Experiment 2

Lipid estimation of blood

Biochemistry and Molecular Biology
Objective
• To determine the amount of total lipids present in serum by
sulfo-phospho-vanillin colorimetric method.
Introduction
• Lipids such as cholesterol, phospholipids and triglycerides are present
in serum, either independently or as lipoproteins.
• They can be measured to examine total lipid level in blood.
• Such assessment is important to understand whether the person is
suffering from hyperlipidemia.
• Unchecked hyperlipidemia often blocks cardiac arteries which is
commonly known as atherosclerosis.
Mechanism
• Unsaturated lipids react with sulphuric acid to produce carbonium
ions.
• These carbonium ions react with phospho-vanillin reagent to give a
pink colour.
• The intensity of the colour generated is proportional to the total lipid
concentration in the sample.

Unsaturated Fat + H2SO4 Carbonium ions

Carbonium ions + Phosphovanillin Chromogen (Colour compound)

Requirements

• Concentrated Sulphuric acid

• Phospho-vanillin reagent:

Mix 20 ml of Vanillin (0.6% w/v in H2O) in 80 ml Phosphoric acid

Procedure
1. Label clean and dry test tubes as Standard (S), and Test (T) and plan them as per
table:
Test
H2SO4 (ml) 2.5
Sample (µl) 100

2. Mix and incubate at 100oC for 10 minutes in a boiling water bath.

• This reaction acts as acid digest of standard and sample for further use.
Use acid digest to set new reaction
1. Cool in iced water and transfer into tubes as per table:
Control Test
Reagent (ml) 1.0 1.0
Sample (µl) -- 50
Water (µl) 50 --

2. Mix and incubate for 15 minutes at 37 oC.

3. Measure the absorbance at 520 nm (quantification test)

Result Interpretations
• Formation of colour indicates presence of lipids in the solution.

• Colour in test will be darker as compared to blank/control.

Precautions

• Pipetting by mouth is strictly prohibited.

• Concentrated sulfuric acid causes severe burns and eye damage.

Applications:
• Able to analyze small volume (100 µl) of sample
• Can be used as a screening method for hyperlipidemia
• Simple, rapid and reliable

Limitations:
• Use of multiple apparatus for a single test makes it difficult for large
scale analysis.
• Involvement of vigorous shaking makes it difficult to use 96 well
plates.
Lipid profile or Lipid test
• Total Cholesterol
Low Density Lipoproteins (Bad Cholesterol)
High-Density Lipoproteins (Good Cholestrol)
• Triglycerides

Optimal Cholesterol Levels

Total cholesterol
LDL (“bad”) cholesterol
HDL (“good”) cholesterol
Triglycerides
About 150 mg/dL
About 100 mg/dL
At least 40 mg/dL in men,
50 mg/dL in women
Less than 150 mg/dL
Centers for Disease Control & Prevention, USA
 Useful Terminology
• Platelets: Smallest of blood cells, bind to the site of the damaged vessel, thereby
causing a blood clot
• Thrombus: Blood clot formed inside the vessels
• Embolus: A thrombus that breaks loose and travels from one location to another
• Embolism: Blocked artery caused by a foreign body, such as a blood clot or an air
bubble
• Blood Flow: Circulatory system, vessels (arteries, veins, capillaries)
• Plaque formation: deposits of fatty substances, cholesterol, cellular waste products,
calcium, and fibrin
• Stroke: a blood vessel that carries oxygen and nutrients to the brain is either blocked by
a clot or bursts
• Ischemia: Blood flow (and oxygen) is restricted or reduced in a part of the body
Supplementary information
• Acid digestion: Decomposition of a sample in strong acids (nitric,
sulfuric, perchloric) in order to obtain the sample in liquid form.
Report for the experiment: Address the following questions
and submit online by Jan 31, 2023 (20:00 hrs)

1. Interpret the results for all the objectives of this experiment.

2. Summarize your learnings from this experiment.

3. List and explain the potential future applications you have identified
for your learnings?
 Assessment for the experiment: Address the following
questions and submit online by Jan 31, 2023 (20:00 hrs)
Answer length: 75-100 words Total score: 5

1. Can you employ this method to accurately measure LDL, VLDL, Cholesterol and Triglycerides in blood
samples?

2. What is fatty liver?

3. What is cardiac fibrosis?

4. What is the relationship between polyunsaturated fatty acids and inflammation?

5. How does statin work?

6. Coconut oil gets frozen in winter, however, mustard oil does not. What is the chemistry behind this
phenomenon?
Format for Report and Assessment
Course name: Biochemistry & Molecular Biology Date: 18 January 2023

Student name: ………………………………….……... Student ID ………..…………………

Experiment Title: Serum Glucose estimation by GOD-POD method

Mention above information on a word file and the type your answers, first for experiment report and then
assessment questions (new page)

Font: Times new roman Font size: 12 Line Space 1.5 Justified

File Name: Arvind U20210017 Experiment 2

References
• Lipid Panel | Johns Hopkins Medicine
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068759/
• https://www.cdc.gov/cholesterol/index.htm
• https://thrombosis.org/
• https://www.sigmaaldrich.com/IN/en/support/calculators-and-apps