Practical 2: Investigation of Total Protein and Albumin

Question 1

What sample do we use when measuring proteins?

Answer 1

Serum

Question 2

What is serum

Answer 2

The fluid that remains after plasma has clotted

Question 3

Why is it important to note that serum is the fluid that remains after plasma has clotted?

Answer 3

It means fibrinogen and most of the clotting factors have been removed

Question 4

Why do we measure proteins?

Answer 4

They give us a general insight into a person’s health

Question 5

What plasma proteins are not synthesised in the liver?
(3)

Answer 5

Immunoglobulins

Haemoglobin

Hormonal proteins

Question 6

What are the roles of plasma proteins?
(8)

Answer 6

Transport

Enzyme catalysis

Homeostatic control

Hormonal regulation

Blood coagulation

Immunity

Growth and repair

Heredity

Question 7

Why would calcium levels be measured
(3)

Answer 7

To screen/monitor for bone diseases

To screen/monitor for diseases of the parathyroid gland

To screen/monitor for diseases of the kidney

Question 8

What is free/ionised calcium?

Answer 8

Calcium not attached to proteins in the blood

Question 9

What percentage of calcium in the blood is attached to proteins

Answer 9

About 50%

Question 10

What is corrected calcium concentration?

Answer 10

An estimate of the total concentration of calcium as if the albumin concentration was normal

Question 11

Why is a corrected calcium concentration needed?
(3)

Answer 11

50% of calcium in the blood is bound to plasma proteins

The majority of protein in plasma is albumin

A low albumin would give a low calcium concentration however this doesn’t accurately reflect the calcium concentration i.e. it might not actually be low

Question 12

How does corrected calcium concentration work?

Answer 12

For every 1g/L that the albumin concentration is below the mean, the calcium concentration is 0.02 mmol/L below what it would be if the albumin concentration was normal

Question 13

What method is used to measure total protein

Answer 13

Biuret

Question 14

What does a total serum protein test measure
(2)

Answer 14

The total amount of protein in the blood

The amounts of albumin and globulin in the blood

Question 15

Why are total serum protein tests carried out?

Answer 15

They are used in the diagnosis and treatment of a variety of diseases involving the liver, kidney and bone marrow as well as other metabolic or nutritional disorders

Question 16

What is the principle behind the Biuret method of measuring total serum protein
(3)

Answer 16

Cupric ions (copper ions) in an alkaline solution react with the peptide bonds of proteins and polypeptides containing at least two peptide bonds

This react produces a violet coloured complex

The absorbance of this complex at 546nm is directly proportional to the concentration of the protein in the sample

Question 17

What absorbance is the biuret reagent read at

Answer 17

546nm

Question 18

What colour is the polypeptide-biuret complex?

Answer 18

Violet

Question 19

How must a blood sample be collected and prepared for the total serum protein test
(4)

Answer 19

Sample = serum, heparinized plasma or EDTA plasma

Normal procedures for collecting and storing serum

Serum will be stable for 3 days between 2+ and +8 degrees Celsius

Serum will be stable for 6 months at -20 degrees Celsius

Question 20

At what temperature should a total serum protein test be carried out at

Answer 20

Room temperature or 37 degrees

Question 21

What is your blank for the total serum protein test?

Answer 21

Water

Question 22

How many standards should you use for the total serum protein test

Answer 22

Three

Question 23

How many controls should you use for the total serum protein test

Answer 23

2

Question 24

How much incubation is needed for the total serum protein test

Answer 24

20 minutes before reading absorbance

Question 25

What method of calculations is needed to determine the concentration of protein in your sample

Answer 25

Unitary method

Question 26

What is the equation for the unitary method

Answer 26

(Absorbance of sample/Absorbance of standard) x Concentration of standard

Question 27

What method is used to determine the concentration of albumin?

Answer 27

Bromocresol Green Method

Question 28

Albumin makes up what percentage of total protein

Answer 28

Between 55 and 65%

Question 29

How long is the half life of albumin

Answer 29

2 to 3 weeks

Question 30

What are the functions of albumin? (3)

Answer 30

To maintain the water balance in serum and plasma To transport and store a wide variety of ligands e.g. fatty acids, calcium, bilirubin and hormones such as thyroxine To provide an endogenous source of amino acids

Question 31

What is hypoalbuminaemia associated with? (7)

Answer 31

Analbuminaemia Impaired albumin synthesis in the liver Livre disease Malnutrition or malabsorption Generalised shock Burns or dermatitis Kidney disease and intestinal disease

Question 32

What is hyperalbuminaemia associated with?

Answer 32

Little diagnostic relevance except perhaps dehydration

Question 33

What is the principle behind the Bromocresol green method of determining albumin concentration? (3)

Answer 33

Its based on the quantitative binding of albumin to the indicator bromocresol green The albumin-BCG-complex absorbs maximally at ~600nm (630nm) The absorbance is directly proportional to the concentration of albumin in the sample

Question 34

How should a sample be prepared and collected for BCG test?

Answer 34

The same as for total serum protein

Question 35

At what absorbance do you measure the albumin concentration

Answer 35

630nm

Question 36

What blank is used for the albumin concentration

Answer 36

Water and reagent blank

Question 37

For how long do you incubate the albumin test

Answer 37

10 mins at 20->25 degrees Celsius

Question 38

How do you calculate the albumin concentration from your absorbances

Answer 38

Unitary method

Question 39

How do you calculate total globulins?

Answer 39

Total protein-albumin i.e. everything other than albumin (fibrinogen not included as serum is the liquid part of clotted blood)