*3.3. LCAT-Dependent Enhancement of CUC Is Suppressed in rHDL Containing Elaidic Acid-PC*

To investigate the effects of elaidic acid in HDL-PL on HDL size and functionality, discoidal rHDL containing various molar percentages of POPC and elaidic acid-PC (0–100% of total PC) were prepared and particle size and CUC were assessed. Native PAGE analysis showed that particle sizes did not differ significantly between rHDLs (Figure 2A).

Similarly, contrary to our expectation, the elaidic acid-PC content in rHDL did not affect CUC (Figure 2B), although these results might have been due to the limitations of CUC analysis using only rHDL.

Under physiological conditions, LCAT is known to bind discoidal small HDLs (preβ-HDL) [26,27] and is important for HDL maturation [28]. In peripheral tissues, free cholesterol effluxes from cells by the ATP-binding cassette transporter A1 (ABCA1) to preβ-HDL and is esterified by LCAT. Due to their hydrophobic chemical properties, cholesterol esters (CE) move to the core of the HDL [29], making it larger and more spherical mature. Recently, it has been reported that rhLCAT increased CE and enhanced cholesterol efflux and the maturation of HDL in vivo [30]. Therefore, we hypothesized that the addition of rhLCAT to rHDL would enable CUC analysis under near-physiological conditions.

**Figure 2.** Elaidic acid-PC contents in rHDL do not affect particle size and CUC. (**A**) rHDL containing different POPC and elaidic acid-PC molar percentages was prepared and native polyacrylamide gel electrophoresis (PAGE) analysis was performed in a 4–20% polyacrylamide gel to assess the particle size. Standard proteins of known hydrodynamic diameters were used for this analysis. Samples (1.0 μg proteins) were separated by non-denaturing gel electrophoresis and stained with Coomassie Brilliant Blue. (**B**) rHDL containing different POPC and elaidic acid-PC molar percentages was prepared and CUC assay was performed. Values are expressed as the mean ± SD (*n* = 6). CUC, cholesterol uptake capacity; A.U., arbitrary units; NS, not significant. Data analyzed by one-way ANOVA with Dunnett's multiple comparisons test.

To investigate the effects of LCAT on CUC, rHDL containing POPC was prepared and the CUC assay was performed in the presence of rhLCAT or rhLCAT pre-incubated with NEM, which inhibits LCAT activity [31–34]. The addition of rhLCAT to rHDL significantly enhanced CUC, and LCAT-dependent enhancement of CUC was suppressed by NEM (Figure 3A).

**Figure 3.** LCAT-dependent enhancement of CUC is suppressed in rHDL containing elaidic acid-PC. (**A**) rHDL containing POPC was prepared and a CUC assay was performed in the presence of rhLCAT or rhLCAT pre-incubated with NEM. Values are expressed as the mean ± SD (*n* = 6). LCAT, lecithin cholesterol acyltransferase; apoA1, apolipoprotein A1; NEM, N-ethylmaleimide; CUC, cholesterol uptake capacity; A.U., arbitrary units. \*\*\* *p* < 0.001. NS, not significant. Data analyzed by one-way ANOVA with Tukey's multiple comparisons test. (**B**) rHDL containing POPC, DOPC, and elaidic acid-PC was prepared and a CUC assay was performed in the presence of rhLCAT. Values are expressed as the mean ± SD (*n* = 6). \* *p* < 0.05, \*\* *p* < 0.01. NS, not significant. Data analyzed by one-way ANOVA with Dunnett's multiple comparisons test.

Next, to investigate the effects of elaidic acid in HDL-PL on CUC in the presence of LCAT, rHDL containing POPC, DOPC, and elaidic acid-PC were prepared and the CUC assay was performed in the presence of rhLCAT. Although PL contain saturated fatty acids mainly in the sn1 position [35–37], we used a PC containing elaidic acid in both the sn1 and sn2 positions as elaidic acid-PC. To confirm the effect of sn1 substitution by monounsaturated fatty acids, DOPC, which contains oleic acid in both the sn1 and sn2 positions, and POPC, which contains palmitic acid in the sn1 position and oleic acid in the sn2 position, were used as controls. Although rhLCAT-dependent enhancement of CUC was observed in all rHDLs, the CUC of rHDL containing elaidic acid-PC was significantly lower than that of rHDL containing POPC or DOPC (Figure 3B). These findings indicate that LCAT plays a crucial role in the enhancement of CUC, and elaidic acid has a negative effect on CUC in the presence of LCAT.

### *3.4. LCAT-Dependent Cholesterol Esterification Is Suppressed in rHDL Containing Elaidic Acid-PC*

Previous studies have shown that conversion of free cholesterol on HDL to CE by LCAT increases the capacity of HDL to remove additional cholesterol and maintains the gradient for cholesterol efflux from cells [29,30]. Therefore, we speculated that elaidic acid in HDL-PL inhibited LCAT-dependent cholesterol esterification on HDL and affected CUC. To evaluate LCAT-dependent cholesterol esterification, a fluorescence-based assay for LCAT activity was developed according to a previous study [24]. First, we prepared rHDL containing both BODIPY-cholesterol and POPC as a proteoliposome substrate and confirmed that the fluorescent signal was detected in the same size as rHDL by native PAGE analysis (Figure 4A).

**Figure 4.** Development of a fluorescence-based assay for LCAT activity. (**A**) rHDL containing both BODIPY-cholesterol and POPC was prepared and native PAGE analysis was performed in a 4–20% polyacrylamide gel. Standard proteins of known hydrodynamic diameters were used for the analysis. Samples (1.0 μg proteins) were separated by non-denaturing gel electrophoresis and stained with Coomassie Brilliant Blue (left). The same Native PAGE gel was analyzed with a ChemiDoc Touch MP (Bio-Rad) set at 488 nm for excitation and 520 nm for emission (right). (**B**) rhLCAT or rhLCAT pre-incubated with NEM was incubated with rHDL containing BODIPY-cholesterol and POPC for 10–90 min at 37 ◦C. The extracted lipids were dissolved in 30 μL of chloroform and applied to the TLC plate. The TLC plate was placed into a closed glass tank, saturated by a developing solvent (petroleum ether, diethyl ether, and acetic acid in mole portions of 230:60:3). After 25 min, the plate was removed and the cholesterol spots (Position A) and esterified cholesterol spots (Position B) were detected using a ChemiDoc Touch MP set at 488 nm for excitation and 520 nm for emission. In order to visualize spots clearly, cholesterol spots were exposed for 0.2 s and esterified cholesterol spots were exposed for 3.0 s. (**C**) The TLC plate was exposed for 0.2 s and cholesterol spots and esterified cholesterol spots were quantified by densitometry analysis using ImageJ® software. Cholesterol esterification rate was calculated as the percentage of cholesterol esterified during HDL incubation at 37 ◦C in 10 min. Values are expressed as the mean ± SD (*n* = 5). \*\* *p* < 0.01. Data analyzed by unpaired Mann–Whitney test.

Second, rhLCAT or rhLCAT pre-incubated with NEM was incubated with rHDL for 10–90 min, both BODIPY-cholesterol and esterified BODIPY-cholesterol were separated by TLC, and fluorescent signals were detected. Fluorescent intensities of esterified BODIPYcholesterol increased depending on the incubation time in the presence of rhLCAT, and this trend was suppressed by pre-incubation of rhLCAT with NEM (Figure 4B). Quantitative analysis also showed that the LCAT-dependent cholesterol esterification rate was suppressed by NEM (Figure 4C). We concluded from these results that the fluorescence activity assay for LCAT developed properly.

Finally, to evaluate the effect of elaidic acid in HDL-PL on LCAT-dependent cholesterol esterification, rHDL containing both BODIPY-cholesterol and POPC or elaidic acid-PC were prepared as proteoliposome substrates and a fluorescence activity assay for LCAT was performed. Although the fluorescent intensities of esterified BODIPY-cholesterol increased depending on the incubation time in the presence of LCAT in both rHDLs (Figure 5A), the cholesterol esterification rate of rHDL containing elaidic acid-PC was significantly lower than that of rHDL containing POPC (Figure 5B), demonstrating that elaidic acid suppresses esterification of cholesterol on HDL when incorporated into HDL-PL.

**Figure 5.** LCAT-dependent cholesterol esterification is suppressed in rHDL containing elaidic acid-PC. (**A**) rHDL containing both BODIPY-cholesterol and POPC or elaidic acid-PC were prepared and incubated with rhLCAT for 10–90 min at 37 ◦C. The extracted lipids were dissolved in 30 μL of chloroform and applied to the TLC plate. The TLC plate was placed into a closed glass tank, saturated by a developing solvent (petroleum ether, diethyl ether, and acetic acid in mole portions of 230:60:3). After 25 min, the plate was removed and the cholesterol spots (Position A) and esterified cholesterol spots (Position B) were detected using a ChemiDoc Touch MP. In order to visualize the spots clearly, cholesterol spots were exposed for 0.2 sec and esterified cholesterol spots were exposed for 3.0 sec. (**B**) The TLC plate was exposed for 0.2 sec and cholesterol spots and esterified cholesterol spots were quantified by densitometry analysis using ImageJ® software. Cholesterol esterification rate was calculated as the percentage of cholesterol esterified during HDL incubation at 37 ◦C for, 10–90 min. Values are expressed as the mean ± SD (*n* = 5). \* *p* < 0.05 \*\* *p* < 0.01. Data analyzed by unpaired Mann–Whitney test.

#### **4. Discussion**

In this study, we demonstrated that CUC, a novel indicator of HDL functionality, was inversely associated with the proportion of elaidic acid in HDL-PL. In vitro analysis using rHDL showed that rhLCAT enhanced CUC, and LCAT-dependent enhancement of CUC was suppressed in rHDL containing elaidic acid in PC compared to rHDL containing oleic acid, a cis analogue of elaidic acid. Moreover, we found that LCAT-dependent cholesterol esterification was also suppressed by elaidic acid.

PL are major components of the HDL lipidome, accounting for 40–60% of total HDL lipids, followed by cholesteryl esters (30–40%), triglycerides (5–12%), and free cholesterol (5–10%) [20]. In the present study, we found that HDL-PL levels were strongly significantly correlated with CUC, which agrees with previous studies that showed a significantly positive correlation between CEC and HDL-PL levels [38]. In vitro analysis using rHDL also showed that CEC at a fixed rHDL protein concentration increased in parallel with increasingly enriched PL [39]. Since cholesterol interacts with PL [40], the latter are indispensable components for maintaining cholesterol in lipid membranes. We believe that our results reflect the intrinsic mechanism of the affinity between PL and cholesterol.

Previously, we showed that serum elaidic acid levels were elevated in middle-aged patients with CAD and/or metabolic syndrome in Japan [13]. We also showed that elevated serum elaidic acid levels were associated with the incidence of target vessel revascularization (TLR) in the same-age Japanese generation with CAD [14]. Dietary TFA are reported to be associated with increased LDL-C and TG, as well as reduced HDL-C [41], suggesting that the adverse effects of TFA on lipoprotein quantity and function may contribute to the increase in CVD events. Nevertheless, the effects of TFA on HDL functionality have not been completely elucidated.

In this study, both CUC and HDL-PL levels were significantly higher in the Rev.(–) group than in the Rev.(+) group. Accompanied by the increase in HDL-PL levels, the elaidic acid levels in HDL-PL also tended to be higher in the Rev.(–) group than in the Rev.(+) group. However, this trend was not statistically significant. To investigate the effect of the elaidic acid composition of HDL-PL on CUC, we examined the relationship between the proportion of elaidic acid in HDL-PL (elaidic acid in HDL-PL/HDL-PL ratio) and CUC, and found a negative correlation. By contrast, oleic acid, a cis analogue of elaidic acid, showed no such relationship. These results suggest that not only the amount of PL but also the composition of PL is a factor in determining CUC, and that the increased proportion of elaidic acid in HDL-PL has a negative effect on CUC.

In the present study, we found that the addition of rhLCAT to rHDL enhanced CUC, and LCAT-dependent enhancement of CUC was suppressed in rHDL containing elaidic acid in PC when compared to rHDL containing oleic acid, a cis analogue of elaidic acid. A previous study showed that the incorporation of structurally linear elaidic acid into PL reduces the fluidity of lipid membranes [19]. Therefore, elaidic acid could reduce the surface fluidity of HDL and attenuate CUC in the presence of LCAT. Additionally, the present study showed that LCAT was less reactive to PC containing elaidic acid than PC containing oleic acid, and affected the efficiency of cholesterol esterification in rHDL. A previous study showed that cholesterol esterification contributed to HDL maturation and increased the capacity of HDL to remove cholesterol [29,30]. Therefore, elaidic acid may affect the esterification of cholesterol in addition to membrane fluidity, thereby inhibiting cholesterol uptake and maturation of HDL. Although the mechanism by which elaidic acid affects LCAT reactivity has not been fully elucidated, considering that substrates of PL need to move into the active site of LCAT from HDL through the path that is made by the interaction between LCAT and apoA1 [42], elaidic acid may decrease the surface fluidity of HDL and reduce the efficiency of providing substrates of PL to LCAT through the path.

In this study, we did not perform a detailed structural analysis to elucidate how rHDL, which contains elaidic acid-PC, undergoes structural changes upon reaction with rhLCAT. Recently, the binding mode of LCAT and HDL was analyzed using negative stain electron microscopy (EM), validated with hydrogen–deuterium exchange mass spectrometry (HDX-MS) and crosslinking coupled with mass spectrometry (XL-MS) [42]. Adaptation of these techniques for rHDL analysis may reveal more detailed effects of elaidic acid-PC on LCATdependent HDL maturation in the future.

Recently, much attention has been focused on restoring or regulating HDL function to prevent atherosclerosis. Previously, we found that EPA enhanced CEC when it was incorporated into HDL [11,12]. In the present study, we found that elaidic acid incorporated in HDL-PL negatively affected CUC. In view of these results, modulation of the PL acyl groups may be an effective strategy to improve HDL function. CUC was significantly enhanced in the presence of rhLCAT. Recently, therapeutic concepts for coronary heart disease and atherosclerosis using recombinant LCAT protein or an LCAT activator have been

proposed, and dose-dependent increases in HDL-C along with the enhancement of cholesterol efflux or in vivo reverse cholesterol transport (RCT) have been demonstrated [30,43]. Considering the enhancement of CUC in the presence of rhLCAT, as shown in this study, CUC may change in response to these molecules, in a manner similar to cholesterol efflux.

#### *Study Limitations*

This study has several limitations. First, because CUC is determined by a cell-free assay, CUC does not reflect the ABCA1 mediated cellular binding of apoA1 and the unidirectional export of cholesterol and PL to lipid-free/-poor apoA1 [7], which is considered as the first step of reverse cholesterol transport. Hence, the effect of elaidic acid in HDL-PL on cholesterol efflux remains to be elucidated. Second, we assessed rHDL containing only elaidic acid in PC for in vitro analysis. Since the concentrations of elaidic acid in vivo are much lower than those of other fatty acids, HDL containing such a highly enriched elaidic acid does not exist in vivo. However, considering the inverse association between CUC and the proportion of elaidic acid in HDL-PL observed in the correlation study using serum samples, we believe that our results reflect the intrinsic effect of elaidic acid on HDL. Further elucidation is required to address this issue. Third, although we used PC, which contains elaidic acid in both the sn1 and sn2 positions, as elaidic acid-PC, it is not consistent with a previous study that showed that PL contains saturated FA at position sn1 and unsaturated FA at position sn2 [35–37]. However, a previous study that assessed the membrane fluidity by steady-state fluorescence polarization of the probe diphenylhexatriene (DPH) showed that lipid membranes made from trans-containing PC (trans-PC) were less fluid than lipid membranes made from cis-containing PC (cis-PC), regardless of the position of incorporation (sn1 only, or both sn1 and sn2 of the glycerol backbone) [19]. Hence, we believe that the type of elaidic acid-PC used in our rHDL analysis did not affect the conclusions of this study. Fourth, we assessed rHDL containing the same amount of apoA1 for in vitro analysis. Since the interaction of LCAT to apoA1 enhances the enzymatic activity of LCAT [42], the amount of apoA1 per HDL particle and post-translational modifications of apoA1 such as nitration [44] may affect the LCAT-dependent cholesterol esterification and CUC. To address this issue, a comprehensive analysis using rHDL containing different amounts and qualities of apoA1 is needed.

Lastly, we did not assess the effects of polyunsaturated fatty acids, which may enhance lipid membrane fluidity. Further studies, such as comprehensive lipid profile assessment of HDL and analysis of rHDL composed of other types of phospholipids are needed to generalize the present findings.

#### **5. Conclusions**

The present study revealed that CUC is affected by the HDL-PL level. Moreover, CUC was negatively associated with the proportion of elaidic acid in HDL-PL, suggesting that the composition of HDL-PL is also a determinant factor of CUC. In vitro analysis using rHDL showed that CUC was positively affected by LCAT-dependent cholesterol esterification, whereas the incorporation of elaidic acid in HDL-PL attenuated the cholesterol esterification efficiency by LCAT in addition to decreasing the fluidity of the HDL surface as reported previously, thereby inhibiting the process of cholesterol uptake and maturation of HDL. Further analysis to elucidate the regulatory mechanisms of CUC will lead to new diagnostic and therapeutic strategies for atherosclerosis and cardiovascular disease.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/nu13093112/s1, Figure S1: Correlations between CUC and elaidic acid or oleic acid in HDL-PL; Table S1: Detailed laboratory data; Table S2: Detailed baseline patient characteristics.

**Author Contributions:** Conceptualization, T.I. (Takuya Iino), R.T., M.N. (Manabu Nagao), Y.I.; methodology, T.I. (Takuya Iino), M.S., A.H. and K.M., Y.I.; software, T.I. (Takuya Iino) and M.S.; validation, T.I. (Takuya Iino), R.T., M.N. (Manabu Nagao), M.S., Y.I., M.N. (Makoto Nishimori), S.Y., Y.S., T.I. (Tatsuro Ishida), and K.-i.H.; formal analysis, T.I. (Takuya Iino); investigation, T.I. (Takuya

Iino); resources, M.S.; data curation, T.I. (Takuya Iino); writing—original draft preparation, T.I. (Takuya Iino); writing—review and editing, R.T., M.N. (Manabu Nagao); visualization, T.I. (Takuya Iino); supervision, R.T., T.I. (Tatsuro Ishida) and K.-i.H.; project administration, R.T., T.I. (Tatsuro Ishida) and K.-i.H.; funding acquisition, R.T., Y.I., T.I. (Tatsuro Ishida) and K.-i.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** Institutional funding by Sysmex Corporation and Grant-in-Aid for Scientific Research (C) 19K08490 and 20K17081 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

**Institutional Review Board Statement:** 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).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The Division of Evidence-based Laboratory Medicine, Kobe University Graduate School of Medicine, was established by an endowment fund from the Sysmex Corporation. Takuya Iino, Amane Harada, Katsuhiro Murakami, Yasuhiro Irino are employees of the Sysmex Corporation.
