Macrolide Antibiotic Cholesterol Complex Fluorescence Detection

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Solution Overview

Problem

Current methods for measuring cholesterol levels, particularly in high-density lipoprotein (HDL) particles, are affected by the activity of enzymes like LCAT, which can be inhibited by substances like iodoacetate, leading to artifacts and inaccurate results.

Innovation Solution

The method involves using macrolide antibiotics to form complexes with cholesterol, allowing for the measurement of free cholesterol and total cholesterol levels through fluorescence spectroscopy, while inhibiting LCAT activity using inhibitors or low temperatures to prevent interference.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If iodoacetate is added to inhibit LCAT activity in cholesterol measurement, then LCAT interference is reduced, but the activity of cholesterol oxidase and peroxidase is altered, leading to measurement artifacts

Engineering Contradiction:

ImproveLCAT inhibition effectivenessVSAvoidcholesterol measurement accuracy

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent uses an antibody specific to LCAT as an intermediary substance to inhibit LCAT activity. The antibody binds to LCAT and prevents its enzymatic activity without interfering with the cholesterol oxidase or peroxidase enzymes. This resolves the contradiction by providing selective inhibition of LCAT while preserving the functionality of other enzymes in the assay system.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If enzymatic reactions are used to measure cholesterol levels, then cholesterol quantification is achieved, but enzyme activity alterations cause artifacts and reduce measurement reliability

Engineering Contradiction:

Improvecholesterol quantification accuracyVSAvoidassay result consistency

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent extracts or removes the problematic iodoacetate inhibitor from the assay system and replaces it with an antibody-based inhibition method. This extraction of the harmful element (iodoacetate that alters enzyme activity) while retaining the beneficial function (LCAT inhibition) resolves the contradiction between measurement precision and reliability.

Inventive Principle:

Principle #2Taking out (Extraction)

3Difficulty of detecting and measuring

If macrolide antibiotics are used to complex with cholesterol for fluorescence measurement, then optical detection is enabled, but differentiation between free and esterified cholesterol requires additional enzymatic steps

Engineering Contradiction:

Improvecholesterol detection capabilityVSAvoidassay procedure complexity

Core Design Contradiction:

Difficulty of detecting and measuringVSDevice complexity

Solution Approach 1:

The patent segments the cholesterol measurement into two distinct parts: free cholesterol measurement using macrolide antibiotic complexation with fluorescence detection, and total cholesterol measurement using the same method after enzymatic hydrolysis of esterified cholesterol. This segmentation allows each measurement type to be optimized independently, resolving the contradiction between detection capability and procedure complexity.

Inventive Principle:

Principle #1Segmentation

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

This approach provides accurate and artifact-free measurements of cholesterol levels and LCAT activity by distinguishing between free and esterified cholesterol, offering a practical and reliable method for assessing cholesterol concentrations in biological samples.

Implementation Method 1

measuring the level of fluorescence emitted upon formation of a complex

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

formation of complexes between cholesterol and macrolide antibiotics that can be quantified using optical spectra

Methodology Applied

Scientific EffectComplexation:

Implementation Method 3

LCAT activity, which catalyzes conversion of free cholesterol to cholesteryl esters

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 4

cholesterol esterase, also known as cholesterol ester hydrolase (CEH)

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Implementation Method 5

cholesterol oxidase and peroxidase along with reagents for detecting hydrogen peroxide

Methodology Applied

Scientific EffectOxidation: Oxidation

Implementation Method 6

cholesterol oxidase and peroxidase along with reagents for detecting hydrogen peroxide

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Data Source

PatentUS10495652B1Determination of LCAT

Publication Date: 2019.12.03 ROAR HOLDING LLC

AI summary

Advantage is taken of macrolide antibiotics' complexation with free cholesterol to yield fluorescent complexes to determine the levels of free cholesterol, total cholesterol, or lecithin: cholesterol acyl transferase (LCAT) in serum or plasma or fractions thereof.