**1. Introduction**

High-density lipoprotein (HDL) is a multifunctional lipoprotein that protects against atherosclerosis. Although the detailed mechanisms are yet to be elucidated, a key function of HDL to protect cardiovascular events is suggested to be the efflux of cholesterol from macrophages in the arterial wall, which could be measured as cholesterol efflux capacity (CEC).

Previous studies have demonstrated a negative correlation between CEC and the probability of coronary artery disease (CAD) independent of HDL cholesterol (HDL-C) concentration [1–3]. However, since CEC assays require radiolabeled cholesterol and cultured cells and time consuming procedures [4,5], application of CEC in clinical settings

**Citation:** Iino, T.; Toh, R.; Nagao, M.; Shinohara, M.; Harada, A.; Murakami, K.; Irino, Y.; Nishimori, M.; Yoshikawa, S.; Seto, Y.; et al. Effects of Elaidic Acid on HDL Cholesterol Uptake Capacity. *Nutrients* **2021**, *13*, 3112. https:// doi.org/10.3390/nu13093112

Academic Editor: Hayato Tada

Received: 27 July 2021 Accepted: 1 September 2021 Published: 4 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

is challenging. To overcome the technical limitations related to CEC, we recently established a simple, high-throughput, cell-free assay system to evaluate cholesterol uptake capacity (CUC) as a novel concept for HDL functionality. We have reported an inverse association between CUC and the recurrence rate of coronary lesions after revascularization in patients with optimal control of low-density lipoprotein cholesterol (LDL-C) concentrations [6,7]. However, the regulatory mechanism of CUC remains unclear.

Several studies have shown that the ability of HDL to accept cellular free cholesterol is related to the amount of phospholipids (PL) present in the particle [8,9], and that PL containing unsaturated fatty acids in their acyl groups increase the fluidity of the HDL surface and improve cholesterol efflux when incorporated into HDL [10]. In addition, we recently reported that oral administration of purified eicosapentaenoic acid (EPA) generates EPA-rich HDL particles, which exhibit cardioprotective properties via the production of anti-inflammatory lipid metabolites and an increase in cholesterol efflux [11,12]. These results indicate the importance of acyl groups of PL in HDL functionality.

Trans-fatty acids (TFA) are unsaturated fatty acids with at least one unsaturated double bond in the trans structure, whose excess intake is considered to be associated with an increased risk of cardiovascular disease (CVD) [13–17]. Previous studies have shown that TFA taken orally are incorporated into PL in plasma [18], where they reduce the fluidity of lipid membranes [19]. Considering that PL are the major lipid component of HDL [20], these results indicate the possibility that TFA are incorporated into the PL of HDL and affect its functionality. However, the relationship between CUC and TFA incorporated into HDL phospholipids (HDL-PL) has not yet been investigated. Therefore, the present study aimed to clarify the effect of TFA in HDL-PL on CUC.

#### **2. Materials and Methods**

#### *2.1. Subjects*

The Kobe Cardiovascular Marker Investigation (CMI) registry is a single-center registry of patients referred to Kobe University Hospital with cardiovascular disease, which is used to identify blood-based biomarkers that are useful in predicting cardiovascular disease. The study protocol was in accordance with the ethical guidelines of the 1975 Declaration of Helsinki. The study was approved by the Ethics Review Committee at Kobe University (Japan) and was registered in the UMIN Clinical Trials Registry (identification number 000030297). Written informed consent was obtained from all patients prior to enrollment in the study.

Serum samples were collected from patients who underwent coronary angiography (CAG) or percutaneous coronary intervention (PCI) and stored at 80 ◦C until measurement. The inclusion criteria for this study were patients with a history of PCI and follow-up CAG with or without revascularization between July 2015 and February 2019. Exclusion criteria were patients who did not have frozen serum samples for any reason.

#### *2.2. Preparation of the apoB-Depleted Serum*

Serum samples were thawed on ice and incubated with the same volume of 22% polyethylene glycol (PEG) 4000 to remove apolipoprotein B (apoB)-containing lipoproteins. Briefly, each serum sample was mixed with a PEG solution and kept at room temperature for 20 min. The samples were then centrifuged at 860× *g* for 15 min to precipitate all apoB-containing lipoproteins, and the supernatant was collected as apoB-depleted serum. A previous study that used gel filtration chromatography showed that cholesterol and PL colocalized in the same fraction as HDL in apoB-depleted serum [21]. Therefore, we used apoB-depleted serum for the HDL-PL analysis.
