Direct measurement of high-density lipoprotein cholesterol in serum with polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin

Abstract

We have developed an automated method for measuring high-density lipoprotein (HDL)-cholesterol in serum without prior separation, using polyethylene glycol (PEG)-modified enzymes and sulfated alpha-cyclodextrin. When cholesterol esterase and cholesterol oxidase enzymes were modified with PEG, they showed selective catalytic activities towards lipoprotein fractions, with the reactivity increasing in the order: low-density lipoprotein < very-low-density lipoprotein approximately chylomicron < HDL. In the presence of magnesium ions, alpha-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and very-low-density lipoprotein, without the need for precipitation of those lipoprotein fractions. The combination of PEG-modified enzymes with alpha-cyclodextrin sulfate provided selectivity for the determination of HDL-cholesterol in serum in the presence of a small amount of dextran sulfate without the need for precipitation of lipoprotein aggregates. The results of the HDL-cholesterol assayed in serum by this direct method correlated well with those obtained by precipitation-based methods and also that by an ultracentrifugation method.

Lipid profile and long‐term outcome in premature myocardial infarction

“…Total cholesterol (TC), HDL cholesterol (HDL-C) and triglycerides (Tg) were measured enzymatically as described previously. [17][18][19] LDL cholesterol (LDL-C) was calculated according to the Friedewald formula. In cases of severe hypertriglyceridaemia (Tg ≥ 400 mg/dL), LDL-C was measured directly.…”

Multiple bioimaging modalities in evaluation of an experimental osteonecrosis induced by a combination of lipopolysaccharide and methylprednisolone

“…Half of the plasma was then stored at −70°C for evaluating pre-thrombotic status, including t-PA/PAI-I (ratio of tissue type plasminogen activator to plasminogen activator inhibitor) by enzyme-linked immunosorbent assay technique using corresponding mouse monoclonal anti-human antibodies (Manufacturer's Datasheet, XITANG Biotechnology, Shanghai, China) and APTT (activated partial thromboplatin time) by an automatic blood coagulation analysis device (SysmexCA-50, Japan). The other half was stored at − 70°C for evaluating lipid transportation, including LDL/HDL (ratio of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol) [12,13].…”

New homogeneous HDL‐cholesterol assay without the influence of high TG sample using the selective detergent to lipoproteins

“…One was a conventional reagent kit (Lot No. 304AAK: Kyowa Medex Co., Ltd., Osaka, Japan) that selectively measured only HDL-cholesterol using polyethylene glycol and cyclodextrin sulfate together with chemically modified cholesterol oxidase and esterase, as described previously (5). The other was the new reagent kit (Lot No.…”

“…Conventionally, it had been measured by the UC method or by the precipitation method. However, homogeneous HDL-cholesterol assay without pretreatment has recently been developed and has been rapidly popularized (4)(5)(6)(7)(8)(9). Several homogeneous HDL-cholesterol assays, based on different principles, are available commercially, but they all give inaccurate results when high-TG samples are tested (10,11).…”