Practical 2: Investigation of Total Protein and Albumin Flashcards
What sample do we use when measuring proteins?
Serum
What is serum
The fluid that remains after plasma has clotted
Why is it important to note that serum is the fluid that remains after plasma has clotted?
It means fibrinogen and most of the clotting factors have been removed
Why do we measure proteins?
They give us a general insight into a person’s health
What plasma proteins are not synthesised in the liver?
(3)
Immunoglobulins
Haemoglobin
Hormonal proteins
What are the roles of plasma proteins?
(8)
Transport
Enzyme catalysis
Homeostatic control
Hormonal regulation
Blood coagulation
Immunity
Growth and repair
Heredity
Why would calcium levels be measured
(3)
To screen/monitor for bone diseases
To screen/monitor for diseases of the parathyroid gland
To screen/monitor for diseases of the kidney
What is free/ionised calcium?
Calcium not attached to proteins in the blood
What percentage of calcium in the blood is attached to proteins
About 50%
What is corrected calcium concentration?
An estimate of the total concentration of calcium as if the albumin concentration was normal
Why is a corrected calcium concentration needed?
(3)
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
How does corrected calcium concentration work?
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
What method is used to measure total protein
Biuret
What does a total serum protein test measure
(2)
The total amount of protein in the blood
The amounts of albumin and globulin in the blood
Why are total serum protein tests carried out?
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
What is the principle behind the Biuret method of measuring total serum protein
(3)
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
What absorbance is the biuret reagent read at
546nm
What colour is the polypeptide-biuret complex?
Violet
How must a blood sample be collected and prepared for the total serum protein test
(4)
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
At what temperature should a total serum protein test be carried out at
Room temperature or 37 degrees
What is your blank for the total serum protein test?
Water
How many standards should you use for the total serum protein test
Three
How many controls should you use for the total serum protein test
2
How much incubation is needed for the total serum protein test
20 minutes before reading absorbance
What method of calculations is needed to determine the concentration of protein in your sample
Unitary method
What is the equation for the unitary method
(Absorbance of sample/Absorbance of standard) x Concentration of standard
What method is used to determine the concentration of albumin?
Bromocresol Green Method
Albumin makes up what percentage of total protein
Between 55 and 65%
How long is the half life of albumin
2 to 3 weeks
What are the functions of albumin?
(3)
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
What is hypoalbuminaemia associated with?
(7)
Analbuminaemia
Impaired albumin synthesis in the liver
Livre disease
Malnutrition or malabsorption
Generalised shock
Burns or dermatitis
Kidney disease and intestinal disease
What is hyperalbuminaemia associated with?
Little diagnostic relevance except perhaps dehydration
What is the principle behind the Bromocresol green method of determining albumin concentration?
(3)
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
How should a sample be prepared and collected for BCG test?
The same as for total serum protein
At what absorbance do you measure the albumin concentration
630nm
What blank is used for the albumin concentration
Water and reagent blank
For how long do you incubate the albumin test
10 mins at 20->25 degrees Celsius
How do you calculate the albumin concentration from your absorbances
Unitary method
How do you calculate total globulins?
Total protein-albumin
i.e. everything other than albumin (fibrinogen not included as serum is the liquid part of clotted blood)
