Small Dense LDL Cholesterol Quantification Enzyme Reagent
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Solution Overview
Problem
Conventional methods for measuring small, dense LDL cholesterol are inconvenient, time-consuming, and lack specificity, particularly when using autoanalyzers, as they require expensive facilities and pretreatment of specimens, and often fail to accurately measure cholesterol due to reliance on turbidity measurements.
Innovation Solution
A method utilizing cholesterol esterase, cholesterol oxidase, and phospholipase to selectively eliminate cholesterol from LDL other than small, dense LDL, followed by the use of surfactants to separate and measure cholesterol in small, dense LDL without pretreatment, using enzymes and surfactants like polyoxyethylene-polyoxypropylene copolymers to differentiate and quantify small, dense LDL cholesterol.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (ultracentrifugation, electrophoresis, HPLC) are used to measure small, dense LDL cholesterol, then measurement accuracy is improved, but device complexity and operational costs increase significantly
Solution Approach 1:
The patent replaces complex mechanical separation systems (ultracentrifugation, electrophoresis, HPLC) with a biochemical reaction system using phospholipase, cholesterol esterase, and cholesterol oxidase enzymes. This substitution allows small, dense LDL cholesterol measurement to be performed using simple autoanalyzer equipment with basic spectrophotometric detection, eliminating the need for expensive specialized facilities while maintaining measurement accuracy.
2Measurement precision
If conventional methods are used for small, dense LDL measurement, then measurement accuracy is improved, but measurement time increases significantly
Solution Approach 1:
The patent employs a continuous multi-enzyme reaction system where phospholipase first hydrolyzes phospholipids in LDL particles, then cholesterol esterase hydrolyzes cholesterol esters, and finally cholesterol oxidase converts cholesterol to cholestenone with hydrogen peroxide production. This continuous biochemical cascade allows rapid sequential processing of all LDL fractions, enabling complete measurement within minutes using standard autoanalyzer throughput, significantly reducing measurement time compared to conventional methods.
3Ease of operation
If turbidity-based absorbance measurement is used, then operational simplicity is improved, but measurement specificity deteriorates
Solution Approach 1:
The patent utilizes a colorimetric reaction system where cholesterol oxidase converts cholesterol to cholestenone, producing hydrogen peroxide that reacts with chromogenic substrates to generate colored products. This color change provides a specific optical signal that can be measured by standard spectrophotometric detection in autoanalyzers, maintaining operational simplicity while achieving high measurement specificity for cholesterol in small, dense LDL particles, eliminating the non-specificity of turbidity-based methods.
4Measurement precision
If pretreatment procedures are implemented for small, dense LDL separation, then measurement specificity is improved, but ease of operation deteriorates
Solution Approach 1:
The patent employs a universal reagent system containing multiple enzymes (phospholipase, cholesterol esterase, cholesterol oxidase) and surfactants that can process various specimen types (serum, plasma, whole blood) without requiring prior separation or pretreatment. The enzymatic reactions universally target cholesterol in small, dense LDL particles across different specimen matrices, eliminating the need for complex pretreatment procedures while maintaining high measurement specificity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables rapid, convenient, and specific measurement of small, dense LDL cholesterol directly in an autoanalyzer without pretreatment, improving accuracy and reducing operational costs by selectively isolating and quantifying cholesterol in small, dense LDL.
Implementation Method 1
the reaction rate of an enzyme against LDL other than small, dense LDL increases to a greater extent than that of an enzyme against small, dense LDL because of the use of phospholipase reacting specifically with a specific substrate
Implementation Method 2
when cholesterol in a sample containing various lipoproteins is measured using cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase
Implementation Method 3
cholesterol oxidase or cholesterol dehydrogenase
Implementation Method 4
cholesterol in LDL other than small, dense LDL could be eliminated and led outside of the reaction system with the use of catalase or 4 aminoantipyrine
Implementation Method 5
when a specific surfactant reacting with lipoproteins other than LDL such as HDL and VLDL was added before or after the reaction to lead LDL other than small, dense LDL outside of the reaction system
Data Source
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AI summary
A method for fractional measurement of small, dense LDL, which is adaptable for an autoanalyzer, and a reagent for measurement, are provided, making it possible to conduct rapid and convenient analysis with good sensitivity without pretreatment of a specimen. The method for quantitatively determining small, dense LDL cholesterol in a sample comprises the steps of: (1) eliminating cholesterol in LDL other than small, dense LDL in the presence of phospholipase; and (2) quantitatively determining cholesterol in lipoproteins remaining in step (1) above.