MLS 11 - CLINICAL CHEMISTRY 1 LECTURE

MODULE 5: PROTEINS
Lecturer: Ma’am Grace Hope Gallego

Note:
OUTLINE OF TOPICS ● The function of proteins is dependent on its constituent amino acids or kung ano ang
I. Introduction to Amino Acids XV. Protein Structure amino acid na present dira
II. Peptide Bonds A. Primary ● Amino acids are organic compounds that contains amino group and carboxyl group
III. Basic Structure of Amino Acids B. Secondary ● Amino acids are peptides of carbohydrates and forms a chain to form polypeptide
IV. Amino Acid Metabolism C. Tertiary chains/proteins link by peptide bonds
V. Essential and Non-Essential Amino Acids D. Quaternary
VI. Analysis of Amino Acids XVI. Charge & Isoelectric Point
VII. Different methods for the Analysis of Amino XVII. Protein Solubility PEPTIDE BONDS
Acids XVIII. Classification by Function
VIII. Aminoacidopathies A. Enzyme Peptide Bonds
A. Phenylketonuria (PKU) B. Hormones
B. Tyrosinemia C. Transport Proteins ● Are covalent bonds formed when the N-terminal end amino group (-NH2) and the
C. Alkaptonuria (AKU) D. Immunoglobulins C-terminal end carboxyl group (-COOH) bond to the alpha-carbon with the amino
D. Maple Syrup Disease (MSUD) E. Structural Proteins group of one amino acid is linked with the carboxyl group of another
E. Isovaleric Acidemia F. Storage Proteins ● For short, the bond that exists between amino acids
F. Homocystinuria G. Energy Source
G. Citrullinemia H. Osmotic Force
1. Type I XIX. Classification by Protein Structure
2. Type II XX. Plasma Proteins
H. Argininosuccinic Aciduria A. Pre-albumin
I. Cystinuria B. Albumin
IX. Proteins C. Globulin
X. Protein Synthesis XXI. Other Protein of Clinical Significance
XI. Anabolism & Catabolism A. Too many to count lol
XII. Protein Digestion XXII. Proteins in Other body Fluids
XIII. Nitrogen Balance XXIII. Total Protein Abnormalities
XIV. Nitrogen Content XXIV. Laboratory Analysis

BASIC STRUCTURE OF AMINO ACIDS

AMINO ACIDS
What makes an amino acid unique from another amino acid?
A. Amino Acids
● Thy are the building blocks/precursors of proteins Ans: R-group
● They function for growth, repair, and maintenance of all cells in the body
● Determines the biologic activity or proteins R-Group
● Amino acid side chains, this is where amino acids differ from one another

Prepared by: MANINGO C.M., MATULAC, S.A., NOBLES, S.K., PE, J.K., PECATE, M.S.
BSMLS 3-D2
2022-2023
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 AMINO ACIDS METABOLISM
Examples: Examples:
● About half of the 20 amino acids needed by humans cannot be synthesized at a rapid
● Alanine - deaminated to pyruvate ● Leucine
enough rate to support growth; they must be supplied in food
● Arginine - converted to alpha-keto glutarate ● Lysine
● Amino acids are released by the normal breakdown of body proteins
● Aspartate - converted to oxalo-acetate ● They degrade acetyl-CoA
● Proteins provide 12-20% of the total body energy requirement
● pyruvate or citric acid cycle intermediate or acetoacetyl-CoA
NOTE:
● Proteolytic Enzymes - involved in the break down/proteolysis of amino acids
● Example: Pepsin and Trypsin - proteolytic enzymes necessary to break large NOTE:
molecules of proteins to amino acids ● Some amino acids can be both ketogenic and glucogenic
● After the breaking down of proteins into amino acids, amino acids will now then be
rapidly absorbed from the intestines to the bloodstream. ESSENTIAL AND NON-ESSENTIAL AMINO ACIDS
● We need to express proteins into amino acids so that our intestines may absorb them
to be transported into the bloodstream, and blood will carry these amino acids into Essential Amino Acids
the storage form/function ● Cannot be made by our own body and must come from dietary intake
● PVT. TIM HALL
Amino Acid groups can be removed from amino acid by either:
Non-Essential Amino Acids
Deamination ● Amino acids that can be generated by the body
● There is removal of the amino group from the amino acid ONLY.
● Enzyme catalyst of deamination process are called deaminases Essential Amino Acids Non-Essential Amino
● Occurs primarily in the liver. Deamination also occurs in the kidneys but only in small Acids
amounts Arginine Alanine
Histidine Asparagine
Transamination Isoleucine Aspartic Acid
● There is also removal of amino groups in amino acids but its difference from Leucine Cysteine
deamination is that, there is transfer of amino groups into ketoacids Lysine Cystine
● The resultant ketoacid can enter into a common metabolic pathway with CHO & fats.
Methionine Glutamine
● Enzymes involved: Transaminases
Phenylalanine Glycine
Threonine Proline
NOTE:
Tryptophan Serine
● Why is it necessary to transfer amino groups to ketoacids?
○ The purpose is that, we can generate amino acid version of keto acids Valine Tyrosine

Glucogenic Amino Acid Ketogenic Amino Acids

● Generates precursors (amino acid) of glucose ● Generates ketone bodies
● Can be converted into glucose as sources of
energy (amino acid → glucose as source of
energy)

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 Classification of Amino Acids DIFFERENT METHODS FOR THE ANALYSIS OF AMINO ACIDS
Non-Polar AA Uncharged AA Acidic Charged AA Basic Charged AA
Glycine Serine Aspartic Acid Histidine A. Thin Layer Chromatography
Alanine Threonine Glutamic Acid Lysine ● Screening Test
Valine Tyrosine Arginine ● The application of either one-dimensional separations depends on the
Leucine Asparagine purpose of the analysis
● Solvents that can be used:
Isoleucine Cysteine
○ Acetic Acid
Phenylalanine Glutamine
○ Water & Ethanol
Tryptophan
○ Ammonia & Water
Methionine
Proline 1. One Dimensional TLC
● Used if we want to search for a particular category of amino acid
TWO NEW AMINO ACIDS ● Used is we want to know if it is a single amino acid or branched amino acid
Selenocysteine Pyrrolysine
● Not encoded directly in the genetic ● Naturally occurring 2. Two Dimensional TLC
code genetically-encoded AA ● Used we we want to know what amino acid is present in the sample

NOTE:
ANALYSIS OF AMINO ACIDS ● To confirm that amino acid is indeed present in the sample, we use Ninhydrin as a
● 6 to 8 hours fasting dye/stain.
○ So that protein will not be affected by dietary conditions ● In a presence of blue color from ninhydrin indicates presence of amino acids in the
○ If heparin is used as anticoagulant, plasma from the blood should be sample
separated immediately
● Avoid hemolysis AMINOACIDOPATHIES
● Perform Deproteinization within 30 minutes
○ Deproteinization - removal of proteins Aminoacidopathies
○ PFF - Precipitation of protein to form protein-free filtrate ● Occurs when there are problems in the breakdown of amino
● Other samples that you can use for AA analysis: acids
○ Urine & amniotic fluid ● Caused by the deficiency or lack of enzymes necessary
○ Urine - ideal sample used for screening tests for the metabolism of amino acids
○ For Urine: ● These conditions are characterized by the accumulation of
■ Random sample - screening purposes amino acids in the body which is toxic for the body
■ 24 Hour Urine sample - for quantification of proteins in urine
● Add thymol to preserve proteins in 24 hour urine 1. Phenylketonuria (PKU)
● We can store urine samples in freezing temperatures of -20°C ● Inherited autosomal recessive trait
to -40°C ● Absence of activity of phenylalanine hydroxylase
(PAH)
○ PAH is necessary to break phenylalanine into tyrosine (
○ If there is absence of PAH, phenylalanine will accumulate in the blood
and will appear in the urine
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 ● Urine: “musty odor”
● Retarded mental development & microcephaly for infants
○ Microcephaly - tiny head
● Tests to detect PKU:
○ Kuvan - screening test
○ Guthrie Test - Semi quantitative test to determine the level of
phenylalanine
■ Bacterial inhibition assay
■ Measures the ability of PKU to facilitate bacterial growth
○ HPLC - reference method for quantitative serum phenylalanine
■ For new-born screening
2. Tyrosinemia
● Inborn metabolic disorders
● Defect on tyrosine catabolism
● Excretion of tyrosine and tyrosine catabolites in urine
● Types: Type I, type II, type III
● Characterized by: Jaundice (build up of tyrosine in the liver, pwede mn sa
kidney)
● Type I: Fumarylacetoacetate hydrolase
○ Cabbage-like urine
○ defect / insufficient
● Type II: Tyrosine aminotransferase
○ Tyrosine cannot be broken down into smaller molecules, thus cannot
be excreted in the kidney and will accumulate.
● Type III: 4-Hydroxyphenylpyruvate dioxygenase
● Common among adults and can be prevented by having low-protein diet
○ NTBC - drug that prevents the formation of maleylacetoacetic acid
and fumarylacetoacetic acid, which can be converted to
succinylacetone
● Test to detect/ monitor Tyrosinemia:
○ Tandem-Mass Spectroscopy - used as confirmatory test to detect
tyrosinemia, specifically the presence of the metabolite of succinyl
acetone
3. Alkaptonuria (AKU)
● Transmission of HGD gene, which causes the lack of the enzyme
homogentisate oxidase
○ Homogentisate oxidase breaks down homogentisic acid, and the
absence of this enzyme will cause homogentisic acid to accumulate in
the blood and appear in urine (Alkaponuria)
● Urine: brownish-black when it mixes with air due to oxidation
● Test to detect AKU
○ Addition of Ferric Chloride in urine; once
urine turns black, it is indicative of
positive homogentisic acid in the
urine/alkaptonuria
○ greyish/blackish/blueish deposits in the
eye, blackish/reddish earwax
○ Thickening of the cartilage in the ears
4. Maple Syrup Disease (MSUD)
● Absence or greatly reduced activity of the enzyme a-ketoacid decarboxylase
○ a-ketoacid decarboxylase - metabolizes leucine, isoleucine, and
valine
○ Leucine, Isoleucine and Valine are branched chain amino acids
○ Accumulation of ketoacids in the blood, urine, and spinal fluid
○ MSUD is hereditary
● Urine: Maple syrup or burnt sugar odor of the urine, breath, and skin
● Test to detect MSUD
○ Modified Guthrie Test & Tandem Mass Spectroscopy Test
■ Test for newborn screening
NOTE:
● Infants that have this condition seem to be normal upon birth, but within a
week, the baby will show signs such as vomiting, lethargy, or loss of appetite
for milk/breast milk, or worse, death
● As maple syrup disease progresses, it can cause severe mental retardation,
seizure for some, hypoglycemia, acidosis
● Characteristic odor of urine: burnt sugar odor of the urine, breath, and skin
5. Isovaleric Acidemia
● A hereditary disorder caused by a mutation of the
gene, particularly deficiency in isovaleryl-CoA
dehydrogenase, preventing normal metabolism of
the branched-chain amino acid
● Isovaleryl-CoA dehydrogenase is necessary for the
breakdown of leucine.
● Since isovaleryl-CoA dehydrogenase is absent,
leucine will be broken down and will start to build
up in the bloodstream.
● This will cause the appearance of symptoms.
● Main Symptom: sweaty-feet odor caused by the build-up of isovaleric acid
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 ■ Patients that have this type have hyperammonemia.
6. Homocystinuria ■ Signs and Symptoms
● A rare metabolic disorder characterized by the accumulation or increased ● Lack of appetite
amount of homocysteine in the urine ● Vomiting
● This is due to the deficiency of the enzyme cystathionine Beta-synthase ● Lethargy
● Cystathionine Beta-synthase is an enzyme ● Seizures for some
responsible for the breakdown of ● Even comatose
methionine to cysteine ● Severe brain damage or even death if not treated earlier
● Deficiency of of cystathionine
Beta-synthase will cause elevation on the ○ Type II Citrullinemia
levels of methionine and homocysteine in ■ Adult-onset
the plasma and of course, in the urine ■ Citrin deficiency
● This disorder affects the eyes, CNS in the ■ Caused by the mutation of the gene SLC25A13
brain, bones, and blood vessels. ■ SLC25A13 gene carries instructions for the generation of Citrin
● Abnormalities affecting the eyes is the ■ Citrin is necessary because it is the carrier of the amino acid
first outward sign of homocystinuria glutamate to the mitochondria. At the same time, it also transports
● Tests to detect Homocystinuria: amino acid aspartate from
○ Guthrie Test mitochondria out of the cell
○ HPLC ■ Aspartate participates in the urea
○ MS/MS (tandem Mass Spectroscopy) cycle.
○ LC-MS/MS to detect and measure the levels of methionine and ■ Deficiency of the gene causes the
homocystinuria buil-up of ammonia
■ Affects the brain
7. Citrullinemia ■ Very common in Japanese people
● Disorders of the urea cycle NOTE:
● Urea cycle - is a process involving the removal of excess nitrogen generated by the ● Both Type 1 & 2 Citrullinemia involves problems in
body; Breaking down of nitrogen to urea to be eliminated. the urea cycle, that is, the conversion of nitrogen
● Nitrogen that enters the body should be equal to the nitrogen eliminated. Once and urea, which causes build-up of nitrogen in the blood in the form of ammonia,
nitrogen is not eliminated, it will become toxic to the body which is a neurotoxin toxic to the body.
● Two Types:
○ Type I Citrullinemia 8. Argininosuccinic Aciduria
■ Most common form of the disorder ● Also involves problems in the urea cycle
■ defective/deficiency/absence of the enzyme argininosuccinate ● Lack the enzyme argininosuccinate acid
synthetase (ASS) lyase
■ Argininosuccinate synthetase (ASS) - enzyme responsible for the ● Deficiency of this enzyme prevents the
removal of nitrogen in the body; participates in the urea cycle conversion of argininosuccinic acid into
■ Lack of the enzyme will cause the nitrogen to not be converted to arginine
urea, so nitrogen will start to build up in the blood. Nitrogen ● There will be problems with the breakdown
accumulated in the body is in the form of ammonia. and removal of the nitrogen in the body
■ Ammonia is a neurotoxin that damages the neurons of the brain, and nitrogen will then start to build up in
inhibiting their normal functions the body in the form of ammonia
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 ● Ammonia is a neurotoxin which causing damages /inhibition in the function PROTEINS
of the neurons the brain
Proteins
● Signs & Symptoms: ● Are macromolecules (large)
○ Vomiting ● Polymers of amino acids
○ Refusal to eat/ loss of appetite ● Most of them are synthesized in the liver
○ Lethargy ○ Except for immunoglobulins which are synthesized in the plasma cells
○ Coma ● Typical protein contains 200 to 300 amino acids
○ smaller (peptides)
9. Cystinuria ○ larger (titin, in muscles)
● Mutation in the SLC3A1 & SLC7A9 genes
● Deficiency of these genes causes abnormal transport of cysteine in the PROTEIN SYNTHESIS
kidneys
● Cysteine will then start to build up in the kidneys, leading to cystinuria
● With Cystinuria, there is increasing amount of undissolved cysteine in the
urine, along with arginine, lysine, and ornithine
● Defect in the amino acid transport system rather than a metabolic enzyme
deficiency
● They will start to accumulate in the urinary system which result to the
formation of kidney stones/ Appearance of Cystine crystals in the urine
● Treatment:
○ High fluid intake
○ Penicillamine - if water intake is not effective
○ Percutaneous Nephrolithotripsy - if kidney stones persists and if
water intake and penicillamine is not effective ★ TRANSCRIPTION
■ Removal of kidney stones through a tube that sucks the stones ● Takes place in the nucleus
out of the kidneys ● DNA-encoded genes are first used to
produce mRNA
● First part of the central dogma of
molecular biology
● DNA → RNA

Steps in Transcription

1. Initiation
● Beginning of transcription
a. During this process, RNA polymerase binds to
the promoter region of the gene.
b. This will signal the DNA to unwind so that
RNA polymerase will “read” each base of
protein to make a copy of mRNA.
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 2. Elongation ● degradation of major nutrient to building block molecules
● Addition of nucleotides to mRNA strands
Stage 2: Energy Release
3. Termination ● conversion of building block molecules from stage 1 to a small # of
● Once mRNA strand is complete, mRNA will detach from the DNA simpler molecules
● Most important product: Acetyl-CoA
★ TRANSLATION Stage 3: Energy Storage
● Transcripted RNA will leave the nucleus and enter the ribosome where ● Oxidation of carbon atoms of the acetyl group to CO2
translation occurs ● Transfer of H+atoms to coenzymes NAD+ & FAD
● Synthesis of CHON or protein from an mRNA template ● Significant amount of energy is released when NADH & FADH2 are
● RNA → PROTEINS oxidized by the electron transport system
● Portion of energy generated: for ATP synthesis
ANABOLISM & CATABOLISM

Metabolism
● Sum of all the enzyme-catalyzed reactions in a living organism
● Each biochemical pathway consists of several reactions that occur sequentially
● Product of one reaction is the substrate for the one that follows

A. Anabolic Pathway
● Small precursors to large molecules
● Building block molecules are incorporated into larger, more complex
molecules
● Requires energy
● Example:
○ Amino acids → polypeptide
○ Fatty acids → lipids
○ Monosaccharides → Polysaccharides
B. Catabolic Pathway
● Large complex molecules to smaller, more simpler molecules Anabolic Hormones Catabolic Hormones
● Some catabolic pathways release free energy (energy production) Thyroxine Glucagon
○ Energy produced here is used in the anabolic pathway Growth Hormone Cortisol
● Fraction of the energy; captured and used to drive anabolic reactions Insulin
● Examples: Testosterone
○ Proteins → amino acids
○ Polysaccharides → Monosaccharides Anabolic Hormones
○ Lipids → Fatty acids ● Hormones involved in the synthesis of proteins
● From smaller → larger molecules
Stages of Catabolic Pathway

Stage 1: Digestion
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Catabolic Hormones Proteolytic Enzymes and their Actions
● Involves in the synthesis/ production of proteins Secreted by Enzyme Action
● Breakdown of proteins Stomach Pepsin ● Converts proteins into small peptides
● From larger → smaller molecules Pancreas Trypsin ● Specifically acts on peptide bonds
contributed by basic amino acids like
MNEMONICS: (ABCD) arginine, lysine, and histidine
● Anabolism → Build ● Activates trypsinogen to trypsin
● Catabolism → Destroy ● Procarboxypeptidase to carboxypeptidase
● Pro-elastase to elastase
● Pro-aminopeptidase to amino peptidase
PROTEIN DIGESTION Chymotrypsin ● Specifically acts in peptide bonds contributed
by aromatic amino acids like phenylalanine,
A. Gastric Phase (Stomach) tyrosine, trypsine
Steps: Carboxypeptidase ● Carboxy terminal amino acids
1. Chief cells release pepsin (inactive form)
Elastase
2. Activation of pepsinogen by stimulation of pepsin and gastric acidity
Small Intestine Aminopeptidase ● Amino terminal amino acids
3. Gastrin hormone stimulates secretion of pepsinogen and HCl
Dipeptidase ● Acts on dipeptides and releases free amino
● Hydrochloric acid (HCl) denature proteins by unfolding protein,
acids
causing the destruction of its structure
● After unfolding due to the action of HCl, pepsin will break the unfolded
protein into even smaller fragments.
4. Breaking down of proteins into larger polypeptides
B. Pancreatic Phase (Pancreas)
Steps:
1. When chyme reaches the duodenum, pancreas release proteolytic enzymes
● Release of Succus entericus (proteolytic enzyme) to release
proteases and peptidases
● Chyme is acidic but once it reaches the duodenum, it is neutralized by
bicarbonate buffers to resume the digestion process
2. Endopeptidases (trypsin, chymotrypsin and elastase) cleave protein in their
internal sites
3. Exopeptidase (carboxypeptidase) cleave one amino acid from the
carboxyl-terminus of the polypeptide
4. Secretin hormone stimulate pancreas to produce protein-free electrolyte
solution rich in bicarbonate
C. Pancreatic Phase (Small Intestine)
● Mediated by peptidases produced by the mucosal cells
○ Aminopeptidases and dipeptidases hydrolyze residual peptides
● AAs are transported across the mucosal cell membrane
○ Gamma-glutamyl cycle

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 NITROGEN BALANCE
Positive Nitrogen Balance
● Involves Anabolic State (synthesis of
proteins)
● Intake of nitrogen exceeds their loss
as net protein synthesis proceeds
● Pregnant women, growing children,
and adults recovering from major
illness
NOTE:
● One of the significant differences between proteins from carbohydrates is the
Negative Nitrogen Balance presence of nitrogen.
● On average, the content of serum protein is approximately 16%.
● Catabolic State of Nitrogen
● Nitrogen content is related to the protein content in the body.
(breakdown of proteins)
PROTEIN STRUCTURE
● More nitrogen is excreted than
incorporated into the body
Four distinct levels of protein structure:
1. Primary
● Excessive tissue destruction, such as
2. Secondary
burns, wasting disease, continual
3. Tertiary
high fevers, or starvation
4. Quaternary

Nitrogen Balance Protein Structure

● Is achieved if nitrogen intake is equivalent to nitrogen output Primary ● Represents the number and types of amino acids in the specific
● There is equilibrium between protein intake and protein elimination. amino acid sequence
● In order to function properly, proteins must have the correct
Pathways Involved in the Conversion of Intracellular proteins to Free Amino Acids sequence of amino acids
1. Lysosomal Pathway ● Easiest structure to denature
● Degradation of both intracellular and extracellular proteins but majority are ● Peptide Bonds
extracellular proteins (proteins outside the cell) Secondary ● Regularly repeating structures stabilized by hydrogen bonds
● Some degradation of intracellular proteins but only in small amounts between the amino acid within the protein
● Principal Function: Breakdown/Degrade large proteins into the building ○ Alpha helix
block amino acids ○ Beta-pleated sheets
Tertiary ● Overall shape or conformity of the protein molecule
2. Cytosolic Pathway ● Three-dimensional (3D)
● Involves degradation of ONLY intracellular proteins (proteins found inside the ● Stabilized by:
cell) ○ Hydrophobic effect
○ Ionic attraction
NITROGEN CONTENT ○ Hydrogen bonds
○ Disulfide bonds
Elements that consists proteins (CHONS) Quaternary ● The shape or structure that results from the interaction of more
1. Carbon than one protein molecule, or protein subunits
2. Oxygen ● Most difficult structure to dismantle
3. Hydrogen ● Stabilized by:
4. Nitrogen ○ Hydrogen bonds
5. Sulfur ○ Electrostatic interactions

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NOTE: PROTEIN SOLUBILITY
Proteins will denature via the addition of:
1. Heat ● Soluble proteins have a charged on their surfaces
2. Strong acids ● Protein has its lowest solubility at its pI
3. Strong bases ● Solubility of proteins in blood requires a pH on the range of 7.35 to 7.45
4. Addition of enzymes
5. Addition of urea
6. Exposure to UV light Factors affecting solubility of proteins:
1. Type of amino acid present
CHARGE & ISOELECTRIC POINT 2. pH
● Proteins contain many ionizable groups on the side chains of their amino acids as on
their N- and C- terminal ends. CLASSIFICATION BY FUNCTION
● Because of this, proteins can be positively and negatively charged. 1. Enzyme
○ By nature, proteins are amphoteric molecules. 2. Hormones
● There are time that proteins have no charge (isoelectric point) 3. Transport Proteins
4. Immunoglobulins
[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒] 5. Structural Proteins
Henderson-Hasselbalch Equation: 𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔 [𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑎𝑐𝑖𝑑] 6. Storage Proteins
7. Energy Source
8. Osmotic Force
● ⇧ Increases in pH ⇨ there will be deproteinization of proteins
○ Carboxyl groups are converted to carboxylate ions A. ENZYMES
○ Ammonium groups are converted to amino groups ● Catalysts of chemical reactions inside the body
● Normally found inside the cells (intracellular)
Isoelectric Point (pI) ○ Released in the bloodstream in cases of tissue damage
● pH at which an amino acid or protein has no net charge ● Examples: transaminases
● Point at which the number of positively charged groups equals the number of
negatively charged groups B. HORMONES
● Proteins differ in their pI values, but for most proteins, it occurs in the pH range of ● Chemical messengers
5.5 to 8. ● Capable of controlling of a specific cell or organ
● Examples: insulin, testosterone
pH above (>) pI Net negative charge
pH below (<) pI Net positive charge C. TRANSPORT PROTEINS
● Involved in the movement of certain molecules
NOTE: ● Transport movement of ions, small molecules, or macromolecules
● Majority of proteins are negatively charged at neutral pH ● Examples: Hemoglobin and albumin

D. IMMUNOGLOBULINS
● Produced by B cells (plasma cells) in the bone marrow
● Functions for the immune response to identify foreign substances infecting the body
● Examples: IgG, IgA, & IgM
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 EDEMA
E. STRUCTURAL PROTEIN ● The abnormal accumulation of fluid in the tissues
● Structures of cells and tissues ● Excessive or Greater leakage of water towards the
● Examples: collagen and elastin tissues due to decreased oncotic/osmotic pressure
attributed to the decrease in plasma albumin
F. STORAGE PROTEIN concentration
● Serves as reservoirs/reserves of metal ions & amino acids ● Protein is decreased because there is greater loss of
● Released and used later without harm occurring to cells during time of storage protein in the urine
● Examples: Ferritin ● People who are edematous are advised by their doctors to
lessen fluid intake, that’s why patients that are
G. ENERGY SOURCE undergoing dialysis are oftentimes dehydrated.
● Reserve source of energy for tissues and muscles NOTE:
● Alternative source of energy since CHO is the immediate source of energy ● Normally, proteins should not be found in the urine because they are large molecules
and should be filtered by the glomerulus
H. OSMOTIC FORCE ● Whenever there is damage to the kidneys (e.g. renal failure), there will be presence of
● Involved in the distribution of H2O protein in the urine, resulting to loss of protein ⇨ decreased plasma protein
throughout the body compartments
● Their colloid osmotic force, due to their Renal Failure ⇨ Loss of Protein in urine ⇨ Decreased plasma albumin ⇨ Decreased
sizes, does not allow proteins to cross the osmotic pressure
capillary membranes
CLASSIFICATION BY PROTEIN STRUCTURE
NOTE:
● Plasma proteins (mostly albumin) ● Database (Manual)
distributes water throughout the ● Database (Automated)
compartments of the body ● Simple Proteins
● Normally, blood vessels are permeable to nutrients (water, glucose, amino acids, ● Conjugated Proteins
oxygen, carbon dioxide, and other nutrients)
○ These nutrients possesses small amounts of oncotic pressure A. Simple Proteins
● On the other hand, proteins, unlike the nutrients, cannot penetrate the capillary wall ● Contains peptide chains composed of only amino acids
so they stay inside the blood vessels because proteins are large in size. ● Can be (based in shapes)
● This large size of proteins contributes to their high oncotic force ○ Globular
■ Rounded
IMPORTANT! ■ Symmetrical that’s why they are water-soluble (hydrophilic)
○ The oncotic pressure that our proteins possess controls the release of other ■ Globulin, Albumin, Immunoglobulins
smaller nutrients towards the tissue compartments ○ Fibrous
○ Therefore, oncotic pressure prevents the excess leakage of these nutrients ■ Filamentous (elongated)
and are the ones responsible for balancing and controlling the movement of ■ Asymmetrical - insoluble in water (hydrophobic)
these nutrients out of the capillary walls and towards the tissues. ■ Troponin and Collagen
○ In short, oncotic pressure generated by albumin pulls fluid back to the PLASMA PROTEINS
capillaries to prevent excess/further leakage of water to the tissues.
● Proteins in the body that are easily observe during electrophoretic procedure
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 ● Albumin
○ protein that is fastest to migrate towards the anode during electrophoresis C. Globulin
procedure in consideration with the five protein fractions
○ Protein with the highest concentration in the blood

A. Pre-Albumin
● Have the LEAST concentration in the plasma
● “Transthyretin” or “Thyroxine-Binding Pre-Albumin”
● Migrates before albumin in the classic serum/plasma protein electrophoresis
● Transports triiodothyronine
● Each fraction consists of a number of different CHONs with different functions
● Binds with retinol-binding protein (RBP)
○ A1 - Globulin (alpha 1)
○ Pre-albumin binds to RBP. After the binding of RBP to P-A, RBP can be binded ○ A2 - Globulin (alpha 2)
with Vitamin A
○ β - Globulin (beta)
○ Therefore, if there is a problem with Pre-albumin, vitamin A transport is also
○ 𝜸 - Globulin (gamma)
affected throughout the body.

DECREASE IN PRE-ALBUMIN
● Hepatic damage
○ Since most of the protein are synthesized in the liver except for
immunoglobulin
○ Any condition involving damage to the liver affects the levels of proteins, and
of course, our pre-albumins
● Acute-phase inflammatory response
● Tissue necrosis

B. Albumin 1. A1 - Globulin
● Present in HIGHEST concentration in the plasma a. A1 - Antitrypsin
● Responsible for nearly 80% of the colloid osmotic pressure of the intravascular fluid ● Protease-inhibitor
○ Ones albumin levels decrease, oncotic pressure is greatly affected ○ Prevents proteolysis - breakdown of protein into amino acids
● Buffers pH ● Positive acute phase reactant
● A negative acute-phase reactant protein ○ Increase in plasma protein concentration during inflammation,
○ Negative acute-phase reactant proteins (such as albumin) are proteins that infection, and trauma
decrease during inflammation, infection, and trauma ● Intrinsic factor in the homeostatic mechanism modulating
● Functions of Albumin endogenous proteolysis
○ Transports: ● ⇧ INCREASED during:
■ Thyroid hormones & other hormones ○ Pregnancy, Inflammatory conditions, Use of Contraceptives
■ Iron
■ Fatty acids 2. A2 - Globulin
● Abnormally high albumin levels are seldomly clinically important a. A2 - Macroglobulin
○ It is only alarming if the albumin levels are abnormally low ● Largest major non-immunoglobulin protein
● Protease inhibitor
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 ● ⇧ INCREASED during: 10 fold increase during nephrotic syndrome ● Immunologic tests should be done to detect these proteins
● Inactivates proteases
b. Haptoglobin A. MYOGLOBIN
● Migrates in the a2-region ● single -chain, globular, 153 amino acids
● Combines with hemoglobin to preserve body iron & store protein ● Contains heme (iron-containing) prosthetic group
● ⇧ INCREASED during: Stress, Infection, Acute inflammation, or Tissue ● Primary O2 - carrying protein
Necrosis ● Used in conjunction with troponin to diagnose or rule out heart attack
● AMI (Acute Myocardial Infarction): increase within 2-3 hours of onset, reaches peak
3. β - Globulin level in 8-12 hours
a. Transferrin / Siderophilin ○ NOTE:
● Transports ferric ions from the iron stores of intracellular or mucosal ■ AMI is caused by the reduction of blood supply in the heart that can
ferritin be due to blockage of blood vessel
● Has an antibacterial effect by complexing iron ■ In 18-20 hrs after myocardial infarction, the myoglobin level goes
○ Transferrin is antibacterial because it binds to iron which is back to its baseline.
also essential for bacterial growth ● Useful marker in monitoring the success or failure of reperfusion
○ Mang-aagaw ng iron ○ Reperfusion means restoration of blood flow in the heart

Note:
❖ Myoglobin is a protein that are found in the muscles of the animal
➢ Functions as oxygen storage unit
■ Carries oxygen to muscles
❖ Approximately 2% of muscle proteins are in the form of myoglobin
❖ Found in both striated skeletal muscle and cardiac muscle
❖ Similarity to Hemoglobin: (Myoglobin and Hemoglobin) Has the ability to reversibly
bind oxygen
➢ Reversibly means anytime oxygen are readily to let go by the
hemoglobin/myoglobin (naol ready to let go hshshs)
■ Myoglobin requires a very low oxygen tension to let go oxygen
● Very easy lang kay myoglobin i let go si oxygen nga gin
kaptan nya kaina (Gallego, 2022).
■ Hemoglobin requires greater oxygen tension to release the bound
oxygen
● TN: wag tularan si hemoglobin
➢ Myoglobin binds to oxygen → carries to the tissue requiring oxygen
❖ Since myoglobin is found in the cardiac muscle, it is considered to be a cardiac
marker.
OTHER PROTEINS OF CLINICAL IMPORTANCE ➢ E.g. In heart attack or myocardial infarction
➢ The advantage of myoglobin in other cardiac markers is that myoglobin is
● Proteins that are not usually detected by standard electrophoresis because they are released earlier from damaged cells than other cardiac markers.
present in very low levels in the blood ■ For example, the cardiac markers are myoglobin, creatinine kinase,
CK-MB, and troponin.
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 ● Among those 4, the myoglobin is released early. ➢ Advantage: Troponin I and Troponin T has great specificity in myocardial
➢ Myoglobin can give us an early detection of acute myocardial infarction or damage
heart attack. ■ Troponin I is most common in the laboratory
❖ Myoglobin is a small molecule and it is freely filtered by the kidney, which cas be ■ Troponin I and Troponin T has begun to replace the CK and CK-MB
easily eliminated from the body. because they are more specific in heart injury
❖ Bale ang myoglobin dasig siya mag taas during myocardial infarction and dasig ● Remains elevated for a longer period of time
man siya mag nubo after pila ka hours. (awts naol dasig mag abot kag dasig man ➢ Considered to be STAT
madura HAHAHA) ➢ Goes back to baseline from 5-10 days and is dependent on the size of the clot
❖ Increased level of myoglobin is toxic to the kidney can cause damage into it
CARDIAC TROPONIN
Causes of Myoglobin Elevation NORMAL INCREASED Possible Interpretation
● Acute MI Troponin CK, CK-MB, and myoglobin Either a lesser degree of
● Angina w/o infarction injury is present or that the
○ There is a decreased blood supply injury took place more than
● Rhabdomyolysis 24 hours in the past
○ Something to do with muscle damage First Troponin Test After subsequent 6 to 12 Heart injury likely occurred
● Muscle trauma hours troponin was tested within couple of hours prior
● Renal failure again to the first test and had not
● Myopathies had time to increase
● Vigorous exercise CK-MB and Troponin CK Due to another cause, such
● Intramuscular injection as skeletal muscle injury
● Open heart surgery
● Seizures (tonic-clonic) C. BRAIN NATRIURETIC PEPTIDE & N-TERMINAL BRAIN NATRIURETIC PEPTIDE
○ Tonic- stiffening of muscle; clonic- twitching of muscles ● Natriuretic peptides are a family of structurally related hormones that
● Electric shock include:
● Arterial Thrombosis ○ Atrial natriuretic peptide - produced in the kidneys
● Certain toxins ○ B-type (or brain) natriuretic peptide (BNP) - produced in the brain
○ C-type natriuretic peptide - produced in the endothelium
B. CARDIAC TROPONIN ○ Dendroaspis natriuretic peptide
● Includes NOTE: these are secreted by cardiomyocytes, in response to stretching (increased
○ Troponin I (cTnI) ventricular blood volume). In short these are cardiac hormones.
○ Troponin T (cTnT)
● Specific to the heart muscles D. FIBRONECTIN
● “Gold Standard” in the diagnosis of acute syndrome (ACS) ● Composed of two nearly identical subunits
● TcTnI and cTnT have begun to replace the CK and CK-MB ● Found in the plasma and on cell surfaces
● cTnT Reference Interval: <0.1 ng/mL (mg/L) ● Synthesized in the liver by hepatocytes, endothelial cells, peritoneal
● cTnI Cut-off concentration for immunoassays: 0.1 to 3.1 ng/mL (mg/L) macrophages, and fibroblasts
NOTE: NOTE: It is an adhesive glycoprotein, important in tissue repair and regulating the
❖ Troponin is one of the cardiac markers attachment and motility of the cell. Daw glue ba.
➢ Protein that are present in thin filaments of the striated muscles

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 ● Fetal fibronectin (fFN)
○ Predict the short-term risk of premature delivery
○ Produced at the boundary between the amniotic san and the decidua
○ Maintains the adherence of placenta to the uterus
NOTE: may be detected using amniotic fluid or placenta tissue during early
pregnancy, not after 24 weeks or 6 months. Shouldn’t be detectable on 22-36 weeks
of pregnancy, if there is an increase in fFN levels it may be a marker of impending
labor or preterm labor. May mean there is disturbance at the uteroplacental
junction.
E. ADIPONECTIN
● 247-amino acid fat hormone produced by adipocytes
● Composed of an N-terminal collagen-like domain & a C-terminal globular
domain
● Inverse correlation between BMI and adiponectin (low BMI, high adiponectin;
high BMI, low adiponectin)
risks in low adiponectin: increased risk of heart disease, developing type 2
diabetes mellitus, or result to obesity
F. B-TRACE PROTEIN
● 168-amino acid, low molecular-mass protein in the lipocalin protein family
● Accurate marker of CSF leakage
● Potential marker for detecting impaired renal function
● Promising marker in the diagnosis of perilymphatic fluid fistulas
NOTE: important component of the CSF. Higher concentration in the CSF, low
concentration in the blood. If there’s an increase in BTP level in the blood, it may
suggest a problem with the blood-brain barrier.
G. CROSS-LINKED C-TELOPEPTIDES
● Proteolytic fragments of collagen I formed during bone resorption (turnover)
● Biochemical marker for bone resorption
● More useful in monitoring the response to antiresorptive therapy
NOTE: one of the products of the breakdown of collagen.
○ Bone resorption - a process in which the osteoclasts break down the
tissues in the bone, resulting in the release of minerals, particularly
calcium. Then, calcium is transported into the blood.
○ If there are high levels of this, it could suggest that osteoclasts break
down the bones at a quick pace
○ Antiresorptive therapy - prevents the rapid breakdown of the bones
■ 3-6 months after the therapy, cross-linked C-telopeptide levels
will drop by 35-55% from the baseline level, indicative of an
effective therapy
● Limitations of measuring CTX values:
○ The need to establish a baseline level
○ May vary due to patient’s diet, exercise, and time of the day
○ Cannot replace bone mineral density to diagnose osteoporosis
H. CYSTATIN C
● Low molecular-mass protein with 120 amino acids
● A cysteine proteinase inhibitor (prevents proteolysis)
● Produced and destroyed at a constant rate
● Recently proposed new marker or the early assessment of changes to the
GFR
● Excellent indicator of reduced kidney function
NOTE: it is freely filtered by the glomerulus and is almost fully reabsorbed and
catabolized by the proximal tubule for recycling and brought back to the body to
function. Associated with glomerular filtration rate (GFR). Increased levels suggest
problems in the filtration system of the kidneys
● Recently proposed as a new sensitive endogenous serum marker for the GFR
● Not affected by muscle mass, gender, age, or race unlike creatinine, nor are
they generally affected by most drugs, infections, diet, or inflammation
● May be used as an alternative to creatinine & creatinine clearance to screen
for & monitor kidney dysfunction
I. AMYLOID
●Formed due to an alteration in the B-pleated
sheets
● In the picture, it is stained with Congo-red. If
viewed under a polarized microscope, a positive
test shows a yellow-green birefringence.
● Insoluble fibrous protein aggregates formed due
to an alteration in their secondary structure known
as I-pleated sheets.
● Amyloid 1342 (A1342) and Tau protein tests are not currently part of a typical
patient assessment but can be used as supplemental tests to help
differentiate a diagnosis of Alzheimer’s disease from other forms of dimentia.
J. AMYLOID β42 (Aβ42) & TAU PROTEIN
● Help differentiate diagnosis of Alzheimer’s disease from other forms of
dementia
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 ● Low Aβ42 along with Tau reflects an increased likelihood of Alzhemier’s
disease (not a confirmation, only shows likelihood)
● If there are no abnormal levels, the dementia or being forgetful in not
because of Alzheimer’s disease
Amyloid Alzheimer’s Disease
● Formation of beta-amyloid plaques
○ Beta-amyloid: end product of breakdown of APP (Amyloid Precursor
Protein) produced in the brain. The enzyme cuts APP into half, and is flushed
out into the form of beta-amyloid.
● Accumulation of Tau Protein
NOTE:
● Between a normal person and one who has Alzheimer’s disease, the difference is in
the enzyme.
● In those with Alzheimer’s disease, the two enzymes which replaced alpha-secretase
chop APP into proteins that the brain can’t metabolize.
● The proteins will stick/accumulate with each other.
● If it leads to a chronic situation, there will be buildup or accumulation.
● Hard plaques will be formed in the synapses of the neurons. These plaques
(accumulation of proteins in the brain) block the communication between neurons.
● This in turn results in cell death in the brain which leads to memory loss.
● The Tau protein will begin to form because of the presence of beta amyloid plaque.
The Tau protein is a hallmark of Alzheimer’s disease. Presence of Tau in the brain will
result in hyperphosphorylation.
● Tau will be broken down and lose their shape which leads to degeneration of Tau
protein.
● They will accumulate/tangle with each other.
● This will grow in size to as large enough to block the chemical and electrical signals Risk factors for kidney disease:
(communication) leading to death of the neuron (if a neuron is involved with
memory, this results in forgetfulness).
● Behavior and language (unable to speak) can also change.
● If it persists for long-term, the degeneration of Tau continues in the brain until such
time that the person forgets how to swallow or eat. Eventually, it results in death.
Amyloidosis
● Rare condition
● General term
○ There are lots of diseases which results from amyloidosis
● Deposition of amyloid proteins deposited in organs and/or tissues
○ Deposition of amyloid fibrils can infiltrate organs of the body, heart, blood
vessels, kidney, liver, and brain.
● Inherited due to different disease
○ Complications produced are many.
PROTEINS IN OTHER BODY FLUIDS
● Proteins are substances that are essential for your body to function properly. Protein
is normally found in the blood. Detecting protein in other body fluids such as urine
and CSF is also of importance for it can suggest some diseases present in the
patient's body.
A. URINARY PROTEINS
● Plasma proteins appear in the urine because they have passed through the renal
glomerulus and have not been reabsorbed by the renal tubules
○ Protein present in the urine comes from the blood. The main protein found in
the blood is in the form of albumin.
○ Healthy kidneys remove extra fluid and waste from the blood. Protein and
other important nutrients are returned to the circulation.
○ If kidney function is not normal, this allows proteins, specifically albumin, to
escape the filter and thus, be present in the urine (proteinuria or
albuminuria).
○ Sign of nephrotic syndrome or early sign of kidney disease because it is not
normal to have protein in the urine.
○ Creatinine is a waste product whose levels in the urine are higher than
proteins. Otherwise, in the urine, if protein levels are higher than creatinine
(similar to blood), it means kidney damage.
○ Diabetes
○ High blood pressure
○ Family history of kidney disease
● Urine usually presents a fluid with much lower protein concentration than plasma.
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 ● Urine present in part, and ultrafiltrate of plasma, with most of the plasma proteins, ● Formed in the choroid plexus of the
particularly the larger proteins, removed by the combined action of glomerular ventricles by ultrafiltration of the blood
filtration and tubular uptake of protein. plasma.
● The urinary epithelium adds a few proteins, such as Tamm-Horsfall protein, that ● Reference interval (10-40 y.o.): 15-45
are not present in plasma. mg/dL
● Analysis of urinary proteins is performed mainly to diagnose kidney disease or ● Abnormal increase total CSF proteins:
disorders with paraprotein production. ○ Increased permeability of the
● Four basic causes of increased urinary protein excretion (proteinuria) are known: capillary endothelial barrier (viral,
○ glomerular injury bacterial, and fungal meningitis)
○ tubular injury ○ Hyperthyroidism
○ overflow proteinuria of low-molecular-weight proteins, and; ○ Fluid is leaking from the CNS
○ postrenal proteinuria. ● Albumin: reference permeability CHON
● Qualitative test for proteinuria. Lots of factors can affect the results for the test ○ Total protein conc. in the CSF is compared with total protein in the
body. It should be 0.2-0.5%
Reagent Strip: ○ CSF reference protein is albumin. This is not synthesized to any degree
in the CSF. Presence of albumin is abnormal.
1. 24-hour Urine Collection ● Reference Value for CSF albumin-serum
● Quantitative level: best measurement!! albumin ratio: less than 2.7-7.3
● Allows for circadian rhythmic changes in excretion at certain times of the ● CSF is the extracellular fluid around the brain
day. and the spinal column.
● Reported as: weight of protein per 24 hours ● It is secreted by the choroid plexuses, along
● Reference value: 100-250 mg every 24 hours the walls of the ventricles of the brain, and is
reabsorbed into the blood through the
Other Precipitation Methods Reagents: arachnoid villi.
● TCA ● It usually has total protein concentrations
● Sulfosalicylic acid about 100-fold lower than those of plasma
● Benzethonium and a different protein composition.
● Laboratory testing for CSF is usually obtained by a physician performing a
NOTE: spinal tap in the lumbar region.
● Precipitants are used until now, specifically TCA and SSA. These precipitants
are usually acid. ● The protein concentration of CSF is lowest in ventricular fluid and is slightly
● Semi-quantitative and uses random urine higher along the spinal cord, where specimens are usually collected.
● Since CSF is mainly an ultrafiltrate of plasma, relatively small plasma
B. CSF PROTEINS (CSF) proteins, such as:
● Normal: clear colorless. Traumatic collection: red 1. Prealbumin
○ Other causes of red CSF: traumatic tap, multiple sclerosis, obstruction, 2. Albumin, and;
neoplasm, cerebral infarction. 3. Transferrin, typically predominates.
○ Normally, there should be no RBC and WBC in the CSF. Less than 5 ● No protein with a size larger than that of IgG is present in sufficient amount
cells/uL. to be visible in an electrophoretic pattern.

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 TOTAL PROTEIN ABNORMALITIES ● In addition to dehydration, hyperproteinemia may be the result of excessive
production, primarily of the y-globulins. This protein is an intact
● A total protein test measures the amount of protein in your blood. Proteins are immunoglobulin molecule or, occasionally, K or X light chains only.
important for the health and growth of the body's cells and tissues. ● The most common disorder is multiple myeloma, in which the neoplastic
plasma cells proliferate in the bone marrow. Paraprotein in multiple myeloma
A. HYPOPROTEINEMIA may reach a serum concentration of several grams per deciliter.
● A total protein level less than the reference interval, occurs in any condition
where a nitrogen balance exists NOTE:
● Causes: excessive loss, decrease intake ● In multiple myeloma and Waldenstrom’s macroglobulinemia, gamma globulins are
● One cause of a low level of plasma proteins is excessive loss, decreased involved.
intake either because of malnutrition or through intestinal malabsorption ● In active multiple myeloma, levels of chemical messengers particularly IL-6 are
as seen in sprue; or as a result of decreased synthesis is also seen in liver elevated. This blocks production of albumin.
disease (site of all nonimmune protein synthesis) or in inherited ● Low albumin = more aggressive/active multiple myeloma. There will be increased
immunodeficiency disorders; or accelerated catabolism of proteins. globulins and total protein, but decreased albumin.
● If there is excessive loss of proteins, there will be excretion of proteins in the ● Non-hodgkin lymphoma: Cancer cells are producing large amounts of abnormal
urine because of: proteins in the form of macroglobulin. Paraprotein present is IgM.
○ Renal disease like inflammation or damage
○ Leakage into GI tract due to inflammation in digestive system NOTE:
○ Loss of blood because of an open wound, internal bleeding, burns, ● The fat kids are in a state of malnutrition.
systemic inflammation. This is a form of hypoproteinemia
○ Hypoproteinemia: Malnutrition, Malabsorption because the food mainly eaten by the
children are only “camote.” No source of
B. HYPERPROTEINEMIA meat.
● An increase in total plasma proteins is not an actual disease state but is the ● Kwashiorkor is a hypoproteinemia (low
result of the underlying cause, dehydration. protein because of malnutrition).
● Hyperproteinemia is increased abnormal conc. of proteins in the blood ● The children become bloated (low protein
● Increased are the monoclonal proteins (paraproteins) which can also be -> decreased oncotic pressure -> leakage
found in the urine of fluid from the capillaries to the tissues).
● Other causes: There is bloating of the stomach and
○ Gamma-globulin cheeks, which is an indication of edema.
● Intact immunoglobulin molecule or, occasionally, κ or λ light chains only as LABORATORY ANALYSIS
seen in multiple myeloma
● Paraproteins: A. TOTAL NITROGEN ANALYSIS
○ Usually IgG, IgA, or , κ or λ light chains ● Acceptable Specimens: plasma & urine
○ IgD and IgE paraproteins rarely occurs ● Useful in assessing nitrogen balance
○ Waldenstrom’s macroglobulinemia ● Chemiluminescence
○ Inflammation, infection, hepatitis b or c, HIV, multiple myeloma ○ Sample, in the presence of O2, is heated to a high temperature
● Dehydration results from a variety of conditions, including vomiting, diarrhea, NOTE:
excessive sweating, diabetic acidosis, and hypoaldosteronism. ● One of the components of proteins is nitrogen
● When excess water is lost from the vascular system, the proteins, because of ● How is this test performed?
their size, remain within the blood vessels.
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 ○ Sample has bound-nitrogen in it. First step in TNA is to oxidize D. BIURET METHOD
bound-nitrogen to produce nitric oxide. ● Most widely used method
○ After oxidation, nitric oxide is mixed with ozone (O3) and there will be ● Recommended by IFCC expert panel
formation of nitrogen dioxide molecule and reach its excited state ○ TAKE NOTE! International Federation of Clinical Chemistry (IFCC)
○ Once energy gradually decreases and the molecule will moves from excited ● Cupric ions complex w/ groups involved in the peptide bonds
state to ground state, there will be emission of chemiluminescent light ● Violet-colored chelate: presence of at least 2 peptide bonds
○ The amount of chemiluminescent light is proportional to the amount of ○ Or Pink - reddish violet
nitrogen
■ ⇧ nitrogen = ⇧ intensity of chemiluminescence; NOTE:
■ ⇩ nitrogen, = ⇩ intensity of chemiluminescence ● Reagent has has two components: Sodium Potassium Tartrate and Potassium iodide
○ Potassium iodide acts as an antioxidant
B. TOTAL PROTEIN ANALYSIS ● Intensity of the color of solution is directly proportional to the concentration
● Serum is most often used; fasting is NOT needed
● Interferences includes; E. DYE BINDING
○ Lipemia ● Based on the ability of most proteins in serum to bind dyes
○ Hemolysis ● Stains used:
● Lower levels: seen in pregnancy & as the person ages ○ Bromophenol blue
○ Ponceau S
Reference Interval for Serum Protein ○ Amido black 10B
● Ambulatory Adults: 6.5 - 8.3 g/dL (65 to 83 g/L) ○ Lissamine green
○ Sudden change in position ○ Coomassie brilliant blue
● Recumbent position: 6.0 - 7.8 g/dL (60 to 78 g/L)
○ Lying down F. A/G RATIO
● A reversal or significant change in the ratio of albumin and total globulin, the
NOTE: A/G ratio, is found in diseases of the kidney and liver
● Hemolysis is an interference because as RBCs lyse, enzymes (proteins) will be ● To determine the A/G, total protein and albumin are measured and globulins
released which will falsely increase the results of analysis are calculated by: Total Protein - Albumin = Globulins
● If a person suddenly changes their position, it affects the levels of proteins because
there is sudden shifting of water from the tissues into the blood vessels G. SALT FRACTIONATION
● Done using precipitation
○ Done through the addition of sodium salts/salts to precipitate
C. KJELDAHL METHOD globulins
● Classic Method for total CHON quantitation ● Separation of globulins and albumin by salting out
● Determines nitrogen ● Albumin remains in the solution on the supernatant
● Not used in the laboratory anymore because it is very time-consuming and ● On the other hand, Globulins are present in the precipitate
tedious to use as routine test
● An average of 16% nitrogen mass in CHON is assumed to calculate the CHON H. ALBUMIN
concentration ● Dye-binding procedure - most widely used
● Precipitants: organic acid (TCA or tungstic acid) ● pH of solution is adjusted so that albumin will become positively charged
● The amount of albumin is calculated by measurement of the absorbance of
the albumin-dye complex
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 Dyes used:
● Methyl Orange - nonspecific Reference value for Each Fraction:
● 2,4’-hydroxyazobenzene-benzoic acid (HABA) - low sensitivity but more ● Albumin: 53%-65% of the total CHON (3.5-5.0 g/dL) - most concentrated
specific for albumin ● a1-Globulin: 2.5-5% (0.1-0.3 g/dL) - least concentration
● Bromocresol green (BCG) - not affected by interfering substances such as ● a2-Globulin: 7-13% (0.6-1.0 g/dL)
bilirubin and salicylates but it can also bind with hemoglobin ● β - Globulin: 8-14% (0.7-1.11 g/dL)
● Bromocresol purple (BCP) - specific only for albumin ● 𝜸 - Globuli: 12-22% (0.8-1.6 g/dL)

I. TOTAL GLOBULINS Disease Electrophoretic Pattern Observations

● Direct colorimetric method using glyoxylic acid added to sample with globulin
Normal ● Normal levels of
● Produces purple color
proteins
J. ELECTROPHORESIS
● Performed when an abnormality is found in the total protein or albumin
● Separates proteins on the basis of their electric charge densities
● Cations ⇿ cathodes
● Anions ⇿ anodes
● The velocity of the movement also depends on the:
○ Electric field strength
○ Size and shape of the molecules
○ Temperature 2. Multiple ● M-spike on gamma
○ Characteristics of the buffer Myeloma globulin
● Increased a1-globulin
Standard Method: and β - Globulin
1. Apply serum sample close to the cathode end of that is saturated with
alkaline buffer (pH 8.6)
2. Support medium is connected to two electrodes
3. All major serum proteins carry a net negative charge at pH 8.6 and
migrate toward the anode
● Serum proteins appear in 5 bands (in order from the anode to
cathode):
○ Albumin travels farthest to the anode
○ A1-globulins (A1-Antitrypsin (major A1))
○ A2-globulins (A2-Macroglobulin (major A2))
○ β - Globulin (beta)
○ 𝜸 - Globulin (gamma)
4. After separation, the protein fractions are fixed using an acid solution
(e.g. acetic acid)
● Acid denatures proteins so proteins are immobilized
5. Proteins are then stained.

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3. A1- Antitrypsin ● low levels of 6. Liver Cirrhosis ● Beta-gamma bridging
Deficiency a1-globulins (almost ● Infectious hepatitis
absent ○ Gamma
globulins
increases as
hepatocellular
damage worsen
● Obstructive jaundice

4. Nephrotic ● damage to nephrons,

Syndrome causing:
○ decrease in
albumin and K. HIGH-RESOLUTION PROTEIN ELECTROPHORESIS
gamma-globuli ● Can be further separated into as many as 12 bands
○ increase in ● Uses higher voltage coupled with a cooling system and a more concentrated
alpha and buffer
beta-lipoprotein ● Support medium: agarose gel
● Useful in detecting small monoclonal bands and differential unusual bands or
prominent increase of normal bands
● Width of the band represents the concentration of protein (wider the band,
the more concentrated it is)

5. Inflammation ● Trauma
or Acute Phase ○ Vehicular
Reactant accidents where
there is great
loss of blood
● Burns
● Infarction
● Liver Disease
● Malignancy
● Decreased albumin
● Increased a1, a2, and
beta globulin L. CAPILLARY ELECTROPHORESIS
● Collection of techniques in which the separation of molecules takes place in
silica capillaries
● Size of capillaries: 30 to 50 cm long
● Internal Diameter: 25 and 100 um

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 ● Electroosmotic flow (OF)
● Electrophoretic Mobility

M. ISOELECTRIC FOCUSING (IEF)

● Zone electrophoresis that separates proteins on the basis of p.i.
● 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

Clinical Applications:
● Phenotyping of a1-antitrypsin deficiencies
● Determination of genetic code
● Variants of enzymes and hemoglobin
● Detection of paraprotein in serum and oligoclonal bands in CSF
● Isoenzyme determinations
○ LD1, LD2, LD3 are isoenzymes of the enzyme LDH

N. IMMUNOCHEMICAL METHODS
● Specific proteins may be identified by immunochemical assays in which the
reaction of the protein (antigen) and its antibody is measured.

~ END OF MODULE 5: PROTEINS~

“I won’t sugarcoat. Kadamoooo 😭”

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