A homogeneous assay to determine high-density lipoprotein subclass cholesterol in serum.

Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL2-C and HDL3-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL2, HDL3, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL3-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL2, LDL, and VLDL increased, but the size of HDL3 fraction particles remained constant, allowing only HDL3-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.

An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine.

Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 µl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice.

Remnant lipoprotein cholesterol homogenous assay (RemL-C) is closely associated with very-low-density lipoprotein remnants: comparison with the immunoseparation assay.

BACKGROUND: Remnant-like particle-cholesterol (RLP-C) is recognized as a risk factor for cardiovascular disease. As an alternative to the immunoseparation assay widely used for the measurement of RLP-C, a new remnant lipoprotein-C homogenous assay (RemL-C) is available. In light of its homogeneity as an assay method, we speculated that this homogeneous assay (RemL-C) is closely associated with very-low-density lipoprotein(VLDL) remnant including intermediate-density lipoprotein(IDL). We examined the characteristics of the homogeneous assay for reacting with VLDL remnants. METHODS AND RESULTS: VLDL1, VLDL2, and IDL were separated by ultracentrifugation in the fasting serum of subjects including hypertriglyceridemia and uremic patients usually having higher levels of remnants. While RemL-C and RLP-C were mainly recovered in VLDL1 and both assays were strongly correlated with serum TG and VLDL1, the RemL-C assay was more closely correlated with VLDL2 and IDL levels than the RLP-C assay. RemL-C levels were significantly correlated with IDL-C, whereas RLP-C levels had only borderline associations with IDL-C (r= 0.56 Vs. 0.31). CONCLUSIONS: The remnant lipoprotein cholesterol homogenous assay is more closely associated with VLDL2 and IDL than the immunoseparation assay.

Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10.

A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55 degrees C, respectively. The enzyme acted on 3beta-hydroxysteroids such as cholesterol, pregnenolone, and beta-sitosterol at high rates, but on dehydro-epi-androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (K(m)=30 microM).

A new method of measuring the antioxidant activity of polyphenols using cumene hydroperoxide.

BACKGROUND: It is well known that polyphenols lower the risk of chronic diseases such as heart disease and cancer. We describe a new method using cumene hydroperoxide (CHP) to determine the antioxidant activity of polyphenols. CHP is considered to be a model reactive lipid peroxidation product. METHODS: The method was based on two reactions: the reduction of a known concentration of CHP by polyphenols and the reaction of the remaining CHP with a methylene blue derivative (10-N-methylcarbamoyl-3,7 dimethylamino-10H-phenothiazine) in the presence of haemoglobin. Methylene blue was formed as a result of the two reactions and then measured by absorbance at 675 nm. We named this assay the 'cumene hydroperoxide/haemoglobin.methylene blue (CHP/Hb.MB) method'. We examined 13 polyphenols and nine compounds known to be antioxidants. RESULTS: Among the 13 polyphenols, milicetin, with the largest number of hydroxyl groups, had the highest antioxidant activity, followed by cyanidin, pelargonidin and quercetin. It is suggested that the increase in the number of hydroxyl groups induced a higher antioxidant activity. Within-run coefficients of variation were 3.6% and 3.5% at the mean antioxidant activity of 50 micromol/L and 151 micromol/L, respectively. CONCLUSION: We conclude that this CHP/Hb.MB assay is capable of measuring the antioxidant activity of polyphenols.

Development of a homogeneous assay to measure remnant lipoprotein cholesterol.

BACKGROUND: Quantification of triglyceride-rich lipoprotein (TRL) remnants is useful for risk assessment of coronary artery disease and the diagnosis of type III hyperlipoproteinemia. Although an immunoseparation procedure for remnant-like particle cholesterol has been evaluated extensively in recent years, available methods for measuring TRL remnants have not achieved wide use in routine laboratory practice, suggesting a need for a homogeneous assay that can measure TRL remnant cholesterol in serum or plasma without pretreatment. METHODS: We screened for suitable surfactants that exhibited favorable selectivity toward the VLDL remnant (VLDLR) fraction, including intermediate-density lipoproteins (IDLs). We investigated the principal characteristics of this assay by gel filtration of lipoproteins and their particle size distribution. We developed a simple assay and evaluated its performance with the Hitachi-7170 analyzer. RESULTS: Polyoxyethylene-polyoxybutylene block copolymer (POE-POB) exhibited favorable selectivity toward VLDLR and IDL fractions. POE-POB removed apolipoprotein (apo) E and apo C-III from IDL particles in the presence of cholesterol esterase (CHER), and the particle size distribution of IDLs became smaller after the reaction. These results revealed that IDL particles are specifically modified in the presence of CHER and POE-POB, making their component cholesterol available for enzymatic assay. Addition of phospholipase D improved the reactivity toward chylomicron remnants (CMRs). We found a high correlation [y = 1.018x- 0.01 mmol/L, r = 0.962 (n = 160)] between the proposed assay and the immunoseparation assay in serum from healthy individuals. CONCLUSION: The homogeneous assay described in this report can measure TRL remnant cholesterol, including CMRs, VLDLRs, and IDLs, with high sensitivity and specificity.