Lesson 15 - Lipid Methodologies

Lipids are insoluble in

Water

Lipids are soluble in

Organic solvents and serum

Examples of organic solvents

Alcohol, ethyl ether, petroleum ether, chloroform

What type of lipids are soluble in the aqueous environments of the blood?

Lipoproteins

Purpose of lipoproteins

• Enhance solubility of lipids in water

• Protect and stabilize lipids

• Allow lipids to be transported efficiently throughout the body to be used for:

  • Cellular structure

  • Energy storage

  • Hormone synthesis

What would happen if there is no solubility mechanism?

Lipids would clump together and form solid masses

Types of lipids in man

• Phospholipids

• Cholesterol

  • free or esterified

• Triglycerides

• Non-esterified fatty acids

• Fat-soluble vitamins

  • Vitamin A

  • Vitamin D

  • Vitamin E

  • Vitamin K

What are important indicators of Coronary Heart Disease Risk (CHD Risk)

Lipids and lipoproteins

A blood test that measures the levels of different types of lipids in the blood

Lipid profile

Types of lipids included in a lipid profile

• Phospholipids

• Cholesterol

  • Total cholesterol

  • HDL

  • LDL

• Triglycerides

What is Total Lipid Determination?

It is the general measure of the total lipids in the blood

What reagent is used in Total Lipid Determination

Bloor’s reagent

Composition of Bloor’s reagent

Alcohol and ether

It is a rough estimate of lipid levels

Total Lipid Determination

It is a more specific measurement of lipid levels

Lipid profile

What are the methods for determining total lipids?

1. Gravimetric Method

2. Colorimetric Method

3. Turbidimetric Method

Principle of Gravimetric Method

• Extraction of lipid using Bloor’s reagent

• Aliquot of the extract is dried and lipids are weighed

What sample is used in Total Lipid Determination?

Serum

In Total Lipid Determination, what is added to serum to extract all fatty material?

Bloor’s reagent

What is the principle of Colorimetric Method?

• Separated lipids are OXIDIZED using dichromate solution

• Reduced chromium ions are measured using a spectrophotometer

What i

What are the critical factors in obtaining reliable data on lipid status?

Patient preparation and sampling

What are the consideration or criteria of lipid samples for optimum results?

1. Px should remain on a regular diet for at least 3 days prior to blood collection

2. No significant weight change

3. Px should fast for at least 12 hours before blood collection

4. Water is allowed but no food should be consumed

5. Dehydration

6. Medications

7. Ethanol consumption

Effects of weight loss in blood samples

• Increased mobilization of stored fats

• Temporarily affects cholesterol and triglycerides

What lipid is affected by fasting/food intake?

Triglycerides

Triglycerides rise significantly within ____ after eating

2 hours

Purpose of a 12-hour fast

• Ensures body has time to process, absorb, and clear any dietary fats

• Prevents presence of chylomicrons

Other than triglycerides, what lipids may be present when a patient does not fast?

Presence of elevated amounts of chylomicrons leading to a turbid or cloudy samples

What is the most common mistake seen in the lab

Patients consume food during fasting period

Volume of water allowed during fasting

at least 1-2 glass or sips of water

This can concentrate blood and affect accuracy of lipid measurements

Dehydration

Examples of medications that may alter lipid values

• Oral contraceptions

• Antihypertensives

• Some vitamins

  • Fish oil supplements rich in omega-3 fatty acids can lower triglycerides

  • Vitamin D

Ethanol consumption should be restricted/eliminated at least _____ prior to blood sample collection

2 days

What increases when a patients consumes ethanol?

Serum triglycerides

Chronic ethanol consumption is associated with

• Hepatic lipid accumulation

• Elevated serum triglycerides

Why is ethanol consumption restricted or eliminated when assessing lipid levels?

The liver will prioritize breakdown of ethanol, disrupting the normal processing of fats in the body

Composition of phospholipids

Esters of glycerol containing:

• 2 fatty acyl

• Phosphatidic acid

Composition of cholesterol

Unsaturated steroid alcohol based on cyclopentanoperhydrophenanthrene (CPP) nucleus

Composition of triglycerides

Esters of

• Glycerol

• 3 fatty acids

Characteristics of triglycerides

• Constitute about 95% of the adipose tissue

• Main storage form of lipids in man

Lipoproteins

Transport vehicles of insoluble lipids in the plasma

Most abundant form of lipids in the human body

Phospholipids

Types of phospholipids

• Lecithin/Phophatidylcholine

• Sphingomyelin

• Cephalins

Components of lecithin

Phosphatidylcholine

Components of sphingomyelin

• Ceramide

• Phosphorylcholine

Components of cephalins

• Phosphatidylethanolamine

• Phosphatidylserine

Characteristics of lecithin, sphingomyelin, and cephalins respectively

• Lecithin

  • make up about 70% of total phospholipids

• Sphingomyelin

  • found in the brain and nerve tissues

• Cephalins

  • make up about 10% of total phospholipids

Functions of lecithin, sphingomyelin, and cephalins respectively

• Lecithin

  • fat transport in plasma

  • vital for the brain and cell membrane structure

• Sphingomyelin

  • nerve insulation

• Cephalins

  • involved in cell membrane structure and blood clotting

Sequence of enzymes used in Choline-Containing Phospholipids assay

1. Phospholipase D

2. Choline oxidase

3. Horseradish peroxidase

What does the Choline-Containing Phospholipids Assay measure?

• Lecithin

• Lysolecithin

• Sphingomyelin

Enzyme used to release choline

Phospholipase D

Enzyme used to oxidize choline

Choline oxidase

Enzyme used to generate a measurable color change

Horseradish peroxidase

Lecithin:Sphingomyelin ratio indicating a mature fetal lung

equal to or greater than 2:1

Second most abundant lipid in man (phospholipids are the most abundant)

cholesterol

What is cholesterol a precursor of?

• Bile acid

• Steroid hormone

Characteristics of cholesterol esters

• makes up ¾ of total cholesterol

• found in plasma and serum

Characteristics of free cholesterol

• makes up ¼ of total cholesterol

• found in serum, plasma, RBC

Normal value for total cholesterol based on NCEP

• Normal

  • < 200 mg/dL

• Borderline high

  • 200-239 mg/dL

• High

  • equal to or greater than 240 mg/dL

Chemical methods used in cholesterol analysis

• Leibermann-Buchardt Reactions

• Salkowski Reaction

• Isotope Dilution Mass Spectrometry

Single-step method in cholesterol analysis

Direct colorimetric

Two-step method in cholesterol analysis

• Extraction

• Colorimetric

Three-step method in cholesterol analysis

• Saponification

• Extraction

• Colorimetric

Four-step method in cholesterol analysis

• Saponification

• Extraction

• Precipitation

• Colorimetric

Reagents used in Liebermann-Burchardt Reactions and their purpose

• Acetic acid

  • main solvent

  • maintains moderate acidic environment

• Acetic anhydride

  • dehydrating agent

  • helps activate cholesterol

  • reacts with sulfuric acid to form acetyl sulfate

• Sulfuric acid

  • strong acid catalyst

  • protonates cholesterol reacts

  • mild oxidizer