MLS 414 LAB – CLINICAL CHEMISTRY

AMINO ACIDS AND PLASMA PROTEINS

2ND SEMESTER | MIDTERM | DR. JHANNEAL BIANCA RAMIREZ, RMT, ASCPI, MD

OUTLINE
I. Total Protein Methods
A. Kjeldahl Method
B. Biuret Method
C. Dye Binding
D. Refractometry
II. Fractionation, Identification, and Quantification of
Specific Proteins
A. Salt Fractionation
B. Dye Binding
III. Total Globulins
IV. Electrophoresis
● Serum proteins are precipitated with an organic acid
V. Serum Protein Electrophoresis
(TCA or Tungstic Acid).
VI. Selected Densitometric Patterns of Protein
Electrophoresis ○ Start with the precipitation of proteins. Proteins will
VII. High Resolution Protein Electrophoresis be precipitated with the presence of tungstic acid.
VIII. Other Methods ● The non protein nitrogen is removed with the
IX. Proteins in Other Body Fluids supernatant.
A. Urinary Protein ○ Pag naa na si tungstic acid, the non-protein
B. Urine Protein Methods nitrogens will also be removed with the supernatant
C. CSF Protein ● Protein pellet is digested in the sulfuric acid with heat
(340 - 360 degrees celsius)
TOTAL PROTEIN METHODS ○ Proceed with digestion
● Serum (sample of choice) ○ Cupric Sulfate = catalyst (speed the reaction)
● Need not be fasting ○ Potassium Sulfate = increase boiling point
○ Sample can be taken at any time of the day whether ● Sulfuric acid oxidizes the C,H, and S in protein to CO2,
or not the patient had a meal H2O, and SO2.
● Lipemia may interfere ● The nitrogen is converted to ammonium bisulfate
○ Must be contraindicated because it could (NH4HSO4), which is then measured by adding alkali
interferences in the procedures and distilling the ammonia into boric acid solution.
● Hemolysis ● Ammonium borate is then titrated with a standard
○ Elevate Total Protein because off the release of solution of HCl to determine the amount of nitrogen in
RBC proteins in the serum the original protein solution.
KJELDAHL METHOD
● Classic method (determine Nitrogen) 1. Digestion
● Not used in Clinical Laboratory ● Will occur if you mix the sample with sulfuric acid and
○ Time-consuming heat between 340°C - 360°C
○ The procedure is way too tedious for it to be a part ● To speed the reaction, a catalyst will be added. Cupric
of routine activity sulfate is utilized. In some cases, another reagent is
○ Requires many reagents added that is potassium sulfate in order to increase
● Average of 16% nitrogen as in proteins the boiling point to improve the efficiency of the
○ In order to calculate the protein concentration digestion
○ The actual nitrogen content of protein may actually ● Sulfuric acid will oxidize the carbon, hydrogen, and
vary ranging from 15.1% - 16% sulfur in the protein to become CO2, CO, H2O, SO2
○ An error can be introduced if the protein standard ● Nitrogen in the protein will be converted to become your
which was calibrated by the Kjeldahl method that it ammonium bisulfite which is then utilized for
may actually differ in protein composition compared to neutralization and distillation
the sample 2. Neutralization and Distillation
● No proteins of significant concentration in the ● In neutralization, your ammonium bisulfite was highly
unknown specimen are lost in the precipitation step exposed to an acid which is sulfuric acid which needs
which is the first step of the method to be neutralized before proceeding to distillation
● The neutralizing agent is by the addition of an alkaline,
sodium hydroxide

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● Once it is neutralized, proceed with distillation into a
standard boric acid solution
● From ammonium bisulfite, because of the presence of
your boric acid, it now becomes your ammonium
borate
3. Titration
● Ammonium borate will be titrated with standard HCl so
we can be able to determine the number of proteins
that is present on the patient sample
BIURET METHOD
● Most widely used method
● Recommended by IFCC (International Federation of
Clinical Chemistry) for determination of Total Protein
● Cupric ions complex with the groups involved in
peptide bond
○ There will be a violet-colored soln if there are at
least two peptide bonds detected in the sample
● Alkaline medium and at least 2 peptide bonds, a
violet-colored chelate formed.
● Biuret Reagent
○ Potassium hydroxide
■ Provides an alkali medium so the reaction can
take place.
■ Contains sodium potassium tartrate to
complex cupric ions
■ Potassium iodide as antioxidant
○ Sodium potassium tartrate
■ Complex cupric ions to prevent their
precipitation in the alkaline solution
○ Potassium iodide
■ Acts as antioxidant
● Absorbance is measured at 540 nm
● Also determines the size of the protein particles that is
present on the samples
● When small peptides react, the color of the chelate
produced has a different shade than that seen with
larger peptide
● Color varies from pink to a reddish violet
● Color formed is proportional to the number of peptide
bonds and reflects the Total Protein level
○ If pink = lower TP level
○ If reddish violet = higher TP level
● In (abnormally small proteins) multiple myeloma,
c-protein concentration is underestimated due to lighter
shade of color produced
○ Smaller peptides could produce lighter shade
compared to samples with larger protein
● Lipemia in the sample is interferent
○ High lipid content must be rejected as it will interfere
to the procedure
DYE BINDING
● Ability of most proteins in serum to bind dyes
● Most common dye utilized: Coomassie brilliant blue
250 relies on the binding to protein
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○ When it forms complexes with the protein in the
sample, it will be causing a shift of the absorbance of
the dye from 465-595 nm
● A shift absorbance maximum of the dye from 465-595
nm
● Absorbance at 595 nm is used to determine the
protein concentration
● Aside from coomassie brilliant blue, other dyes may
be used such as:
○ Bromothymol blue
○ Ponceau S
○ Amido black 10b
○ Lissamine green
● Although it can be a simple and fast method, caution
must still be applied in utilizing this method because
individual proteins tend to have different affinity to the
dyes to be utilized
Principle of Dye-binding Method in quantifying TP
● Coomassie Brilliant Blue - dye utilized
● With the presence of the protein in the sample, it is going to
form a dye-protein complex
● With this formation, we have a change in the maximum
absorbance of the dye which started at 465 - 490 nm to
shift to its maximum absorbance of 595 nm
● Increased in absorbance at 595 nm, is utilized to determine
the protein concentration of the sample
REFRACTOMETRY
● A quick alternative to chemical analysis of serum total
protein when a rapid estimate is required
● Measurement of RI due to solutes in serum
○ Refractive Index (RI) can be accurate in measuring
the serum protein concentrations, but not in urine
protein measurements because of the excess
amounts of solutes in relation to the protein
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FRACTION, IDENTIFICATION, AND QUANTIFICATION
OF SPECIFIC PROTEINS
● Albumin
● Globulin
○ These 2 proteins can give us useful diagnostic
information to determine the presence of kidney or
liver disease, we refer to it as A/G ratio
○ Get total protein, and determine the levels of either
albumin or globulin (most often its albumin)
○ Then, subtract to the total protein and we can get
the levels of globulin
○ If there’s a reversal or significant change in the ratio
of albumin and total globulin, it will aid us in
determining if the patient has problems in the kidney
or liver
SALT FRACTIONATION
● Fractionation of proteins is commonly done using the
precipitation technique:
● Globulins are separated from albumin by salting out,
using sodium salt to precipitate globulins
○ Globulin - precipitate
○ Albumin - supernatant fluid (measured)
■ Measured thru routine total protein
technique.
● Biuret test
● Dye binding method
○ Salting is no longer used bcoz we have available
methods that can directly react with ur albumin in the
given sample.
DYE BINDING
● Dye-binding procedures
○ most widely used methods for determining Albumin.
● pH of the solution is adjusted so that albumin is
positively charged.
○ If it is in complex with the albumin, maximum
absorbance is going to change
● Albumin is attracted to and binds to an ionic dye
● Amount of albumin
○ (calculated by) absorbance of the albumin-dye
complex
1. Methyl Orange
● nonspecific for albumin
● Beta-lipoproteins, alpha-1 and alpha-2 globulins
also bind to dye
○ α = HDL
○ β = LDL/VLDL
2. 2,4’-hydroxyazobenzene-benzoic acid (HABA)
● low sensitivity but more specific for albumin
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● salicylates, penicillin, conjugated bilirubin, and
sulfonamides interfere albumin to HABA
3. Bromocresol Green (Bcg)
● Not affected by any interfering substances that can
be present in the sample such as bilirubin and
salicylates.
● hemoglobin binds to BCG (for every 100 mg/dL of
Hgb, albumin increased by 0.1 g/dL)
● has been reported to overestimate low albumin
values (nephrotic syndrome or end-stage renal
disease)
4. Bromocresol Purple (Bcp)
● alternate dye, binding specifically to albumin
● not subject to interferences, precise, exhibits
excellent correlation with immunodiffusion reference
methods
● Disadvantage: In renal insufficiency, BCP method
underestimates serum albumin
● Bilirubin interferes with BCP binding to Albumin
TOTAL GLOBULINS
● Albumin can be calculated by subtraction of the globulin
from Total Protein.
● Total Globulin level- direct colorimetric method using
glyoxylic acid.
● Glyoxylic acid, in the presence of Cu2+ and in an acid
medium (acetic acid and H2SO4), condenses with
tryptophan found in globulins to produce purple color.
○ If in case that the methods for albumin
determination is not available, we can still get the
albumin levels by calculation.
○ Read spectrophotometrically, kukunin yung
absorbance and will be utilized to compute the total
globulin levels.
○ Determination of TG based on the presence of
tryptophan is not commonly used because mas easier
gamitin ang dye binding method for albumin
determination.
ELECTROPHORESIS
● separates proteins on the basis of their electric charge
densities.
● protein will move according to their density determined
by pH of a surrounding buffer.
● direction of movement depends on charge
○ Cations → cathode terminal (-)
○ Anions → anode terminal (+)
● the speed of migration can be estimated from the
difference between the pIof the protein and the pH of
the buffer.
● velocity depends on electric field strength, size, and
shape of molecule, temperature and characteristics of
buffer
● Cellulose acetate or agarose gel- support media
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○ After determining the total albumin and globulin * Reference values for each fraction (Bishop, 7th ed.)*
levels, if abnormalities are noticed then we can ● Rank according to % in the total protein
proceed to electrophoresis ○ 1st = Albumin
SERUM PROTEIN ELECTROPHORESIS ○ 2nd = Gamma
● Serum are applied close to the cathode end of a ○ 3rd = Beta
support medium (ph 8.6) ○ 4th = Alpha
● All major serum proteins carry a net negative charge at ○ 5th = Alpha - 1
ph 8.6 and migrate toward the anode.
● 5 bands: SELECTED DENSITOMETRIC PATTERNS OF PROTEIN
○ Albumin- travels farthest to the anode ELECTROPHORESIS
○ alpha-1-globulins ● Reference Pattern
○ alpha-2-globulins
○ beta-globulins
○ Gamma-globulins
● Width of the band = number of proteins present in that
fraction.
● After separation, protein fractions are fixed by
immersing the support medium in an acid solution
(acetic acid)
● Next step, proteins are stained.
○ Dyes
■ Ponceau S
■ Amido black
■ Coomassie blue
● Cleared transparent medium is placed in a scanning
densitometer for reading
● The pattern on the membrane moves past a slit through
which light is transmitted to a phototube to record the
absorbance of the dye that is bound to each protein
fraction ○ All protein fractions are in normal level
○ The absorbance is going to determine the quantity
of the protein that is present ● Monoclonal Increase
● Absorbance is recorded on a strip-chart recorder to
obtain a pattern of the fraction
○ This pattern is going to be the basis as to what type
of protein has caused the abnormality that led to the
px condition
● The scanning densitometer will compute the area under
the absorbance curve for each band and the
percentage of total dye that appears in each fraction.
○ This percentage will be multiplied to the total protein
which is measured separately
● Concentration is calculated as a percentage of the total
protein
● Computation also be made by cutting out the small
bands from the membrane and eluting the dye in 0.1
mol/L NaOH
○ Absorbances are added to obtain total absorbance,
and the percentage of the total absorbance found in
each fraction is calculated

○ Sharp peak in the gamma globulin area

○ Increased production of gamma globulins

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● Alpha-1-Antitrypsin Deficiency
○ Deficiency in alpha-1
● Nephrotic Syndrome
● Cirrhosis
○ Px tend to lose some of their serum albumin and low
molecular weight proteins including some globulin
gamma
○ Increased alpha-2 macroglobulin, beta lipoprotein,
complement components and haptoglobin
● Inflammation
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● Decrease in albumin but increase in alpha-1, alpha-2,
and beta bcs of increased production of acute phase
reactants
■ Alpha-1 = acid glycoprotein, alpha-1-antitrypsin
■ Alpha-2 = ceruloplasmin & haptoglobin
■ Beta = C reactive protein
● Commonly seen in trauma, burns, infarction,
malignancy, and liver disease
● Why acute phase reactants?
○ Since they are increased mainly in the serum within a
few days following trauma or exposure to inflammatory
reagents
○ Fibrinogen, haptoglobin, ceruloplasmin, serum amyloid
a = will increase several folds
○ C reactive protein & alpha-2 macroglobulin will increase
several hundred folds
○ Good indicators of the presence of inflammation
● If gamma is increased = chronic infection
● Distinct bcs of the presence of beta gamma bridge
○ Caused by the presence of fast moving gamma
globulins in the px sample that prevents distinct bands
from appearing between the gamma and beta globulin
fractions therefore, forming a bridge between two
○ Continuous pattern
■ Caused by fast moving
HIGH RESOLUTION PROTEIN ELETROPHORESIS
● Modifying electrophoretic parameters (12 bands)
● Uses higher voltage coupled with a cooling system
and 4 more conc. buffer.
● Agarose gel
○ support medium
● semiquantitative estimates of protein
○ Densitometer
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● useful in detecting small monoclonal bands and
differentiating unusual bands
Prealbumin - fastest to migrate
OTHER METHODS
● Capillary Electrophoresis
○ separation of molecules takes place in silica
capillaries
○ capillaries are typically 30-50 cm long
○ Amount of sample is nm
● Isoelectric Focusing
○ zone electrophoresis that separates proteins
on the basis of pI
○ When a protein is electrophoresed in the gel,
it migrates to a place in the gel where the pH
is the same as its isoelectric point
● Immunochemical Methods
○ specific proteins may be identified by
immunochemical assays in which the
reaction of protein (antigen) and antibody is
measured
○ radial immunodiffusion,
immunoelectrophoresis, immunofixation
electrophoresis, electro immunodiffusion,
immunoturbidimetry, immunonephelometry
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○ Protein serves as the antigen (same with
racket)
PROTEINS IN OTHER BODY FLUIDS
URINARY PROTEIN
● Can originate from the kidneys and the urinary tract,
lso vagina and the prostate
● Proteinuria
○ resulting from glomerular or tubular
dysfunction
○ Useful indicator for problems of glomerular
or tubular portions of the kidney
● Qualitative test: Reagent test strip
○ Dip the strip in the urine
○ Observe for color
○ Protein error of indicators
● Quantitative test: 12-24 hour urine specimen
○ Results are generally reported in terms of
weight of protein for 24hrs by calculating the
amount of protein that is present in the total
volume of the urine collected during that time
● Precipitation methods: Sulfosalicylic Acid (SSA),
Trichloroacetic acid (TCA), Benzethonium chloride
● Chemical Methods: Biuret Method, Folin-Lowry
Method
○ Biuret agent is mainly with the principle of
the complex formed by the cupric ions of the
biuret reagent with the peptide bonds which
gives off a pink to reddish-violet color
○ Folin-Lowry Method, it is going to use
Folin-Ciocalteu′s reagent which is a
phosphotungstomolybdic acid solution or
frequently called as phenol reagent because
it is able to oxidize phenolic compounds
■ The reagent will change in color
from yellow to blue during the
reaction with the following amino
acids mainly tyrosine, tryptophan,
and histidine that is present in the
urine sample
● Dye-binding methods: Coomasie blue, Ponceau S
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URINE PROTEIN METHODS ● Degree of permeability can be evaluated by

measuring the CSF albumin comparing it with the
serum albumin
Method Principal Comment
○ Albumin is a very good indicator of nervous
Turbidimetric Proteins are Rapid, easy to system problems because it is not produced
methods precipitated as use; (problem) in the CSF , but in the liver
(sulfosalicylic fine particles, unequal ○ In the event that albumin is higher in CSF
acid, turbidity is sensitivity for than serum albumin, that is indicating
trichloroacetic measured individual person increased permeability of capillaries or a
acid, or spectrophotometri
problem in the blood-brain barrier
benzethonium cally
chloride)

Biuret Proteins are Accurate

concentrated by
precipitation,
redissolved in
alkali, then
reacted with
Cu2+, Cu2+
forms colored
complex with
peptide bonds

Folin-Lowry Initial biuret Very sensitive

reaction; oxidation
of tyrosine, 5
tryptophan, and
histidine residues
by Folin phenol
reagent (mixture
of
phosphotungstic
and
phosphomolybdic
acids);
measurement of
resultant blue
color

Dye-binding Protein binds to Limited linearity;

(Coomassie blue, dye, causes shift unequal
Ponceau S) in absorption sensitivity for
maximum individual proteins

CSF PROTEIN WHAT TO EXPECT DURING SPINAL TAP

● The presence of proteins in the CSF is indicative of
infection, inflammation, or any other types of nervous
system disorder ○
○ Also, it can indicate a problem in the
capillary-endothelial barrier through which
ultrafiltration occurs
● Reference interval (10-40 years old)
○ 15-45 mg/dL
● CSF is one of the most carefully handled samples in
● Abnormal increased of total CSF Proteins
the lab because of the difficulty of its extraction
○ Conditions will include bacterial ,viral and
● Means of collecting: lumbar of spinal tap
fungal meningitis, traumatic tap during CSF
● The doctor will do the collection by inserting a syringe
collection, multiple sclerosis, obstruction in
between lumbar 4 and 5 vertebra
the neoplasm, and cerebral infarction
1. Lie on table in fetal position

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2. Doctor injects anesthetic to numb lower back

3. Needle inserted between lower back bones
4. Small amount of cerebrospinal fluid drawn

● The CSF must be separated into three tubes ○

○ Tube 1: chemistry / serology
○ Tube 2: microbiology
○ Tube 3: hematology
○ The excess CSF must be kept in another
tube just in case there are additional tests to
be performed in the sample
● The accepted sample must appear clear
● If there is presence of blood, hemolysis,
xanthochromia because of the presence of bilirubin -
it must be rejected
● But if after centrifugation, you have a CSF with clear
supernatant and formation of red cell button in the
bottom - can still be accepted for processing

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