*2.3. Clinical Variables*

Serum levels of hemoglobin A1c (HbA1c), triglyceride (TG), total cholesterol (TC), LDL-C, HDL-C, and high-density lipoprotein triglyceride (HDL-TG) were measured using a standard assay at the Clinical Laboratory of Kobe University Hospital. HDL-PL

levels were assessed by measuring apoB-depleted serum diluted eight times in phosphatebuffered saline (PBS) at SRL, Inc. (Hachioji, Tokyo, Japan) and calibrated using three-fold serially diluted pooled serum.

#### *2.4. CUC Assay*

The development of the CUC assay has been described previously [6,7]. In this study, the assay principle was applied to the HI-1000TM system (Sysmex, Kobe, Japan), which is a fully automated immunoassay system for research applications. In brief, 5 μL of apoB-depleted serum was diluted in a buffer containing PBS and 0.2% R1 reagent of the HDL-C Reagent KL "kokusai" (Sysmex, Kobe, Japan) 200 times, and 10 μL of the diluted apoB-depleted serum was incubated with 90 μL of 1 μM biotin-PEG-labeled cholesterol (the preparation method is described in Appendix A) in reaction buffer (PBS containing 11% glycerol, 1.1% Pluronic F-68 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 0.11 mM methyl-β-cyclodextrin (Merck KGaA, Darmstadt, Germany), 0.055% liposome (Nippon fine chemical, Tokyo, Japan), 0.0047% nonion-K230 (NOF, Tokyo, Japan), 0.37% SF08 (NOF, Tokyo, Japan), and 0.009% oleamide (Kao, Tokyo, Japan)) at 37 ◦C for 1 min. Serum HDL was captured by an anti-apolipoprotein A1 (apoA1) mouse monoclonal antibody clone 8E10 (the preparation method is described in Appendix A) coated on magnetic particles at 37 ◦C for 6 min. After washing the particles with wash buffer (HISCLTM line washing solution containing 0.1% Pluronic F-68 and 138 mM sodium chloride), 100 μL of alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA) in dilution buffer (0.1 M TEA (pH 7.5) containing 10 mg/mL BSA, 5 mg/mL Casein Na, 1 mM MgCl2, and 0.1 mM ZnCl2) was added and incubated at 37 ◦C for 10 min. After washing the particles with wash buffer, the chemiluminescent substrate was added and incubated at 42 ◦C for 5 min, and chemiluminescence was measured as a count. The CUC assay was standardized using the pooled serum.
