [1.169, 2.685]
	Latho	1.2 and 3.0 mg/mL	Any CVD	0.205 [0.154, 0.274]
			CAD	0.140 [0.099, 0.197]
			CeVD	0.643 [0.360, 1.149]
			PAD	0.543 [0.333, 0.893]
	Campe/Latho	1.361 and 4.00	Any CVD	3.235 [2.408, 4.346]
			CAD	4.205 [2.951, 5.990]
			CeVD	1.096 [0.682, 1.762]
			PAD	2.658 [1.536, 4.599]
Excluding those with statin and/or	Campe	3.3 and 6.4 mg/mL	Any CVD	1.528 [1.154, 2.024]
ezetimibe (N = 2,490)			CAD	2.048 [1.411, 2.972]
			CeVD	0.806 [0.524, 1.240]
			PAD	2.326 [1.292, 4.185]
	Latho	1.4 and 3.2 mg/mL	Any CVD	0.546 [0.387, 0.771]
			CAD	0.599 [0.382, 0.941]
			CeVD	0.391 [0.169, 0.901]
			PAD	0.649 [0.352, 1.197]
	Campe/Latho	1.287 and 3.60	Any CVD	1.795 [1.260, 2.557]
			CAD	1.708 [1.086, 2.689]
			CeVD	1.309 [0.748, 2.291]
			PAD	2.666 [1.332, 5.333]

The table gives odds ratios [95% confidence intervals] to obtain outcomes for those at the 75th percentile level versus those at the 25th percentile level of serum biomarkers for cholesterol metabolism (exposure) using nonlinear regression models adjusted for age, sex, diabetes mellitus, hypertension, chronic kidney disease, smoking, BMI, non-HDL-C, HDL-C, and TG.

Abbreviations: Campe, campesterol; Latho, lathosterol; CVD, cardiovascular disease as a composite of CAD, CeVD, and PAD; CAD, coronary artery disease; CeVD, cerebrovascular disease; PAD, peripheral artery disease.

Associations between Biomarkers of Cholesterol Metabolism and CAD

In the total individuals, CAD showed a similar trend as CVD. CAD was determined to have a positive association with the campesterol concentration and Campe/Latho ratio and a U-shaped association with the lathosterol concentration (Fig.3A). Additional analyses in the individuals excluding those using statins and/or ezetimibe showed similar patterns as in the total individuals, although the association between the campesterol concentration and odds of CAD was not apparent in the range of 6–7 mg/mL or higher (Fig.3B).

Fig.3. Association between biomarkers of cholesterol metabolism and coronary artery disease

The associations of serum levels of campesterol and lathosterol and the Campe/Latho ratio with coronary artery disease were analyzed in the total individuals (A) and individuals excluding those using statins and/or ezetimibe (B) via a multivariable-adjusted nonlinear regression model. The curves and shaded areas indicate the means and 95% confidence intervals, respectively. Adjustment was made for age, sex, diabetes mellitus, hypertension, smoking status, body mass index, non-high-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides. Abbreviations: Campe, campesterol; Latho, lathosterol.

Adjusted odds ratios for the presence of CAD in the 75th versus 25th percentile levels of serum biomarkers of cholesterol metabolism were almost comparable to those for the presence of CVD (Table 2).

Associations between Biomarkers of Cholesterol Metabolism and CeVD

The odds of CeVD tended to have inverse associations with serum biomarkers of cholesterol metabolism, but these associations were deemed statistically insignificant (Fig.4A). Additional analyses in the individuals excluding those using statins and/or ezetimibe showed a similar pattern as in the total individuals. Consistent with the data for the total individuals, the associations did not show statistical significance (Fig.4B).

Fig.4. Association between biomarkers of cholesterol metabolism and cerebrovascular disease

The associations of serum levels of campesterol and lathosterol and the Campe/Latho ratio with cerebrovascular disease were analyzed in the total individuals (A) and individuals excluding those using statins and/or ezetimibe (B) via a multivariable-adjusted nonlinear regression model. The curves and shaded areas indicate the means and 95% confidence intervals, respectively. Adjustment was made for age, sex, diabetes mellitus, hypertension, smoking status, body mass index, nonhigh-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides. Abbreviations: Campe, campesterol; Latho, lathosterol.

Associations between biomarkers of cholesterol metabolism and PAD

The odds of PAD showed an inverted U-shaped association with the campesterol concentration and Campe/Latho ratio. The odds of PAD peaked around the campesterol concentration of 6–7 mg/mL and around the Campe/Latho ratio of 5, and these associations were not apparent in the ranges higher than these levels. The association between PAD and the lathosterol concentration was deemed statistically insignificant (Fig.5A). Additional analyses in individuals excluding those using statins and/or ezetimibe showed similar patterns as in the total individuals (Fig.5B).

Fig.5. Association between biomarkers of cholesterol metabolism and peripheral artery disease

The associations of serum levels of campesterol and lathosterol and the Campe/Latho ratio with peripheral artery disease were analyzed in the total individuals (A) and individuals excluding those using statins and/or ezetimibe (B) via a multivariable-adjusted nonlinear regression model. The curves and shaded areas indicate the means and 95% confidence intervals, respectively. Adjustment was made for age, sex, diabetes mellitus, hypertension, smoking status, body mass index, nonhigh-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides.

Abbreviations: Campe, campesterol; Latho, lathosterol.

Adjusted odds ratios for the presence of PAD in the 75th versus 25th percentile levels of campesterol concentration and Campe/Latho ratio were 1.77 [1.17, 2.69] and 2.66 [1.54, 4.60] in the total individuals, respectively, and 2.33 [1.29, 4.19] and 2.67 [1.33, 5.33] in the individuals excluding those using statins and/or ezetimibe, respectively (Table 2).

Discussion

In the present study, we investigated the association between cholesterol metabolism biomarkers and the presence of CVD, either as a composite of CAD, CeVD, and PAD or as the individual vascular disease, using the relatively large sample of the CACHE study. The key findings include the following: 1) higher levels of campesterol, lower levels of lathosterol, and higher Campe/Latho ratios were associated with higher odds of a composite CVD among the total individuals; 2) these associations of the cholesterol metabolism biomarkers with the odds of CAD were distinct, but the associations with the odds of PAD were found weaker than those for CAD, and the associations with the odds of CeVD were statistically insignificant among the total individuals; and 3) most of these associations remained the same even when the individuals using statins and/or ezetimibe were excluded from the analysis.

Previous studies have examined the association between cholesterol absorption and CVD. For example, the POSCH (Program on Surgical Control of Hyperlipidemias) trial has shown that in patients undergoing partial ileal bypass surgery, there is a 35.0% risk reduction in the occurrence of cardiovascular events along with a reduction in the plasma levels of TC¹⁸⁾. A prospective cohort study also showed that lower cholesterol absorption was associated with fewer recurrent cardiovascular events and with better survival in elderly patients⁵⁾. In the IMPROVE-IT (IMproved Reduction of Outcomes: Vytorin Efficacy International Trial) showed the combination of statin and ezetimibe improved cardiovascular outcomes as compared to statin alone¹⁹⁾. On the other hand, a prospective cohort study showed that low serum lathosterol, a cholesterol synthesis marker, correlated with a high risk of cardiovascular events and excess all-cause mortality in patients without lipid-lowering drugs²⁰⁾. These results are consistent with the current findings that CVD has a positive association with cholesterol absorption and a negative correlation with cholesterol synthesis.

In this study, we showed that higher cholesterol absorption was associated with higher odds of CVD, especially CAD. It is conceivable that some patients with CVD started LDL-C-lowering treatment with statin and/or ezetimibe after they had suffered from CVD. These medications can directly affect hepatic cholesterol synthesis and intestinal cholesterol absorption. Therefore, the occurrence of CVD may have induced the changes in cholesterol metabolism. However, this possibility does not fully explain the higher odds for CVD in those with higher levels of cholesterol absorption because essentially the same results were shown even in analyses excluding those on statins and/or ezetimibe. Therefore, these current findings suggest that higher cholesterol absorption may not be the result, but the cause of CVD.

Dietary oxysterols are known as an important residual risk factor for CAD^{21, 22)}. Emerging evidence suggests that oxysterols induce inflammation, oxidative stress, endoplasmic reticulum stress, and apoptosis in the pathogenesis of atherosclerosis^23-26). Since NPC1L1 mediates intestinal absorption of cholesterol and non-cholesterol sterols, including campesterol and oxysterols, a higher cholesterol absorption suggests a higher oxysterol concentration. The CuVIC (Effect of Cholesterol Absorption Inhibitor Usage on Target Vessel Dysfunction after Coronary Stenting) trial, which is a prospective clinical study conducted by some of our investigators, showed that the inhibition of cholesterol absorption decreased serum oxysterol levels and preserved coronary endothelial function in patients with CAD²⁷⁾. Therefore, we speculate that the increased oxysterol levels in patients with CAD could be a potential mechanism that explains the observed association between CAD and cholesterol absorption biomarkers.

This study did not show any significant associations between CeVD and cholesterol biomarkers. This finding may be explained by the presence of stroke subtypes with different pathogeneses. The CeVD in this study did not exclude hemorrhagic stroke. In addition, ischemic stroke included lacunar infarction, atherothrombotic infarction, and cardioembolic infarction. In the Japanese population, a study has shown a significant increase in the incidence of cardioembolic infarction from the 1960s to the late 1990s, while lacunar infarction has declined; this study was done by comparing ischemic stroke subtypes among the three cohorts established at different times in a Japanese community²⁸⁾. This data suggests that the trend of an increased proportion of cardioembolic infarction, rather than atherothrombotic infarction, in Japan may underlie the no significant association between CeVD and cholesterol biomarkers in the present study.

With regard to the associations of cholesterol metabolism biomarkers with PAD, although these associations were less predominant than those with CAD, presumably due to the relatively small number of patients with PAD (N=108) for multivariable-adjusted analysis, the association with campesterol was similar to that with CAD and was statistically significant even in the subgroup analysis excluding statin and/or ezetimibe users. This observation suggests the presence of a common pathophysiology of CAD and PAD in relation to cholesterol absorption. In fact, in patients with severe PAD, tissue and plasma levels of oxysterols are reported to have increased²⁹⁾.

For the clinical translation of these findings, patients with high campesterol and low lathosterol levels may benefit from the inhibition of cholesterol absorption in the prevention of CAD and PAD. There is a lack of direct evidence from interventional studies that ezetimibe alone reduces all-cause mortality in patients with CVD. The IMPROVE-IT¹⁹⁾ and HIJ-PROPER (The Heart Institute of Japan PRoper level of lipid lOwering with Pitavastatin and Ezetimibe in acute coronary syndrome)³⁰⁾ trials showed a reduction in CVD risk in the statin plus ezetimibe group as compared to the statin alone group. It is noteworthy that a subgroup analysis of the HIJ-PROPER trial showed that patients with higher baseline levels of the cholesterol absorption marker sitosterol achieved significant benefit in the statin plus ezetimibe group, which may support this hypothesis.

This study has several limitations. First, this study is a cross-sectional study, and there is a need for further longitudinal studies to be conducted over a long period of time in the future. Second, the CACHE population was composed of data from experts in the fields of cardiology, lipidology, diabetology, endocrinology, nephrology, general internal medicine, and preventive medicine. Therefore, the population was heterogeneous, and this study included patients with various comorbidities and medications. To address this issue, we performed vigorous statistical adjustment for possible confounders. In addition, we conducted subgroup analyses excluding those using statins and/or ezetimibe. On the other hand, the relatively large sample size was one of the strengths of this study; this allowed us to analyze CAD, CeVD, and PAD separately.

In the present study, we have demonstrated the association of CVD with serum biomarkers of synthesis and absorption of cholesterol using the relatively large sample set of the CACHE study. Patients with higher levels of campesterol, lower levels of lathosterol, and higher Campe/Latho ratios had higher odds of CVD, especially CAD, among the total individuals. These associations between CVD, especially CAD, and these biomarkers were found to be statistically significant even in analysis excluding patients using statins and/or ezetimibe. Further studies are needed to clarify the mechanisms underlying these associations.

Data Availability

The datasets generated and/or analyzed in the current study will be made available upon reasonable request.

Acknowledgments

Part of this study was presented at the 53rd Annual Meeting of the Japan Atherosclerosis Society (October 22-23, 2021, Kyoto, Hybrid version) and at the 18th International Symposium of Atherosclerosis (October 24-27, 2021, Kyoto, Hybrid version).

Conflict of Interest

Tetsuya Matoba reported personal fees from Bayer Yakuhin Ltd. and MSD and research grants from Amgen and Kowa. Hiroshi Yoshida reported personal fees from Denka Company Ltd. and Kowa Company Ltd. Tatsuro Ishida reported personal fees from Bayer Yakuhin Ltd. and Kowa Inc. Yasushi Ishigaki reported personal fees from Bayer Yakuhin, Kowa Pharmaceutical Company, MSD, Novartis, Novo Nordisk, Ono Pharmaceutical, Sanofi K.K., and Takeda Pharmaceutica; research grants from Daiichi Sankyo and Takeda Science Foundation; and scholarship grants from MSD and Ono Pharmaceutical. Takeshi Matsumura reported personal fees from Eli Lily Japan KK and Boehringer Ingelheim Japan Inc. and research grants from Shimazu Corporation. Tomoko Nakagami reported personal fees from Sanwa Kagaku Kenkyusho Co Ltd, Novo Nordisk Pharma Ltd. Japan, Eli Lily Japan KK, Sanofi K.K., Sumitomo Pharma, and Boehringer Ingelheim Japan Inc. Shizuya Yamashita reported personal fees from Kowa. Hiroyuki Tsutsui reported lecture fees (Kowa, Teijin Pharma, Nippon Boehringer Ingelheim, Mitsubishi Tanabe Pharma, Pfizer Japan, Ono Pharmaceutical, Daiichi Sankyo, Novartis Pharma, Bayer Yakuhin, Otsuka Pharmaceuticalm, and AstraZeneca), manuscript fees (Nippon Rinsho), research funding (Mitsubishi Tanabe Pharma, Nippon Boehringer Ingelheim, IQVIA Services Japan, MEDINET, Medical Innovation Kyushu, Kowa, Daiichi Sankyo, Johnson & Johnson, and NEC Corporation) and scholarship funds (Abbott Medical Japan, Otsuka Pharmaceutical, Boston Scientific Japan, Ono Pharmaceutical, Bayer Yakuhin, Nippon Boehringer Ingelheim, St. Mary’s Hospital, Teijin Pharma, Daiichi Sankyo, and Mitsubishi Tanabe Pharma). Tetsuo Shoji reported personal fees and research grants from Bayer Yakuhin Ltd. Other authors reported no financial conflicts of interest relevant to this study.

References

• 1)  Tsao CW, Aday AW, Almarzooq ZI, Alonso A, Beaton AZ, Bittencourt MS, Boehme AK, Buxton AE, Carson AP, Commodore-Mensah Y, Elkind MSV, Evenson KR, Eze-Nliam C, Ferguson JF, Generoso G, Ho JE, Kalani R, Khan SS, Kissela BM, Knutson KL, Levine DA, Lewis TT, Liu J, Loop MS, Ma J, Mussolino ME, Navaneethan SD, Perak AM, Poudel R, Rezk-Hanna M, Roth GA, Schroeder EB, Shah SH, Thacker EL, VanWagner LB, Virani SS, Voecks JH, Wang NY, Yaffe K and Martin SS: Heart Disease and Stroke Statistics-2022 Update: A Report From the American Heart Association. Circulation, 2022; 145: e153-e639
• 2)  Altmann SW, Davis HR, Jr., Zhu LJ, Yao X, Hoos LM, Tetzloff G, Iyer SP, Maguire M, Golovko A, Zeng M, Wang L, Murgolo N and Graziano MP: Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science, 2004; 303: 1201-1204
• 3)  Miettinen TA, Tilvis RS and Kesaniemi YA: Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population. Am J Epidemiol, 1990; 131: 20-31
• 4)  Matthan NR, Pencina M, LaRocque JM, Jacques PF, D’Agostino RB, Schaefer EJ and Lichtenstein AH: Alterations in cholesterol absorption/synthesis markers characterize Framingham offspring study participants with CHD. J Lipid Res, 2009; 50: 1927-1935
• 5)  Strandberg TE, Tilvis RS, Pitkala KH and Miettinen TA: Cholesterol and glucose metabolism and recurrent cardiovascular events among the elderly - A prospective study. J Am Coll Cardiol, 2006; 48: 708-714
• 6)  Shoji T, Akiyama Y, Fujii H, Harada-Shiba M, Ishibashi Y, Ishida T, Ishigaki Y, Kabata D, Kihara Y, Kotani K, Kurisu S, Masuda D, Matoba T, Matsuki K, Matsumura T, Mori K, Nakagami T, Nakazato M, Taniuchi S, Ueno H, Yamashita S, Yoshida H and Yoshida H: Association of Kidney Function with Serum Levels of Cholesterol Absorption and Synthesis Markers: The CACHE Study CKD Analysis. J Atheroscler Thromb, 2022; 29: 1835-1848
• 7)  Harris PA, Taylor R, Thielke R, Payne J, Gonzalez N and Conde JG: Research electronic data capture (REDCap)--a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform, 2009; 42: 377-381
• 8)  Harris PA, Taylor R, Minor BL, Elliott V, Fernandez M, O’Neal L, McLeod L, Delacqua G, Delacqua F, Kirby J, Duda SN and Consortium RE: The REDCap consortium: Building an international community of software platform partners. J Biomed Inform, 2019; 95: 103208
• 9)  American Diabetes A: Diagnosis and classification of diabetes mellitus. Diabetes Care, 2014; 37 Suppl 1: S81-90
• 10)  Committee of the Japan Diabetes Society on the Diagnostic Criteria of Diabetes M, Seino Y, Nanjo K, Tajima N, Kadowaki T, Kashiwagi A, Araki E, Ito C, Inagaki N, Iwamoto Y, Kasuga M, Hanafusa T, Haneda M and Ueki K: Report of the committee on the classification and diagnostic criteria of diabetes mellitus. J Diabetes Investig, 2010; 1: 212-228
• 11)  Kashiwagi A, Kasuga M, Araki E, Oka Y, Hanafusa T, Ito H, Tominaga M, Oikawa S, Noda M, Kawamura T, Sanke T, Namba M, Hashiramoto M, Sasahara T, Nishio Y, Kuwa K, Ueki K, Takei I, Umemoto M, Murakami M, Yamakado M, Yatomi Y, Ohashi H and Committee on the Standardization of Diabetes Mellitus-Related Laboratory Testing of Japan Diabetes S: International clinical harmonization of glycated hemoglobin in Japan: From Japan Diabetes Society to National Glycohemoglobin Standardization Program values. J Diabetes Investig, 2012; 3: 39-40
• 12)  Shimamoto K, Ando K, Fujita T, Hasebe N, Higaki J, Horiuchi M, Imai Y, Imaizumi T, Ishimitsu T, Ito M, Ito S, Itoh H, Iwao H, Kai H, Kario K, Kashihara N, Kawano Y, Kim-Mitsuyama S, Kimura G, Kohara K, Komuro I, Kumagai H, Matsuura H, Miura K, Morishita R, Naruse M, Node K, Ohya Y, Rakugi H, Saito I, Saitoh S, Shimada K, Shimosawa T, Suzuki H, Tamura K, Tanahashi N, Tsuchihashi T, Uchiyama M, Ueda S and Umemura S: The Japanese Society of Hypertension Guidelines for the Management of Hypertension (JSH 2014). Hypertens Res, 2014; 37: 253-390
• 13)  Matsuo S, Imai E, Horio M, Yasuda Y, Tomita K, Nitta K, Yamagata K, Tomino Y, Yokoyama H and Hishida A: Revised equations for estimated GFR from serum creatinine in Japan. Am J Kidney Dis, 2009; 53: 982-992
• 14)  Harada-Shiba M, Arai H, Ishigaki Y, Ishibashi S, Okamura T, Ogura M, Dobashi K, Nohara A, Bujo H, Miyauchi K, Yamashita S and Yokote K: Guidelines for Diagnosis and Treatment of Familial Hypercholesterolemia 2017. J Atheroscler Thromb, 2018; 25: 751-770
• 15)  Friedewald WT, Levy RI and Fredrickson DS: Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem, 1972; 18: 499-502
• 16)  Yoshida H, Tada H, Ito K, Kishimoto Y, Yanai H, Okamura T, Ikewaki K, Inagaki K, Shoji T, Bujo H, Miida T, Yoshida M, Kuzuya M and Yamashita S: Reference Intervals of Serum Non-Cholesterol Sterols by Gender in Healthy Japanese Individuals. J Atheroscler Thromb, 2020; 27: 409-417
• 17)  Sonoda M, Shoji T, Kimoto E, Okute Y, Shima H, Naganuma T, Motoyama K, Morioka T, Mori K, Fukumoto S, Shioi A, Koyama H, Emoto M and Inaba M: Kidney function, cholesterol absorption and remnant lipoprotein accumulation in patients with diabetes mellitus. J Atheroscler Thromb, 2014; 21: 346-354
• 18)  Buchwald H, Varco RL, Matts JP, Long JM, Fitch LL, Campbell GS, Pearce MB, Yellin AE, Edmiston WA, Smink RD, Jr. and et al.: Effect of partial ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia. Report of the Program on the Surgical Control of the Hyperlipidemias (POSCH). N Engl J Med, 1990; 323: 946-955
• 19)  Cannon CP, Blazing MA, Giugliano RP, McCagg A, White JA, Theroux P, Darius H, Lewis BS, Ophuis TO, Jukema JW, De Ferrari GM, Ruzyllo W, De Lucca P, Im K, Bohula EA, Reist C, Wiviott SD, Tershakovec AM, Musliner TA, Braunwald E, Califf RM and Investigators I-I: Ezetimibe Added to Statin Therapy after Acute Coronary Syndromes. N Engl J Med, 2015; 372: 2387-2397
• 20)  Weingartner O, Lutjohann D, Meyer S, Fuhrmann A, Cremers B, Seiler-Mussler S, Schott HF, Kerksiek A, Friedrichs S, Ulbricht U, Zawada A, Laufs U, Schulze PC, Scheller B, Fliser D, Bohm M, Sijbrands E and Heine GH: Low serum lathosterol levels associate with fatal cardiovascular disease and excess all-cause mortality: a prospective cohort study. Clin Res Cardiol, 2019; 108: 1381-1385
• 21)  Gargiulo S, Gamba P, Testa G, Leonarduzzi G and Poli G: The role of oxysterols in vascular ageing. J Physiol, 2016; 594: 2095-2113
• 22)  Song J, Wang D, Chen H, Huang X, Zhong Y, Jiang N, Chen C and Xia M: Association of Plasma 7-Ketocholesterol With Cardiovascular Outcomes and Total Mortality in Patients With Coronary Artery Disease. Circ Res, 2017; 120: 1622-1631
• 23)  Gargiulo S, Sottero B, Gamba P, Chiarpotto E, Poli G and Leonarduzzi G: Plaque oxysterols induce unbalanced up-regulation of matrix metalloproteinase-9 in macrophagic cells through redox-sensitive signaling pathways: Implications regarding the vulnerability of atherosclerotic lesions. Free Radic Biol Med, 2011; 51: 844-855
• 24)  Pedruzzi E, Guichard C, Ollivier V, Driss F, Fay M, Prunet C, Marie JC, Pouzet C, Samadi M, Elbim C, O’Dowd Y, Bens M, Vandewalle A, Gougerot-Pocidalo MA, Lizard G and Ogier-Denis E: NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol, 2004; 24: 10703-10717
• 25)  Evans TD, Sergin I, Zhang X and Razani B: Modulating Oxysterol Sensing to Control Macrophage Apoptosis and Atherosclerosis. Circ Res, 2016; 119: 1258-1261
• 26)  Sato K, Nakano K, Katsuki S, Matoba T, Osada K, Sawamura T, Sunagawa K and Egashira K: Dietary cholesterol oxidation products accelerate plaque destabilization and rupture associated with monocyte infiltration/activation via the MCP-1-CCR2 pathway in mouse brachiocephalic arteries: therapeutic effects of ezetimibe. J Atheroscler Thromb, 2012; 19: 986-998
• 27)  Takase S, Matoba T, Nakashiro S, Mukai Y, Inoue S, Oi K, Higo T, Katsuki S, Takemoto M, Suematsu N, Eshima K, Miyata K, Yamamoto M, Usui M, Sadamatsu K, Satoh S, Kadokami T, Hironaga K, Ichi I, Todaka K, Kishimoto J, Egashira K and Sunagawa K: Ezetimibe in Combination With Statins Ameliorates Endothelial Dysfunction in Coronary Arteries After Stenting: The CuVIC Trial (Effect of Cholesterol Absorption Inhibitor Usage on Target Vessel Dysfunction After Coronary Stenting), a Multicenter Randomized Controlled Trial. Arterioscler Thromb Vasc Biol, 2017; 37: 350-358
• 28)  Kubo M, Hata J, Doi Y, Tanizaki Y, Iida M and Kiyohara Y: Secular trends in the incidence of and risk factors for ischemic stroke and its subtypes in Japanese population. Circulation, 2008; 118: 2672-2678
• 29)  Virginio VW, Nunes VS, Moura FA, Menezes FH, Andreollo NA, Rogerio F, Scherrer DZ, Quintao EC, Nakandakare E, Petrucci O, Nadruz-Junior W, de Faria EC and Sposito AC: Arterial tissue and plasma concentration of enzymatic-driven oxysterols are associated with severe peripheral atherosclerotic disease and systemic inflammatory activity. Free Radic Res, 2015; 49: 199-203
• 30)  Hagiwara N, Kawada-Watanabe E, Koyanagi R, Arashi H, Yamaguchi J, Nakao K, Tobaru T, Tanaka H, Oka T, Endoh Y, Saito K, Uchida T, Matsui K and Ogawa H: Low-density lipoprotein cholesterol targeting with pitavastatin＋ezetimibe for patients with acute coronary syndrome and dyslipidaemia: the HIJ-PROPER study, a prospective, open-label, randomized trial. Eur Heart J, 2017; 38: 2264-2276