Background: Recent studies have shown that the cholesterol efflux capacity of HDL is a better predictor of cardiovascular disease (CVD) than HDL cholesterol. However, the standard procedures used for measuring cholesterol efflux capacity involve radioisotope-labeled cholesterol and cultured macrophages. Thus, a simpler method to measure HDL functionality is needed for clinical application. Methods: We established a cell-free assay system to evaluate the capacity of HDL to accept additional cholesterol, which we named cholesterol “uptake capacity,” using fluorescently labeled cholesterol and an anti-apolipoprotein A1 antibody. We quantified cholesterol uptake capacity of apolipoprotein B (apoB)-depleted serum samples from patients with coronary artery disease who had previously undergone revascularization. Results: This assay system exhibited high reproducibility (CV <10%) and a short processing time (<6 h). The myeloperoxidase-mediated oxidation of apoB-depleted serum impaired cholesterol uptake capacity. Cholesterol uptake capacity correlated significantly with cholesterol efflux capacity (r² = 0.47, n = 30). Furthermore, cholesterol uptake capacity correlated inversely with the requirement for revascularization because of recurrence of coronary lesions in patients with optimal control of LDL cholesterol (P < 0.01, n = 156). A multivariate analysis adjusted for traditional coronary risk factors showed that only cholesterol uptake capacity remained significant (odds ratio, 0.48; 95% CI, 0.29–0.80; P = 0.0048). Conclusions: Cholesterol uptake capacity assay evaluates the functionality of HDL in a sensitive and high-throughput manner without using radioisotope label and cells. This assay system could be used for the assessment of CVD risk in the clinical settings.

High-density lipoprotein (HDL) cholesterol efflux capacity (CEC), which is a conventional metric of HDL function, has been associated with coronary heart disease risk. However, the CEC assay requires cultured cells and takes several days to perform. We previously established a cell-free assay to evaluate cholesterol uptake capacity (CUC) as a novel measure of HDL functionality and demonstrated its utility in coronary risk stratification. To apply this concept clinically, we developed a rapid and sensitive assay system based on a chemiluminescent magnetic particle immunoassay. The system is fully automated, providing high reproducibility. Measurement of CUC in serum is completed within 20 min per sample without HDL isolation, a notably higher throughput than that of the conventional CEC assay. CUC decreased with myeloperoxidase-mediated oxidation of HDL or in the presence of N-ethylmaleimide, an inhibitor of lecithin: cholesterol acyltransferase (LCAT), whereas CUC was enhanced by the addition of recombinant LCAT. Furthermore, CUC correlated with CEC even after being normalized by ApoA1 concentration and was significantly associated with the requirement for revascularization due to the recurrence of coronary lesions. Therefore, our new assay system shows potential for the accurate measurement of CUC in serum and permits assessing cardiovascular health.

Recently we established a cell-free assay to evaluate “cholesterol uptake capacity (CUC)” as a novel concept for high-density lipoprotein (HDL) functionality and demonstrated the feasibility of CUC for coronary risk stratification, although its regulatory mechanism remains unclear. HDL fluidity affects cholesterol efflux, and trans fatty acids (TFA) reduce lipid membrane fluidity when incorporated into phospholipids (PL). This study aimed to clarify the effect of TFA in HDL-PL on CUC. Serum was collected from 264 patients after coronary angiography or percutaneous coronary intervention to measure CUC and elaidic acid levels in HDL-PL, and in vitro analysis using reconstituted HDL (rHDL) was used to determine the HDL-PL mechanism affecting CUC. CUC was positively associated with HDL-PL levels but negatively associated with the proportion of elaidic acid in HDL-PL (elaidic acid in HDL-PL/HDL-PL ratio). Increased elaidic acid-phosphatidylcholine (PC) content in rHDL exhibited no change in particle size or CUC compared to rHDL containing oleic acid in PC. Recombinant human lecithin-cholesterol acyltransferase (LCAT) enhanced CUC, and LCAT-dependent enhancement of CUC and LCAT-dependent cholesterol esterification were suppressed in rHDL containing elaidic acid in PC. Therefore, CUC is affected by HDL-PL concentration, HDL-PL acyl group composition, and LCAT-dependent cholesterol esterification. Elaidic acid precipitated an inhibition of cholesterol uptake and maturation of HDL; therefore, modulation of HDL-PL acyl groups could improve CUC.

Background/Objectives: Menstrual blood, a periodic uterine discharge, represents a non-invasive source for an indication of the functional status of the endometrium. While menstrual blood-derived stem cells have been extensively characterized and menstrual blood is considered a diagnostic material for the analysis of gynecologic pathology in research studies, it is not routinely used in clinical settings. To develop novel noninvasive diagnostic tools for endometrial status assessment, we aimed to characterize the morphological and molecular markers of menstrual blood. Methods: Menstrual blood samples were obtained from healthy volunteers and characterized macroscopically and microscopically using smears (May-Grunwald-Giemsa staining), confocal microscopy, and imaging flow cytometry (cluster of differentiation [CD]90, CD45, fibrin). Clot dissociation was performed to analyze the cellular composition of clots. Results: We morphologically characterized menstrual blood cells and identified three uterine-derived cells and cell cluster types (endometrial stromal, endometrial epithelial, and vaginal epithelial). Additionally, we confirmed the specificity of CD90 for endometrial stromal cell populations, which were separately characterized in the supernatant and menstrual blood clots using light and confocal microscopy, and we analyzed the composition of the menstrual blood supernatant and dissociated clots using imaging flow cytometry. Conclusions: The results of this study may serve as a foundation for the development of new non-invasive diagnostic tools for endometrial pathology for the potential support or replacement of highly invasive procedures, such as diagnostic dilation and curettage.

Vascular calcification in atherosclerosis is a key process underlying cardiovascular disease (CVD) and chronic kidney disease (CKD), strongly predicting acute cardiovascular events. Macrocalcification stabilizes plaques, whereas microcalcification destabilizes them, making the assessment of active microcalcifications important for predicting plaque rupture. Although imaging methods such as ¹⁸F-NaF positron emission tomography can detect active microcalcifications, their clinical utility is limited by cost and accessibility. Therefore, there is a potential need for simple, minimally invasive blood biomarkers to assess microcalcification activity. Recent studies have reported that calcified extracellular vesicles (EVs) are crucial mediators of microcalcification and we hypothesized that asymptomatic plaque rupture may lead to the release of calcified EVs. In this study, we developed a fully automated immunoassay to quantify tissue-nonspecific alkaline phosphatase (TNAP) activity on calcified EVs in serum as a new biomarker of vascular microcalcification. The assay demonstrated high reproducibility and rapid turnaround (17 min), requiring only 25 μL of serum. It successfully detected calcified EVs in the culture media of primary human aortic smooth muscle cells that differentiated into osteoblast-like cells under high-phosphorus conditions, as well as in suspensions of their ultracentrifugation-derived precipitates. Feasibility testing revealed increased TNAP activity on calcified EVs in the serum of patients with a history of coronary artery disease and CKD compared with that in non-disease controls, supporting its potential as a biomarker. This study suggested that our assay could serve as a standardized tool for evaluating active microcalcification and asymptomatic plaque rupture, potentially enabling early detection of high-risk patients and informing therapeutic development.