Part 5: Lipids

What does a high TC/HDL ratio mean?

• increased risk of CVD

What ratios and risk scores can be used to evaluate CVD risk?

• TC/HDL

• LDL/HDL

What is the purpose of the TC/HDL ratio (Total cholesterol/HDL cholesterol)?

• estimates balance between total cholesterol and protective HDL cholesterol

What is an ideal TC/HDL ratio?

• < 4.0

What TC/HDL ratio means moderate risk?

• between 4.0 and 5.0

What TC/HDL ratio would mean high risk?

• > 5.0

What does a higher TC/HDL ratio mean clinically?

• more atherogenic particles relative to protective HDL

• increased CVD risk

What is the purpose of the LDL/HDL ratio?

• compares atherogenic LDL cholesterol to protective cholesterol

What is the ideal LDL/HDL ratio?

• < 3.0

What values would be high for LDL/HDL ratio?

• > 3.0

  • increased cardiovascular risk

What does the LDL/HDL ratio mean clinically?

• more specific than TC/HDL in targeting LDL’s role in atherogenesis

What methods can be used to determine Total Cholesterol (TC)?

• Enzymatic Colorimetric Methods (most common)

• Liebermann-Burchard Reaction (old chemical)

• Gas Chromatography (GC) (Reference Labs)

• High-Performance Liquid Chromatography (used in research)

Describe the Enzymatic Colorimetric method used to determine total cholesterol.

• routine use

• high specificity

• automated

• gold standard in clinical labs

Describe the Liebermann-Burchard Reaction method used to determine total cholesterol.

• not used for routine use

• low specificity

• no automation

• historical interest only

Describe the Gas Chromatography method used to determine total cholesterol.

• No routine use

• very high specificity

• no automation

• reference labs only

Describe the HPLC method used to determine total cholesterol.

• no routine use

• very high specificity

• no automation

• research and validation

What method is the most widely used because of its specificity, accuracy, and compatibility with automated analyzers to measure total cholesterol?

• Enzymatic Colorimetric Method (CHOD-PAP method)

Describe the Enzymatic Colorimetric Method.

• series of enzymatic reactions

  • cholesterol oxidase (CHOD)

  • peroxidase

  • 4-aminophenazone

What is the principle of the Enzymatic Colorimetric Method?

• three enzymatic reactions

• convert cholesterol → red/pink colored quinoneimine dye

• measured spectrophotometrically

• red color = cholesterol concentration

What wavelength is the Enzymatic Colorimetric Method measured at?

• 500-550 nm

• commonly 546 nm

How is detection done for the Enzymatic Colorimetric Method ?

• red quinoneimine dye formed has absorbance at 500-550 nm

  • commonly 546 nm

• absorbance is directly proportional to cholesterol concentration in sample

What methods are used in the HDL Cholesterol (HDL-C) Determination Method?

1. Precipitation Methods (old method)

2. Homogenous (Direct) Assays (modern clinical standard)

3. Ultracentrifugation (reference method)

Describe the Precipitation Method used to measure HDL Cholesterol.

• limited routine use

• moderate specificity

• no automation

• manual, affected by TG

Describe the Homogenous Method used to measure HDL Cholesterol.

• routine use

• high specificity

• automated

• modern clinical standard

Describe the Ultracentrifugation Method used to measure HDL Cholesterol.

• no routine use

• very high specificity

• no automation

• reference method

Describe the principle of the HDL-C Precipitation method.

• non-HDL lipoproteins are precipitated using:

  • Heparin-managase chloride

  • phosphotungstic acid-magnusium chloride

  • Dextran sulfate-magnesium chloride

• sample is centrifuged and supernatant is analyzed for cholesterol using an enzymatic method

Describe the principle of the HDL-C Homogenous (direct) assays.

• uses different chemicals to block or modify non-HDL lipoproteins in the serum

  • selective detergents

  • polymers or enzymes

• HDL cholesterol is measured directly using enzymatic colorimetric assay

What methods can be used to determine LDL-holesterol levels?

1. Friedewald Formula (calculated LDL-C)

   1. most common in routine labs

2. Direct (homogeneous) LDL Assay

   1. modern automated

3. Beta Quantification (ultrafugation+ precipitation)

   1. reference method

Describe the Friedewald formula for measuring LDL-cholesterol.

• calculated

• routine use

• moderate accuracy

• inaccurate if TG> 400 mg/dL

Describe the Direct LDL Assay for measuring LDL-cholesterol.

• homogeneous

• routine use

• high accuracy

• useful with high TG or non-fasting sample

Describe the Beta-Quatification for measuring LDL-cholesterol.

• reference lab

• no routine use

• very high accuracy

• time consuming, expensive

What is the formula for the Friedewald equation that calculates LDL-C?

• TC - HDLC - (TG/5)

What must be assumed when using the Friedewald Formula?

• VLDL-C is approx = TG/5 (in fasting samples)

• not accurate if TG > 400 mg/dL

What are the disadvantages of the Friedewald formula?

• needs fasting sample

• innacturate in hypertriglyceridemia, diabetes, liver/kidney disease

What is the principle of the Homogeneous (Direct) assay for measuring LDL-cholesterol.

• reagents selectively block or solubilize non-LDL lipoproteins (HDL,VLDL)

• LDL-C is measured enzymatically

• works EVEN IF TG levels are high

What methods can be used to determine Triglyceride levels?

1. Enzymatic Colorimetric Methods

   1. gold standard in clinical labs

2. Glycerol Blank Method

   1. enzymatic with correction

3. Chemical (non-enzymatic) Methods

   1. historical

4. high-performance liquid chromatography (HPLC)

   1. research

5. Gas Chromatography(GC)

   1. research/fatty acid composition

Describe the enzymatic colorimetric method used to determine triglyceride levels.

• enzymatic

• routine use

• high accuracy

• clinical standard

Describe the glycerol blank method used to determine triglyceride levels.

• enzymatic

• sometimes used routinely

• higher accuracy

• used when free glycerol interference is suspected

Describe the van Handel-Zilversmit method used to determine triglyceride levels.

• chemical

• no routine use

• moderate accuracy

• historical interest

Describe the HPLC method used to determine triglyceride levels.

• chromatographic

• no routine use

• very high accuracy

• research use

Describe the gas chromatography method used to determine triglyceride levels.

• chromatographic

• no routine use

• very high accuracy

• fatty acid composition analysis

Describe the principle of the TG Enzymatic Colorimetric Method

• multi-step enzymatic reaction breaks down TG and measures glycerol by converting it into red or pink quinoneimine dye measured spectrophotometrically

• intensity of red = TG concentration

What wavelength is measured for Triglycerides using the Enzymatic Colorimetric Method?

• 500 - 550 nm

• commonly 546 nm

Why is the Enzymatic Colorimetric Method for TG measurements also called the GPO-PAP method?

• it utilizes enzymatic reactions involving:

  • glycerol-3-phosphate oxidase (GPO)

  • peroxidase

  • 4-aminophenazone

What are important lipid biomarkers?

1. lipoprotein (a)

2. remnant cholesterol

What is lipoprotein(a) Lp(a)?

• LDL-like particle with an attached protein called (apolipoprotein (a)) to apoB-100

• genetically determined

• high levels = atherosclerotic cardiovascular disease (ASCVD)

What does a high Lp(a) of (> 50 mg/dL or >125 nmol/L) mean?

• elevated risk of premature coronary artery disease/stroke

What drug does NOT significantly lower Lp(a)?

• statins

What is remnant cholesterol (RC)?

• cholesterol content of triglyceride-rich lipoproteins

  • VLDL remnants (in fasting state)

  • chylomicron remnants (in postprandial state)

• penetrate arterial walls more easily than LDL and contribute to plaque formation

What is the normal range for remnant cholesterol?

• < 20 mg/dL

• < 0.52 mmol/L

What is the borderline/moderate range for remnant cholesterol?

• 20-30 mg/dL

What is the high range for remnant cholesterol?

• > 30 mg/dL

Describe Lipoprotein (a).

• LDL particle + apolipoprotein(a)

• genetic risk, ASCVD, aortic stenosis

• new therapies in trial

Describe Remnant cholesterol.

• cholesterol in TG-rich lipoproteins

• residual risk, inflammation

• therapies target TG lowering

What is Lipid Fractionation?

• lab technique used to separate and characterize lipoproteins based on chemical/physical properties

• provides qualitative/quantitative information

  • lipoprotein sublass

  • particle size

  • particle number

Why is Lipid fractionation important?

• traditional lipid testing measures cholesterol concentration with lipoproteins but not:

  • number of lipoprotein particles

  • variations in particle size/density

Give an example as to why Lipid fractionation is important.

• two patients may have same LDL-C

  • one may have few large LDL particles

  • another may have small, dense LDL particles

What methods are used in Lipid Fractionation?

• Nuclear Magnetic Resonance (NMR) Spectroscopy

What is the principle of Nuclear Magnetic Resonance (NMR) Spectroscopy?

• analyzes lipoproteins based on magnetic signals generated by lipid methyl groups exposed to a magnetic field

• signals differ based on:

  • lipoprotein size

  • lipoprotein concentration

What does Nuclear Magnetic Resonance (NMR) Spectroscopy Measure?

• LDL particle number (LDL-P)

• HDL particle number (HDL-P)

• VLDL particle number

• particle size

• lipoprotein subclass distribution

What is a cheap alternative for Nuclear Magnetic Resonance (NMR) Spectroscopy?

• ApoB test

  • based on immunoassays and measures number of atherogenic particles