CLINICAL CHEMISTRY – RMT 2024
LEC 9 – PROTEINS (PART 2)
Mr. Fritz Bucao, RMT
October 2022
PROTEIN (METHODS OF ANALYSIS)
Total Nitrogen
● Measures all chemically bound nitrogen in the sample. The
method can be applied to various biologic samples, including
plasma and urine.
→ Plasma measures: Total protein and Non-protein
nitrogenous compounds such as Urea and Creatinine
● Useful in assessing a person’s nitrogen balance
Patients receiving Total Parenteral Nutrition (TPN)
Individuals with neurologic injuries who are sustained on intravenous
fluids for suspended periods of time.
Monitoring their nutritional status is particularly important.
Method Used: Chemiluminescence
The sample in the presence of oxygen, is heated to a high
temperature. Any chemically bound nitrogen is oxidized to nitric
oxide. The nitric oxide is then mixed with ozone (O3) to form an
excited nitrogen dioxide molecule (NO2)
→ When the excited nitrogen dioxide molecule decays to the
ground state, it emits a chemiluminescent light which is
then detected, amplified, and converted to an electronic
signal proportional to the total nitrogen content in the
sample and this signal is compared to the standard for
quantitation.
Total Proteins
Most often used specimen: Serum (instead of Plasma)
Fasting specimen is not needed.
Interferences: Lipemia and Hemolysis
Falsely increase total protein result due to release of RBC protein into
the serum.
Avoid lipemic and hemolyzed sample.
Reference Interval for
6.5 to 8.3 d/dL (65 to
Serum Total Protein for
83 g/L)
Ambulatory Adults
Serum Total Protein Concentration 6.0 to 7.8 d/dL
in the Recumbent Position (60 to 78 g/dL)
Method I: Kjeldahl Method
The classical method for quantitation of total protein, which
determines nitrogen.
In this method, an average of 16% nitrogen mass in protein is
assumed to calculate the protein concentration.
Actual nitrogen content of serum protein varies from 15.1%-16.8%.
Error is introduced if a protein standard is used that differs in
composition from the serum specimen to be analyzed because the
percentage of nitrogen will not be the same.
 Not used in the lab anymore because it is time-consuming and
too tedious
 Requires assumption that no proteins of significant
concentration in the unknown specimen are lost in the
precipitation step.
Procedures:
1. The serum proteins are precipitated with an organic acid such
as TCA or tungstic acid.
2. The protein pellet is digested in H2SO4 (sulfuric acid),
considered as digesting agent with the heat at 340C to 360C
and a catalyst, such as cupric sulfate, to speed the reaction.
Can also use Potassium sulfate to increase the boiling point,
to improve efficiency of digestion
 The sulfuric acid oxidizes the Carbon (C), Hydrogen
(H), and Sulfur (S) in the protein to Carbon dioxide,
Carbon monoxide, Water, and Sulfur dioxide (CO2,
CO, H2O and SO2)
3. The nitrogen in the protein is converted to ammonium
bisulfite (NH4HSO4), which is then measured by adding alkali
and distilling the ammonia into a standard boric acid solution.
 The ammonium borate form is then titrated with a
standard solution of hydrochloric acid to determine
the amount of nitrogen in the original protein
solution.
Clinical Chemistry 1, CDU – BSMT3
NOTE:
At birth, total protein concentration is lower, reaching adult levels by
the age of three.
As the person ages, there is a slight decrease of albumin levels.
Low total protein levels are seen in pregnant people.
Method II: Biuret Method
The most widely used method and the one recommended by the
International Federation of Clinical Chemistry (IFCC) 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 absorbance of colored chelate form is measure
at 540nm
If there are small peptides that react, the chelate color will produce
different shades than that seen of the larger peptides.
 the color will vary from a pink to a reddish-violet
The color formed is proportional to the number of peptide bonds
present.
It will reflect the total protein level. However, in the presence of
smaller proteins such as those seen in patients with multiple
myeloma, the C protein concentration is underestimated due to the
lighter shade of the color produced.
Interference: if lipemic sample is used
Acquired its name due to the substance biuret (NH2CONHCONH2)
which reacts with cupric ions.
Reagents:
1. Alkaline Copper Sulfate,
2. Rochelle Salt or Sodium Potassium Tartrate (NaK
Tartrate)
3. NaOH and Potassium Iodide
Method III: Dye Binding Method
Based on the ability of most proteins in serum to bind dyes,
although the affinity with which they bind may vary.
A dye-binding method, Coomassie brilliant blue 250, relies on the
building of Coomassie brilliant blue 250 to protein, causing a shift in
the absorbance maximum of the dye from 465 to 595 nm
Most common stains used to stain protein bonds after
electrophoresis:
 Bromophenol blue
 Ponceau S
 Lissamine Green
 Coomassie brilliant blue
Although the method is simple and fast, the unequal dye-binding
responses of the individual proteins require caution when applying
to the complex measurement of protein found in the serum.
Fractionation, Identification, and Quantitation of Proteins
• In the assay of totals serum proteins, useful diagnostic
information can be obtained through determining the albumin
fractions and globulins.
• There is a reversal or significant change of albumin and total
globulin. The A-G ratio is found in the diseases of the kidney
and liver.
 To determine the A-G ratio, total albumin and globulin
are measured.
 To get globulins, subtract total protein with albumin.
Salt Fractionation
• process of Precipitation
 Globulins are separated from albumin by salting out
using sodium salt to cause precipitation of globulins
 The albumin that remains in solution in the supernatant
is then measured by any of the routine total protein
methods
• Not used today because there are direct methods already
available that reacts specifically with albumin and mixture of
proteins
Page 1 of 5
Dye Binding Electrophoresis
• The albumin is attracted to the binds to an anionic dye by • “phoresis” → separation, migration, or movement
electrostatic forces “electo” → under the influence of an electric field
• 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
• Most widely used method in determining albumin.
• The pH of solution is adjusted so that the albumin is
positively charged.

Most Common Dyes Used

Methyl Orange  Nonspecific for albumin
 Beta lipoproteins, alpha 1 and 2
globulins will also bind to this dye.

Bromocresol green (BCG)  Not affected by interfering substances

such as bilirubin and salicylates
 However, hemoglobin will bind to BCG.
 For every 100 mg/dL of hemoglobin,
albumin is increased to 0.1 g/dL • When an abnormality is found in total protein or albumin,
2,4-hydroxyazobenzenebenzoic  Has low sensitivity, but more specific electrophoresis is performed.
acid (HABA) for albumin  Also, if an abnormality is seen in electrophoretic
pattern, an analysis of the individual proteins within
Bromocresol purple (BCP)  alternate dye for albumin the area of abnormality is made.
determination
 It binds specifically to albumin. NOTE:
 It is not subjected to most
Electrophoresis separates proteins on the basis of their electric
interferences. It is also precise and
charge densities.
exhibits excellent correlation with
immunodiffusion reference method.
Protein, when placed in an electric current, will move according to
 In patients with renal insufficiency,
their charge density, which is determined by the pH of a surrounding
BCP will underestimate the serum
buffer. The direction of the movement will depend whether the charge
albumin. The serum of this patient
is positive or negative.
appears to contain either a substance
tightly bound to albumin or a
• Cations (positive net charge) migrate to the cathode
structurally altered albumin that affects
(negative terminal), whereas anions (negative net charge)
the binding of BCP. Bilirubin will
migrate to the anode (positive terminal).
interfere with BCP binding to albumin,
• The speed of the migration can be estimated from the
while BCG binding is unaffected.
difference between the pI of the protein and the pH of the
 Most commonly preferred than BCG
buffer.
 The more the pH of the buffer differs from the pI, the
Total Globulins greater is the magnitude of the net charge of that
• Albumin can then be calculated by subtraction of globulin from protein and the faster it will move in the electric field.
total protein.
• Total globulin of serum is determined by a direct colorimetric
method. TYPES
 Glycolic acid is used in the presence of cupric ions
Depending upon the nature of supporting medium
and an acid medium. It condenses with
tryptophan found in globulins to produce a purple • Agar gel electrophoresis (AGE)
color. • PAGE, SDS PAGE, QPNC PAGE (Quantitative Preparative
 Albumin has approximately 0.2% tryptophan, • Continuous PAGE)
compared with 2% to 3% for the serum
• Cellulose acetate electrophoresis
globulins. When calibrated using a serum of
• Capillary electrophoresis
known albumin and globulin concentrations, the
total globulins can be determined. Depending upon the mode of technique
 The measurement of globulins based on
tryptophan content has never come into common • Slide gel electrophoresis
use because of the ease and simplicity of the dye • Tube gel electrophoresis
binding method. • Disc electrophoresis
• Low and high voltage electrophoresis
• 2-dimensional gel electrophoresis
APPLICATION

• Separating serum proteins for diagnostic purposes

• Hemoglobin separation
• Lipoprotein separation and ID
• Isoenzyme separation and their analysis
• Nucleic acid synthesis
• Determination of MW of the proteins
FACTORS AFFECTING ELECTROPHORESIS

• The electric field (Voltage, Current, Resistance)

• The sample (Charge, Size, Shape)
• The buffer (Composition, Concentration, pH, Supporting
medium)
TYPES OF BUFFERS

• Tris buffer
• Glycine buffer
• Sodium barbituric acid
• TAE buffer (Tris acidic EDTA)
TYPES OF STAINS
• Amino block
For serum proteins
• Coomassie brilliant blue
For isoenzyme • Nitrotetrazolium blue

Clinical Chemistry 1, CDU – BSMT3 Page 2 of 5

 • Fat red 7B REFERENCE VALUES FOR EACH FRACTION
For lipoprotein zones • Oil red O Albumin 53% to 65% of the total protein 3.5 to 5.0 g/dL
• Sudan Block B α1-Globulin 2.5% to 5% 0.1 to 0.3 g/dL
α2-Globulin 7% to 13% 0.6 to 1.0 g/dL
For DNA fragments • Ethidium bromide β-Globulin 8% to 14% 0.7 to 1.1 g/dL
For CSA proteins • Silver nitrate γ-Globulin 12% to 22% 0.8 to 1.6 g/dL

SERUM PROTEIN ELECTROPHORESIS

Principle: migration of charged particles in an electric field

• Single most clinical application of Serum Protein Electrophoresis

is for the identification of monoclonal spike of
immunoglobulin and differentiating them from polyclonal
hyper gamma globulinemia.
• Serum sample are applied to cathode. Saturated of alkaline
buffer of the pH of 8.6.
• The support medium is connected with two electrodes in which
the current is passed through the medium to separate proteins.
• All major serum proteins carry a net negative charge at pH 8.6
and migrate toward the anode.
It will appear in 5 bands.

• Albumin – travels farthest to the anode

• Alpha 1 globulins ABNORMAL PATTERNS
• Alpha 2 globulins MONOCLONAL GAMMOPATHY
• Beta globulins • most significant finding from electrophoresis pattern
• Gamma globulins • Disease: Monoclonal immunoglobulin disease

• The width of the band of proteins in a fraction depends on the

number of proteins present in that fraction.
MATERIALS NEEDED
 a polysaccharide made from
Agarose seaweed. Agarose is
dissolved in buffer and
heated, then cools to a
gelatinous solid with a
network of crosslinked
molecules
 Some gels are made with
acrylamide if sharper bands
are required.
Buffer  in this case TBE
 The buffer provides ions in
solution to ensure electrical
conductivity.
 Not only is the agarose
• The result of monoclonal gammopathy in an electrophoretic pattern
dissolved in buffer, but the
will involve a spike increase in the gamma and also in beta,
gel slab is submerged sometimes also in the alpha 2 region. If there is an increase of that,
(submarine gel) in buffer you are signaled to examine the immunoglobulins and observe the
after hardening. clinical signs of myelomatosis.
Power supply and gel  gel chambers come in a • If there is a deficiency in the predominant immunoglobulin, it is seen
chamber variety of models, from in much paler stain in gamma area.
commercial through home-
made, and a variety of sizes NEPHROTIC SYNDROME

1. The comb is removed, leaving little wells and buffer is poured

over the gel to cover it completely.
2. The serum samples are mixed with a dense loading dye so they
sink into their wells and can be seen.
3. The serum samples are put in the wells with a micropipette.
a. Micropipettes have disposable tips and can
accurately measure 1/1,000,000 of a liter.
4. Separated fraction is visualized by using appropriate stains.
• patient loses serum albumin and has low molecular weight proteins
5. Quantification of each fraction is done by either densitometer in the urine. Some IgG of these patients are also loss
or elution followed by a colorimeter or spectrophotometer of • There is an increase of alpha 2 macroglobulin, beta lipoprotein, and
the eluted fraction. complement components of haptoglobin
• There is a decrease relative amount of albumin

Clinical Chemistry 1, CDU – BSMT3 Page 3 of 5

ACUTE INFLAMMATORY RESPONSE • The presence of small spikes in beta region is due to iron deficiency
anemia. Transferrin is responsible for this.
• Rheumatoid arthritis or malignancy will result to a polyclonal
gammopathy classified as chronic inflammation.

HIGH RESOLUTION PROTEIN ELECTROPHORESIS

• occurs with stress, inflammation caused by infection, injury or

surgical trauma
• Has a normal or decreased albumin, increased in alpha 1 and alpha
2, and double increase in gamma globulins

CHRONIC INFLAMMATORY RESPONSE

• As we all know, it has only 5 distinct bands but in HRE, this has 12
bands.
• More specific to what protein
• The modification will use a higher voltage coupled with a cooling
system in an electrophoretic apparatus with a more concentrated
buffer used. The support medium most commonly used is agarose
gel
• To obtain this sample, samples are applied on the agarose gel,
electrophorized in a chamber cooled by a gel block, stain it, then
visually inspect.
• Each band is compared with the same band in a reference pattern
for color, density, appearance, density rates, and appearance of
abnormal bands in regions of density.

• If there is a chronic or inflammatory response, this is correlated with

chronic infection such as autoimmune disease, chronic liver
diseases, cancer.
• Has a normal or decreased albumin, increased in alpha 1 and alpha
2, and double increase in gamma globulins

LIVER DAMAGE/HEPATIC CIRRHOSIS

CAPILLARY ELECTROPHORESIS

• caused chronic alcohol abuse or viral hepatitis.

• Has decreased albumin, alpha 1 and alpha 2, and beta globulins.
Increase in IgA in gamma fraction.

ABNORMAL SERUM ELECTROPHORETIC PATTERNS

Gamma spike Multiple myeloma
Beta-gamma bridging Hepatic cirrhosis
A2 globulin band spike Nephrotic syndrome
Juvenile cirrhosis
A1 globulin flat curve
(AAT deficiency)
Spikes of a1, a2, B globulin bands Inflammation
NOTE: memorize this kay if nay case study sa exam na astang taasa
kay sir said if u see these words kay mao na daw answer :>

• The presence of free hemoglobin will cause blip. Present in the late • A collection of techniques in which the separation of molecules takes
alpha 2 or early beta zone region. place in silica capillaries.

Clinical Chemistry 1, CDU – BSMT3 Page 4 of 5

 • are typically 30 to 50 cm long, with an internal diameter between 25
and 100 μm
• In capillary zone electrophoresis, the capillaries are filled with a
conducting solution, usually an aqueous buffer. The detection end
of the capillary is grounded and the sample injection end is
connected to a high-voltage power supply.
• When a positive voltage is applied, the positively charged buffer
molecules flow to the detection end, which is negative relative to the
injection end. The net flow of buffer is called electro osmotic flow
(EOF). When a sample is injected, all molecules have a tendency to
move toward the detector (negative) end of the capillary due to EOF;
however, the negatively charged molecules in the specimen also
have a tendency to migrate back toward the injector (positive) end.
This is referred to as electrophoretic mobility.
• EOF is usually stronger than electrophoretic mobility
• The separated molecules are detected by their absorbance as they
pass through a small window near the detection end of the capillary.
Use of the capillaries allows heat to be effectively dissipated, which
means that higher operating voltages can be used and, therefore,
analysis times are faster. Additionally, the sample size required is
small (nanoliters).

ISOELECTRIC FOCUSING

• Isoelectric focusing (IEF) is zone electrophoresis that separates

proteins on the basis of Pi
• IEF uses constant power and polyacrylamide or agarose gel
mediums, which contain a pH gradient. The pH gradient is
established by the incorporation of small polyanions and polycations
(ampholytes, or molecules that contain both acidic and basic groups)
in the gel. The varying pIs of the polyions cause them, in the
presence of an electric field, to seek their place in the gradient and
to remain there. The pH gradient may range from 3.5 to 10
• The clinical applications of IEF include phenotyping of α1-antitrypsin
deficiencies, determination of genetic variants of enzymes and
hemoglobins, detection of para proteins in serum and oligoclonal
bands in CSF, and isoenzyme determinations.

CSF PROTEINS
Reference • 14-45 mg/dL
range:
Increased • Bacterial • Multiple sclerosis
levels (↑): • Viral • Obstruction
• Fungal meningitis • Neoplasm
• Traumatic tap • Disk herniation
• Cerebral infarction
Decreased • Hyperthyroidism
levels (↓) • When fluid is leaking from the CNS

Clinical Chemistry 1, CDU – BSMT3 Page 5 of 5