Characterization of novel truncated apolipoprotein A-I in human high-density lipoprotein generated by sequential treatment with myeloperoxidase and chymase.

High-density lipoprotein (HDL) cholesterol is a well-known biomarker, which has been associated with reduction in the risk of cardiovascular diseases (CVD). However, some HDL anti-atherosclerotic functions may be impaired without altered HDL-cholesterol (HDL-C) level via its dysfunctional proteins or other physiological reactions in vivo. We previously showed that activated mast cell-derived chymase could modestly cleave apolipoprotein A-I (apoA-I) in HDL3, and further easily cleave lipid-free apoA-I. In contrast, myeloperoxidase (MPO) secreted by macrophages, the main cell type in atherosclerotic plaques, could oxidize HDL proteins, which might modify their tertiary structures, increasing their susceptibility to other enzymes. Here we focused on the co-modification and impact of chymase and MPO, usually secreted during inflammation from cells with possible co-existence in atheromas, on HDL. Only after sequential treatment with MPO and then chymase, two novel truncated apoA-I fragments were generated from HDL. One fragment was 16.5 kDa, and the cleavage site by chymase after MPO modification was the C-terminal of Tyr100 in apoA-I, cross-validated by three different mass spectrometry methods. This novel apoA-I fragment can be trapped in HDL particles to avoid kidney glomerular filtration and has a specific site for antibody generation for ELISA tests. As such, its quantification can be useful in predicting patients with CVD having normal HDL-C levels.

Variation in presepsin and thrombomodulin levels for predicting COVID-19 mortality.

Coronavirus disease (COVID-19) has caused extensive mortality globally; therefore, biomarkers predicting the severity and prognosis of COVID-19 are essential. This study aimed to evaluate the application of presepsin (P-SEP) and thrombomodulin (TM), which are biomarkers of sepsis and endothelial dysfunction, respectively, in the prognosis of COVID-19. Serum P-SEP and TM levels from COVID-19 patients (n = 183) were measured. Disease severity was classified as mild, moderate I, moderate II, or severe based on hemoglobin oxygen saturation and the history of intensive care unit transfer or use of ventilation at admission. Patients in the severe group were further divided into survivors and non-survivors. P-SEP and TM levels were significantly higher in the severe group than those in the mild group, even after adjusting for creatinine values. In addition, TM levels were significantly higher in non-survivors than in survivors. Changes in the P-SEP levels at two time points with an interval of 4.1 ± 2.2 days were significantly different between the survivors and non-survivors. In conclusion, TM and continuous P-SEP measurements may be useful for predicting mortality in patients with COVID-19. Moreover, our data indicate that P-SEP and TM values after creatinine adjustment could be independent predictive markers, apart from renal function.

Decreased rate of blood donors with high ABO antibody titers in Japan and the underlying factors: Comparisons between 2010 and 2021.

BACKGROUND AND OBJECTIVES: Previously (2007), it was reported that ABO antibody titers in Japanese blood donors had decreased significantly compared to 20 years before. Here we evaluated whether further decrease of antibody titers had occurred in recent years, and the potential factors associated with changes in antibody titers. MATERIALS AND METHODS: Serum/plasma from random blood donors in 2010 and 2021 (2010: 3369, 2021: 5796 donors) was classified into low, middle, and high ABO antibody titers according to the reactivity of diluted serum/plasma (2.5-fold and 20-fold) by an automated microplate system. The rates of low/high titer in the two periods were compared. Logistic regression and age-gender-BMI subgroup analyses were conducted to identify the factors that contributed to changes in antibody titers. RESULTS: Compared to 2010, the rate of donors with high ABO antibody titers was　decreased in 2021 for both anti-A and anti-B (anti-A, 2010: 23.8%, 2021: 19.3%; anti-B, 2010: 23.8%, 2021: 16.4%). In logistic regression analysis, age was found to significantly affect both anti-A and anti-B antibody titers (anti-A, adjusted odds ratio 0.36, 95% CI 0.31-0.41; anti-B, 0.42, 0.37-0.47), and BMI (0.82, 0.73-0.92) and other time-related factors (0.79, 0.71-0.88) significantly affect anti-B antibody titers. Subgroup analysis revealed decreased rate of high anti-B titers in the higher age group in 2021. CONCLUSION: The rate of high ABO antibody titers, especially high anti-B titers, was significantly decreased in 2021, and our results suggested an association with aging and obesity of blood donors as well as other time-related factors.

Improvement in bilirubin influence on cholesterol efflux capacity evaluation using the immobilized liposome-bound gel beads method.

INTRODUCTION: High-density lipoprotein (HDL) has a cholesterol efflux capacity (CEC) that protects against atherosclerosis. Recently, we developed an assay for CEC evaluation, named the immobilized liposome-bound gel beads (ILG) method, which is a highly accurate, simple, and safe method for CEC evaluation because it uses liposomes and BODIPY-labeled cholesterol instead of cultured cells and radioactive substances, respectively. Although the ILG method can be implemented in clinical settings, our previous study revealed that bilirubin causes a positive error in the CEC value. Therefore, in the present study, we attempted to improve the influence of bilirubin levels on the ILG method. METHODS: To investigate why bilirubin caused a positive error in CEC values when using the ILG method, 3D fluorescence spectra of BODIPY-labeled cholesterol and bilirubin were measured. To avoid the fluorescence emitted by bilirubin, CEC was measured using the ILG method with shifting of excitation wavelength for BODIPY-labeled cholesterol quantification. In addition, we used bilirubin oxidase to oxidize bilirubin during the incubation time of the ILG method to weaken bilirubin fluorescence. RESULTS: We found that bilirubin emitted fluorescence at the measurement setting of the ILG method. By shifting the excitation wavelength, the positive error caused by bilirubin was improved by approximately 70%. Furthermore, by utilizing bilirubin oxidase, the false-high values of CEC were improved by approximately 80%. CONCLUSIONS: Bilirubin interferes with CEC assay using BODIPY-cholesterol, but we successfully improved the influence of bilirubin on CEC evaluation using the ILG method. These improvements will promote the clinical application of the ILG method.

Development and validation of novel automatable assay for cholesterol efflux capacity.

During the past decade, evaluation of high-density lipoprotein (HDL) functionality has been well studied for predicting cardiovascular disease (CVD) risk. Cholesterol efflux capacity (CEC) is the strongest candidate as the biomarker out of various HDL antiatherosclerotic functions. However, CEC has not yet been introduced clinically because of several technical issues, including the use of radioactive materials and differentiated cells in the assay. Previously, our laboratory developed a radioisotope- and cell-free CEC assay called the immobilized liposome-bound gel beads (ILGs) method to replace the conventional method. However, the separation process of the supernatant was not suitable for installation in an automatic analyzer. The present study aims to develop a new method that is easier to operate. We assumed that the use of magnetic beads instead of gel beads would enable the skip of the centrifugal process. First, similar to the ILG method, porous magnetic beads were treated with liposomes containing fluorescently labeled cholesterol. Fluorescence was observed inside the magnetic beads, and almost the same amount of liposomes as in the ILG method was immobilized successfully. These immobilized liposome-bound magnetic beads (ILMs) were available for CEC assay when HDL and apolipoprotein B-100-depleted serum (BDS) were used as cholesterol acceptors. The ILM method showed sufficient basic performance and a good correlation with the ILG method. Furthermore, when the CEC of 15 serum samples from healthy subjects was measured, a good correlation between HDL-cholesterol level and the ILG method was confirmed. Thus, it was confirmed that the ILM method was successfully developed and could be automated.

Development of internal standard for lipoprotein subclass analysis using dual detection gel-permeation high-performance liquid chromatography system.

The LipoSEARCH® System is an innovative lipoprotein class analysis method based on gel-permeation high-performance liquid chromatography (HPLC). This system uses a gel permeation column to separate the major lipoprotein subclasses (chylomicron, very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein) in serum according to particle size and splits them into two pathways to measure total cholesterol (TC; esterified + unesterified cholesterol) and triglyceride (TG) concentrations simultaneously to obtain chromatograms for each. These chromatograms were analyzed based on the results of the calibration serum by fitting Gaussian curves to profile the 20 lipoprotein subclasses defined in detail. An important assumption of this HPLC system is its simultaneous detection of two pathways to guarantee the accuracy of each analysis. Therefore, in the present study, we investigated the development of an internal standard that can guarantee the simultaneous detection of this system by adding a pigment to the serum. We focused on quinone pigments with absorption at 550 nm, which is the wavelength used for the enzymatic assay of TC and TG concentrations in the system. As a result, we succeeded in producing overlapping pigment peaks that appeared after the analytical chromatograms in two pathways. It is also suggested that the pigment solution as an internal standard is stable in freezing storage and has little effect on the analysis. The developed internal standard is expected to contribute to the accuracy assurance of lipoprotein analysis by this dual-detection HPLC system.

Effect of myeloperoxidase oxidation and N-homocysteinylation of high-density lipoprotein on endothelial repair function.

Endothelial cell (EC) migration is essential for healing vascular injuries. Previous studies suggest that high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I), the major protein constituent of HDL, have endothelial healing functions. In cardiovascular disease, HDL is modified by myeloperoxidase (MPO) and N-homocysteine, resulting in apoA-I/apoA-II heterodimer and N-homocysteinylated (N-Hcy) apoA-I formation. This study investigated whether these modifications attenuate HDL-mediated endothelial healing. Wound healing assays were performed to analyze the effect of MPO-oxidized HDL and N-Hcy HDL in vitro. HDL obtained from patients with varying troponin I levels were also examined. MPO-oxidized HDL reduces EC migration compared to normal HDL in vitro, and N-Hcy HDL showed a decreasing trend toward EC migration. EC migration after treatment with HDL from patients was decreased compared to HDL isolated from healthy controls. Increased apoA-I/apoA-II heterodimer and N-Hcy apoA-I levels were also detected in HDL from patients. Wound healing cell migration was significantly negatively correlated with the ratio of apoA-I/apoA-II heterodimer to total apoA-II and N-Hcy apoA-I to total apoA-I. MPO-oxidized HDL containing apoA-I/apoA-II heterodimers had a weaker endothelial healing function than did normal HDL. These results indicate that MPO-oxidized HDL and N-Hcy HDL play a key role in the pathogenesis of cardiovascular disease.

Novel cholesterol efflux assay using immobilized liposome-bound gel beads: Confirmation and improvement for application in clinical laboratory.

OBJECTIVES: Cholesterol efflux capacity (CEC), an atheroprotective function of high-density lipoprotein, is expected to be a potential biomarker for cardiovascular disease. However, CEC has not been widely introduced for application in clinical laboratories because of the complexity of the conventional CEC assay using cells and radioactive materials. Previously, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILG), which solves these issues. We aimed to confirm the validation and further improve the ILG method for application in the clinical setting. METHODS: Cholesterol efflux capacity values by the ILG method assayed for shorter incubation time (4 h) were compared to those assayed for 16 h (our previous ILG method). To investigate a reference material that can correct the variation between ILG manufacturing lots, bovine serum albumin, human gamma-globulins, and globulin complexes were evaluated. CEC values were also estimated in plasmas obtained with different anticoagulants, serum treated with freeze-thaw cycles, and serum mixed with several interference substances. RESULTS: The CEC of 4- and 16-h incubation times were well correlated. Globulin complexes may be used as a reference material. Plasma can be used as the specimen. The serum and stored temperature of the specimen did not largely affect CEC. Hemoglobin and chyle did not have an effect on CEC, whereas high-bilirubin serum showed elevated CEC. The effect of bilirubin was nearly canceled by subtracting basal fluorescence intensity. CONCLUSIONS: Present ILG method further fulfills some requirements for application in clinical laboratory. Using this reliable simple method, evaluation for clinical significance of CEC is expected.

Apolipoprotein C-II and C-III preferably transfer to both high-density lipoprotein (HDL)2 and the larger HDL3 from very low-density lipoprotein (VLDL).

Triglyceride hydrolysis by lipoprotein lipase (LPL), regulated by apolipoproteins C-II (apoC-II) and C-III (apoC-III), is essential for maintaining normal lipid homeostasis. During triglyceride lipolysis, the apoCs are known to be transferred from very low-density lipoprotein (VLDL) to high-density lipoprotein (HDL), but the detailed mechanisms of this transfer remain unclear. In this study, we investigated the extent of the apoC transfers and their distribution in HDL subfractions, HDL2 and HDL3. Each HDL subfraction was incubated with VLDL or biotin-labeled VLDL, and apolipoproteins and lipids in the re-isolated HDL were quantified using western blotting and high-performance liquid chromatography (HPLC). In consequence, incubation with VLDL showed the increase of net amount of apoC-II and apoC-III in the HDL. HPLC analysis revealed that the biotin-labeled apolipoproteins, including apoCs and apolipoprotein E, were preferably transferred to the larger HDL3. No effect of cholesteryl ester transfer protein inhibitor on the apoC transfers was observed. Quantification of apoCs levels in HDL2 and HDL3 from healthy subjects (n = 8) showed large individual differences between apoC-II and apoC-III levels. These results suggest that both apoC-II and apoC-III transfer disproportionately from VLDL to HDL2 and the larger HDL3, and these transfers might be involved in individual triglyceride metabolism.

Marked Changes in Serum Amyloid A Distribution and High-Density Lipoprotein Structure during Acute Inflammation.

High-density lipoprotein- (HDL-) cholesterol measurements are generally used in the diagnosis of cardiovascular diseases. However, HDL is a complicated heterogeneous lipoprotein, and furthermore, it can be converted into dysfunctional forms during pathological conditions including inflammation. Therefore, qualitative analysis of pathophysiologically diversified HDL forms is important. A recent study demonstrated that serum amyloid A (SAA) can remodel HDL and induce atherosclerosis not only over long periods of time, such as during chronic inflammation, but also over shorter periods. However, few studies have investigated rapid HDL remodeling. In this study, we analyzed HDL samples from patients undergoing orthopedic surgery inducing acute inflammation. We enrolled 13 otherwise healthy patients who underwent orthopedic surgery. Plasma samples were obtained on preoperative day and postoperative days (POD) 1-7. SAA, apolipoprotein A-I (apoA-I), and apolipoprotein A-II (apoA-II) levels in the isolated HDL were determined. HDL particle size, surface charge, and SAA and apoA-I distributions were also analyzed. In every patient, plasma SAA levels peaked on POD3. Consistently, the HDL apoA-I : apoA-II ratio markedly decreased at this timepoint. Native-polyacrylamide gel electrophoresis and high-performance liquid chromatography revealed the loss of small HDL particles during acute inflammation. Furthermore, HDL had a decreased negative surface charge on POD3 compared to the other timepoints. All changes observed were SAA-dependent. SAA-dependent rapid changes in HDL size and surface charge were observed after orthopedic surgery. These changes might affect the atheroprotective functions of HDL, and its analysis can be available for the qualitative HDL assessment.