ACTIVITY

12

TOTAL PROTEIN DETERMINATION

Unit Intended Learning Outcomes

At the end of the activity, the students are expected to:

1. Demonstrate appropriately the specimen preparation for total protein determination.

2. Perform correctly the laboratory methods of total protein determination.
3. Analyze correctly the test results with clinical signs and symptoms.

Pre-laboratory Discussion

Proteins are macromolecules (a molecule with a molecular mass of several thousand or more). They are
polymers built from one or more unbranched chains of amino acids. A typical protein contains 200 to 300
amino acids, but some are much smaller (peptides) and some are much larger (titin, in muscle) and range in
molecular mass from approximately 6,000 for insulin to several millions for some structural proteins.
Proteins consist of the elements carbon, oxygen, hydrogen, nitrogen, and sulfur. It is the fact that proteins
contain nitrogen that sets them apart from pure carbohydrates and lipids, which do not contain nitrogen
atoms. The nitrogen content of serum protein is, on average, approximately 16%. This measurement of
nitrogen content is used in one method for total protein.

There are four distinct levels of a protein’s structure: primary, secondary, tertiary, and quaternary. Primary
structure represents the number and types of amino acids in the specific amino acid sequence. In order to
function properly, proteins must have the correct sequence of amino acids. Secondary structure is regularly
repeating structures stabilized by hydrogen bonds between the amino acids within the protein. Common
secondary structures are the α-helix, β-pleated sheet, and turns, with most serum proteins forming a helix.
Secondary structures add new properties to a protein such as strength and flexibility. Tertiary structure
refers to the overall shape, or conformation, of the protein molecule. The conformation is known as the
fold, or the spatial relationship of the secondary structures to one another. Tertiary structures are three-
dimensional. Tertiary structure results from the interaction of side chains and is stabilized through the
hydrophobic effect, ionic attraction, hydrogen bonds, and disulfide bonds. The function and physical and
chemical properties of a protein depend on its tertiary structure. Quaternary structure is defined as the
shape or structure that results from the interaction of more than one protein molecule, or protein subunits,
held together by noncovalent
forces such as hydrogen bonds and electrostatic interactions, which are part of the larger protein complex
with a precise three-dimensional configuration.

The plasma proteins are the most frequently analyzed of all the proteins. The major measured plasma
proteins are divided into two groups: albumin and globulins. There are four major types of globulins, each
with specific properties and actions. A typical blood panel will provide four different measurements—total
protein, albumin, globulins, and the albumin/globulin (A/G) ratio. The total protein test is a rough measure
of all of the proteins in the plasma. Total protein measurements can reflect nutritional status, kidney
disease, liver disease, and many other conditions. If total protein is abnormal, further tests must be
performed to identify which protein fraction is abnormal, so that a specific diagnosis can be made.

The specimen most often used to determine the total protein is serum rather than plasma. A fasting
specimen is not needed. Interferences in some of the methods occur in the presence of lipemia; hemolysis
falsely elevates the total protein result because of the release of RBC proteins into the serum. The reference
interval for serum total protein is 6.5 to 8.3 g/dL (65 to 83 g/L) for ambulatory adults. In the recumbent
position, the serum total protein concentration is 6.0 to 7.8 g/dL (60 to 78 g/L). The reduction in serum
albumin reduces blood COP. Therefore, the distribution of fluid shifts toward extracellular compartments. At
birth, the total protein concentration is lower, reaching adult levels by age 3. As a person ages, there is a
slight decrease in albumin levels. Lower total protein levels are also seen in pregnancy.

The classic method for quantitation of total protein is the Kjeldahl method, which determines nitrogen. This
method is not used in the clinical laboratory because it is time consuming and too tedious for routine use. In
this method, an average of 16% nitrogen mass in protein is assumed to calculate the protein concentration.
The actual nitrogen content of serum proteins varies from 15.1% to 16.8%. Therefore, error is introduced if a
protein standard (calibrated with the Kjeldahl method) is used that differs in composition from the serum
specimen to be analyzed, because the percentage of nitrogen will not be the same.

The biuret procedure is the most widely used method and the one recommended by the International
Federation of Clinical Chemistry expert panel for the determination of total protein. In this reaction, cupric
ions (Cu2+) complex with the groups involved in the peptide bond. In an alkaline medium and in the
presence of at least two peptide bonds, a violet-colored chelate (a bound metal in complex) is formed. The
reagent also contains sodium potassium tartrate, to complex cupric ions to prevent their precipitation in the
alkaline solution, and potassium iodide, which acts as an antioxidant. The absorbance of the colored chelate
formed is measured at 540 nm. When small peptides react, the color of the chelate produced has a different
shade than that seen with larger peptides. The color varies from a pink to a reddish violet. The color that is
formed is proportional to the number of peptide bonds present and reflects the total protein level.
Principle

Proteins are composed of several peptide bonds. These peptide bonds are chelated by cupric ions to form a
violet colored compound in an alkaline solution. This method utilizes the principle of Biuret’s reaction. The
absorbance of this complex is proportional to the protein concentration in the sample.

Reference Range

Serum/Plasma: 6.6 – 8.3 g/dL (66 – 83 g/L)

Teaching and Learning Activities

Pre-Analytical Phase

1. Disinfect the working area using a disinfectant.

2. Prepare all the materials/equipment needed.
3. Wear all personal protective equipment (PPE) before starting the activity.

Analytical Phase

Materials

Test tubes
Spectrophotometer
Centrifuge
Water Bath
Serological and Automatic Pipettes

Reagents and Sample

Protein Reagent
Protein Standard – 10 g/dL
Nonhemolyzed specimen

Procedure

1. Remove the amount of reagent to be used for testing and allow to warm to ambient temperature.
2. Prepare two test tubes and label each as Standard and Test or Control (If available).
 REAGENTS STANDARD 10 g/dL TEST SERA/CONTROL

Protein Standard 10 µL -

Test Sera/ Control - 10 µL

Protein Reagent 1.0 mL 1.0 mL

3. Mix gently and incubate the tubes at room temperature for 5 minutes.
4. Read the absorbance of the samples at 550 nm against reagent blank.

Calculation of Results

Absorbance of the Unknown

Concentration of the sample=
X 10 = g/dL
Absorbance of the Standard

Post-Analytical Phase

1. Segregate and dispose all wastes into proper trash bins.

2. Disinfect the working area using 10% sodium hypochlorite.
3. Remove all the PPE properly after the activity.

References

1. Bishop, M.L., Fody, E.P., Schoeff, L.E. (2013). Clinical Chemistry: Principles, Techniques and Correlations
7th ed. Philadelphia: Lippincott Williams and Wilkins.
2. Nuevo, J.J. (n.d.). Clinical Chemistry 1 Laboratory Manual (Outcomes Based Education Format).
3. McPherson, Richard A. and Matthew R. Pincus. Henry’s Clinical Diagnosis and Management by
Laboratory Methods. 22nd edition. Philadelphia: Elsevier Inc., 2011.
Name: Date:

Section: Group: Score:

Review Questions

1. Discuss the principle of Kjeldahl Method for determining serum total protein.

2. Enumerate and discuss the different methods employed in total protein determination.

3. Briefly describe the normal pattern of protein electrophoresis.

4. Enumerate the different conditions associated with hypoproteinemia and hyperproteinemia.

5. Draw the different electrophoretic patterns seen in patients with multiple myeloma, nephrotic
syndrome, cirrhosis, inflammation, and anti-trypsin deficiency.
 ACTIVITY 13

ALBUMIN DETERMINATION

Unit Intended Learning Outcomes

At the end of the activity, the students are expected to:

1. Demonstrate appropriately the specimen preparation for albumin determination.

2. Perform correctly the laboratory methods of albumin determination.
3. Analyze correctly the test results with clinical signs and symptoms.

Pre-laboratory Discussion

Albumin is synthesized in the liver from 585 amino acids at the rate of 9 to 12 g/d with no reserve or
storage. It is the protein present in highest concentration in the plasma. Albumin also exists in the
extravascular (interstitial) space. In fact, the total extravascular albumin exceeds the total intravascular
amount by 30%, but the concentration of albumin (plasma albumin concentration = intravascular albumin
mass/plasma volume) in the blood is much greater than its concentration in the interstitial space. Albumin
leaves the circulation at a rate of 4% to 5% of total intravascular albumin per hour. This rate of movement is
known as the transcapillary escape rate, which measures systemic capillary efflux of albumin. Albumin is
responsible for nearly 80% of the colloid osmotic pressure (COP) of the intravascular fluid, which maintains
the appropriate fluid balance in the tissue. Albumin also buffers pH and is a negative acute-phase reactant
protein.

Another prime function of albumin is its capacity to bind various substances in the blood. There are four
binding sites on albumin, and these have varying specificities for different substances. Albumin transports
thyroid hormones; other hormones, particularly fat-soluble ones; iron; and fatty acids. For example, albumin
binds unconjugated bilirubin, salicylic acid (aspirin), fatty acids, calcium (Ca2+) and magnesium (Mg2+) ions,
and many drugs, and serum albumin levels can affect the half-life of drugs. This binding characteristic is also
exhibited with certain dyes, providing a method for the quantitation of albumin.

The most widely used methods for determining albumin are dye-binding procedures. The pH of the solution
is adjusted so that albumin is positively charged. The albumin is attracted to and binds to an anionic dye by
electrostatic forces. When bound to albumin, the dye has a different absorption maximum than the free
dye.
The amount of albumin is calculated by measurement of the absorbance of the albumin–dye complex. A
variety of dyes have been used, including methyl orange, 2, 4ʹ-hydroxyazobenzene-benzoic acid (HABA),
bromocresol green (BCG), and bromocresol purple (BCP). Methyl orange is nonspecific for albumin; β-
lipoproteins and some α1- and α2-globulins also will bind to this dye. HABA has a low sensitivity but is more
specific for albumin. In addition, several compounds, such as salicylates, penicillin, conjugated bilirubin, and
sulfonamides, interfere with the binding of albumin to HABA. BCG is not affected by interfering substances
such as bilirubin and salicylates; however, hemoglobin binds to BCG. For every 100 mg/dL of hemoglobin,
albumin is increased by 0.1 g/dL.65 BCG has been reported to overestimate low albumin values in patients
when the low albumin level was accompanied by an elevated α-globulin fraction, such as occurs in nephrotic
syndrome or end-stage renal disease. It was found that the α-globulins react with BCG, giving a color
intensity that is approximately one-third of the reaction seen with albumin.

Principle

The capacity of albumin to bind with various substances serves as the basis of several methods of albumin
measurement. Albumin can bind with certain dyes to form a colored complex that is measured
spectrophotometrically. Bromcresol green is mixed with the albumin in the sample. The pH of the reaction is
maintained by citrate buffer. The absorbance of this complex formed in proportional to the albumin
concentration in the sample.

Reference Range

Serum: 3.8 – 5.1 g/dL

Teaching and Learning Activities

Pre-Analytical Phase

1. Disinfect the working area using a disinfectant.

2. Prepare all the materials/equipment needed.
3. Wear all personal protective equipment (PPE) before starting the activity.

Analytical Phase

Materials

Test tubes
Spectrophotometer
Centrifuge
Water Bath
Serological and Automatic Pipettes

Reagents and Sample

Albumin Reagent
Albumin Standard – 6.0 g/dL
Nonhemolyzed specimen

Procedure

1. Remove the amount of reagent to be used for testing and allow to warm to ambient temperature.
2. Prepare two test tubes and label each as Standard and Test or Control (If available).

REAGENTS STANDARD 6.0 g/dL TEST SERA/CONTROL

Protein Standard 10 µL -

Test Sera/ Control - 10 µL

Protein Reagent 1.0 mL 1.0 mL

3. Mix gently and color develops immediately.

4. Read the absorbance of the samples at 550 nm against reagent blank within 15 minutes.

Calculation of Results

Absorbance of the Unknown

Concentration of the sample=
X 6.0 = g/dL
Absorbance of the Standard

Globulins

Total Protein – Abumin = Globulins

Post-Analytical Phase

1. Segregate and dispose all wastes into proper trash bins.

2. Disinfect the working area using 10% sodium hypochlorite.
3. Remove all the PPE properly after the activity.
 References

1. Bishop, M.L., Fody, E.P., Schoeff, L.E. (2013). Clinical Chemistry: Principles, Techniques and Correlations
7th ed. Philadelphia: Lippincott Williams and Wilkins.
2. Nuevo, J.J. (n.d.). Clinical Chemistry 1 Laboratory Manual (Outcomes Based Education Format).
3. McPherson, Richard A. and Matthew R. Pincus. Henry’s Clinical Diagnosis and Management by
Laboratory Methods. 22nd edition. Philadelphia: Elsevier Inc., 2011.
Name: Date:

Section: Group: Score:

Review Questions

1. What physical characteristics of albumin differentiate it from globulins?

2. What is the common and reliable way of quantitating protein fractions in electrophoresis? Give the
reference values for each fraction as a percentage of the total protein.

3. Briefly explain why albumin, globulin and total protein are increased in dehydration but decreased in
third degree burns.
4. Briefly discuss the procedure for polyacrylamide gel electrophoresis and its applications.

5. Enumerate and tabulate the minor proteins and indicate the disease/s associated with each type.