Factors Affecting Cholesterol Levels

Factors Affecting Cholesterol Levels

• Age is directly proportional to cholesterol levels, meaning that cholesterol levels increase with age.
• Sex plays a role in cholesterol levels, with women generally having lower levels than men, except during childhood and early 50s.
• Cholesterol levels are slightly higher during cold seasons or periods.
• Daily intake of fat has a direct impact on cholesterol levels, with increased fat consumption leading to higher cholesterol levels.
• When measuring cholesterol levels, patients should maintain their usual diet for at least 2 weeks and avoid significant weight gain or loss.

Acute Illness and Lipoproteins Measurement

• Measurements of lipoproteins should be delayed for at least 8 weeks after any form of trauma or acute bacterial/viral infection.
• It is recommended to wait for 3-4 months after childbirth to take lipoproteins measurements.

Lifestyle Factors and Lipoproteins

• A sedentary lifestyle is associated with higher lipoproteins levels.
• Poor diet habits contribute to increased lipoproteins levels.

Venous Occlusion and Cholesterol

• Prolonged venous occlusion leads to an increase in cholesterol levels by 10-15%.

Cholesterol Measurement

• Non-enzymatic method uses the Abell-Kendall or Bloors method

Methods

• Liebermann-Burchardt (L-B) Reaction is used to measure cholesterol
• L-B Reaction involves the use of H2SO4, acetic acids, and acetic anhydride to produce a green solution

Abell-Kendall Method

• A 3-step principle
• Step 1: Cholesterol is hydrolyzed with alcoholic KOH
• Step 2: Unesterified cholesterol is extracted with petroleum jelly
• Step 3: Measured using the L-B Reaction

Cholesterol Extraction and Measurement Methods

• There are four methods to extract and measure cholesterol: Bloors Method, 2 Step Principle, Enzymatic Method, and Reference Method.
• Bloors Method involves extracting cholesterol using an alcohol-ether mixture.
• The 2 Step Principle measures cholesterol using the L-B reaction.
• The Enzymatic Method uses cholesterol oxidase to measure cholesterol.
• The Reference Method, also known as GCMS, specifically measures cholesterol and does not detect sterols.
• The Gold Standard Method, used by NIST, is the definitive method for measuring cholesterol using isotope dilution mass spectrometry.
• Isotope dilution mass spectrometry is a highly accurate method for measuring cholesterol, making it the gold standard.

Non-Enzymatic Method for Lipid Extraction

• Lipids are extracted using chloroform.
• Phospholipids are removed by zeolite absorption.

Correction Methods for Endogenous Free Glycerol

• "Double-cuvette blank" method: subtracts glycerol blank measurement without lipase enzyme from total glycerol measurement with complete reagent.
• "Single-cuvette blank" method: measures only endogenous free glycerol by incubating lipase-free reagent, taking a blank reading, adding lipase, incubating, and then taking a final reading to correct for the blank.
• Designated calibration blanking: adjusts calibration set points to net or blank-corrected values to compensate for the average free glycerol content of specimens.

Triglyceride Measurement

• Reference level for triglyceride measurement is less than 176 mg/dL or less than 2.0 mmol/L
• Conversion factor for triglyceride measurement is 0.011

Lipoprotein Precipitation

• Polyanions such as heparin sulfate, dextran sulfate, and phosphotungstate are used to precipitate lipoproteins
• Presence of divalent cations Mg, Ca, and Mn is required for the reaction

Lipoprotein Electrophoresis

• Used to identify rare familial disorders related to lipoproteins
• Indicated when serum triglyceride (TG) levels are greater than 300mg/dL
• Also indicated when fasting serum is lipemic (cloudy or turbid) or when there is significant hyperglycemia (high blood sugar)
• Provides a visual display in detecting unusual or variant patterns of lipoproteins
• Utilizes agarose gel or Polyacrylamide Gel Electrophoresis (PAGE) as the medium for electrophoresis
• Not desirable for precise quantitation, but useful in qualitative analysis

Ultracentrifugation

• Used for preparative purposes to fractionate lipoprotein classes
• Involves sequential density adjustments of serum to separate major and minor lipoprotein classes
• Density-gradient methods can be used in two ways: non-equilibrium and equilibrium
• Density-gradient methods allow for the fractionation of multiple or all lipoprotein classes in a single run
• Methods are useful for quantitative purposes and preparative isolations
• However, the process is tedious, expensive, and technically demanding

HDL-C Estimation Methods

• Precipitation method uses divalent cations and polyanions as precipitation reagents to remove all lipoproteins except HDL, allowing for HDL-C estimation
• Enzymatic method is used to quantitate HDL-C in conjunction with precipitation method
• Magnetic method uses a precipitant complexed to magnetic particles to precipitate HDL-C, which then sediments without requiring centrifugation

Homogenous Method (Direct HDL-C Assay)

• The first reagent in the homogenous method "blocks" non-HDLs using antibodies, polymers, or complexing agents like Cyclodextrin.
• The cholesterol esterase and oxidase enzymes are modified to make them selective for HDL-C.
• A blanking step is used to selectively consume cholesterol from non-HDL species.
• The second reagent measures the accessible HDL-C quantities.
• The homogenous method is highly precise and reasonably accurate.
• However, it lacks specificity for HDL in unusual specimens, such as those with liver and kidney disease.
• The homogenous method does not require pre-treatment of the sample.