high-density-lipoprotein-specific-phospholipid-efflux-assay

High-Density Lipoprotein-Specific Phospholipid Efflux Assay

Plasma levels of high-density lipoprotein (HDL)-cholesterol (HDL-C) is currently a key metric for the clinical assessment of cardiovascular disease (CVD) ...

National Institutes of Health

Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.

Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo.

We describe simple, sensitive and robust methods to monitor lipoprotein remodeling and cholesterol and apolipoprotein exchange, using fluorescent Lissamine Rhodamine B head-group tagged phosphatidylethanolamine (*PE) as a lipoprotein reference marker. Fluorescent Bodipy cholesterol (*Chol) and *PE directly incorporated into whole plasma lipoproteins in proportion to lipoprotein cholesterol and phospholipid mass, respectively. *Chol, but not *PE, passively exchanged between isolated plasma lipoproteins. Fluorescent apoA-I (*apoA-I) specifically bound to high-density lipoprotein (HDL) and remodeled *PE- and *Chol-labeled synthetic lipoprotein-X multilamellar vesicles (MLV) into a pre-β HDL-like particle containing *PE, *Chol, and *apoA-I. Fluorescent MLV-derived *PE specifically incorporated into plasma HDL, whereas MLV-derived *Chol incorporation into plasma lipoproteins was similar to direct *Chol incorporation, consistent with apoA-I-mediated remodeling of fluorescent MLV to HDL with concomitant exchange of *Chol between lipoproteins. Based on these findings, we developed a model system to study lipid transfer by depositing fluorescent *PE and *Chol-labeled on calcium silicate hydrate crystals, forming dense lipid-coated donor particles that are readily separated from acceptor lipoprotein particles by low-speed centrifugation. Transfer of *PE from donor particles to mouse plasma lipoproteins was shown to be HDL-specific and apoA-I-dependent. Transfer of donor particle *PE and *Chol to HDL in whole human plasma was highly correlated. Taken together, these studies suggest that cell-free *PE efflux monitors apoA-I functionality.

ApoA-I-Mediated Lipoprotein Remodeling Monitored with a Fluorescent Phospholipid.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating low-density lipoprotein levels in plasma. While PCSK9 variants are causatively associated with familial hypercholesterolemia (FH), additional genotyping methods for FH targeting PCSK9 variants are required in a clinical setting. Loop-mediated isothermal amplification (LAMP) is a unique amplification method that amplifies a target gene under isothermal conditions (60-65 °C). However, a robust standardized method has not yet been established for LAMP-based genetic screening tests for genetic diseases, including FH. The present study aimed to develop a novel modification of the LAMP method designed to genotype single nucleotide variants (SNVs) and to apply it for the detection of PCSK9 variants.

A novel loop-mediated isothermal amplification-based genotyping method and its application for identifying proprotein convertase subtilisin/kexin type 9 variants in familial hypercholesterolemia.

MicroRNAs (miRNAs) are potential clinical biomarkers for the detection of various diseases. However, their quantification has not been implemented in clinical practice given the inconsistencies in their variable recovery rate and accuracy of the results. Thus, we utilized a technique based on bioluminescent enzyme immunoassay (BLEIA) to perform fully automated miRNA quantification using magnetic particles conjugated with antibodies targeting DNA-RNA hybrids, biotinylated DNA probes specific to miRNAs, and firefly luciferase-labeled streptavidin. This method enabled direct use of diluted serum and automation of all processes within 1 hr. The results revealed a wide linear range between 10 fmol/L and 1 nmol/L, high sensitivity with a detection limit of 6.3 fmol/L or below, high specificity with a false positive rate under 2.4%, and high reproducibility in intra- and inter-experimental observations (under CV 10% and r = 0.9965, respectively). Furthermore, a significant correlation was revealed between quantitative reverse transcription-polymerase chain reaction and BLEIA assay for the quantification of synthetic miRNA (r = 0.9993) and endogenous miRNA in healthy serums (r = 0.8203), respectively. Overall, we developed a fully automated miRNA quantification method based on BLEIA, which can be adopted in a clinical setting. However, further studies are needed to assess its clinical performance.

Fully automated microRNA quantification technique based on bioluminescent enzyme immunoassay.

Familial hypercholesterolemia (FH) is characterized by elevated levels of low-density lipoprotein cholesterol (LDL-C) and cardiovascular disease (CVD). Although the role of LDL-C in FH has been studied, the contribution of high-density lipoproteins (HDL) to CVD in FH remains unknown. This study aimed at highlighting the role of HDL in FH.

Article	Year
Plasma lipoprotein-X quantification on filipin-stained gels: monitoring recombinant LCAT treatment ex vivo. Journal of lipid research\| PubMed ID: 30808683	2019
ApoA-I-Mediated Lipoprotein Remodeling Monitored with a Fluorescent Phospholipid. Biology\| PubMed ID: 31336888	2019
A novel loop-mediated isothermal amplification-based genotyping method and its application for identifying proprotein convertase subtilisin/kexin type 9 variants in familial hypercholesterol... Biochimica et biophysica acta. General subjects\| PubMed ID: 34848321	2022
Fully automated microRNA quantification technique based on bioluminescent enzyme immunoassay. Analytical biochemistry\| PubMed ID: 36063916	2022
High-Density Lipoprotein-Specific Phospholipid Efflux in Familial Hypercholesterolemia. Annals of clinical and laboratory science\| PubMed ID: 39048165	2024

Masaki Sato

Biography

JoVE Journal Publications

Other Publications