CC2 Lab

[TRANS] LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY
ENZYMOLOGY
• Tertiary
• Study of enzymes which involves:
o Secondary structures that
o Activity of enzymes are oriented in a three-
o Chemical reaction it catalyzes dimensional manner
o Clinical use
▪ Clinical enzymology
• Quaternary
 tackles about enzymes of clinical significance
o Combination of the different
 E.g. amylase, lipase, transferases, and other subunits of tertiary structure
miscellaneous enzymes
• Term “enzymology’” – for biochemistry • Some of the enzymes are
actually composed of more than one subunit; while
others only have one
ENZYMES
• Proteins that are composed of amino acids How the Enzyme Works
o Amino acids are composed of:
▪ a central carbon
▪ a functional group (depends
on the type of amino acid)
▪ carboxylic acid group
▪ amino group
o Polypeptide – chain of amino acids
o Peptide bonds – join the amino acids together
o Proteins have different functions:
▪ E.g. structural or catalysts
• Biologic catalysts
• Hasten chemical reactions
o Chemical reactions in the body may take a long time • The enzyme works by lowering the energy barrier for a
to proceed to a reaction. Hence, enzymes speed up reaction to occur.
the process up to a nanosecond. • Graph:
• Not consumed during the reaction o Y axis: Activation energy used for the reaction
o In an enzymatic reaction, the substrate becomes the o X axis: Direction of the reaction
product; whereas the enzyme remains the same. ▪ Going to the right = the reaction occurs
• Does not undergo chemical change after the reaction • Dotted lines:
o (1) Before the reaction occurs, there should be an
4 Domains of Structure of Proteins energy input in the system.
• Primary ▪ ANALOGY: The paper breaks into several
pieces because there is an energy introduced in
o The structure of the the system that will tear apart the paper.
enzyme on an amino
acid by amino acid level o (2) The energy barrier in the presence of an enzyme
o The amino acid becomes low.
sequence present in a
▪ The activation energy needed for the reaction to
particular enzyme
occur decreases in the presence of an enzyme.
• Secondary  Thus, the reaction becomes easier.
o How polypeptides are ▪ ANALOGY: The paper can break itself into
twisting pieces given enough time, but it can be easily
o E.g. alpha helix & beta breakdown into several pieces with the help of
pleated sheet the hand (enzymes).
ANTOYAN. CORTEZ. DUMAGONOT. IBONES. JUNIO. LAMOSTE. LATONIO. MAHINAY. MERCADO. NARAGA. OLIVA. PAÑA. BSMLS 3 1
RELATOR. SARGADO. VILLAREAL. VILLENA. AQUINO. CATAPANG. NAVARRO. CAGAS
LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY
Enzyme Location Nomenclature of Enzymes
• Found in all body tissues • 3 ways of naming enzymes:

o The human body is a walking chemical laboratory o Substrate + -ase
wherein each of the cell has its own chemical o Reaction it catalyzes
laboratory. o Enzyme Commission (E.C.) Nomenclature
o Since there are several reactions that need to occur
for a cell to live, there should be enzymes. Substrate + -ase
• Appear in serum/plasma: • Depending on the substrate upon which the enzyme

o Following cellular injury acts upon
• Examples of substate and its corresponding enzyme:
▪ Enzymes appear in an abnormal or
o Lipid = Lipase
pathophysiological circumstance.
o Ester = Esterase
▪ Example 1: Creatinine Kinase – MB (CK-MB)
 Specific to the cardiac muscle tissue ▪ Substrate: molecules that contain ester bond
 Found inside the muscular cardiac muscle o Protein = Protease
tissue of the heart o Starch = Amylase
 A heart injury such as acute myocardial ▪ Named as such because the two components of
infarction (heart attack) can cause damages starch are amylose and amylopectin.
to the cardiac muscle tissue which releases
CK-MB in the bloodstream. Reaction it catalyzes
 The presence of an increased CK-MB in the • Oxidase
serum or plasma means that there is an injury
to the tissue where the enzyme is found. o Oxidation – Removing of electron/s from a molecule
▪ Example 2: Lactate Dehydrogenase (LDH) • Reductase
 Since the LDH is found inside the RBCS, an o Reduction – Addition of electron
increased LDH may indicate intravascular or
extravascular hemolysis. • Hydrolase
o In smaller amounts, degraded cells o Hydrolysis – Addition of water to breakdown a
o For physiologic processes, like coagulation molecule
▪ Enzymes are needed in the serum or plasma • Dehydrogenase

such as in the case of coagulation. o Removal of H+, transferring them to a coenzyme
Two Sites of Enzyme • Decarboxylase
Active Site o Removal of Carboxyl groups
• Where the substance on which the enzyme acts • Deaminase

(substrate) or interacts with particular charged amino o Removal of Amino groups
residue.
• The site where the substrate will fit into to undergo an Enzyme Commission (E.C.) Nomenclature
enzymatic reaction for it to become a product.
• Standard process for naming enzymes
• It is the sub-department of International Union for
Biochemistry (IUB)
• 4 types:
o Numerical code
▪ E.C. 3.2.1.1. (for amylase)
▪ 1st digit = class
▪ 2nd digit = subclass
▪ 3rd and 4th = sub subclass & serial number
Allosteric Site o Systematic name (full name)
• Cavity other than the active site ▪ 1,4-D-Glucan Glucanohydrolase (for amylase)
• May bind regulator molecules o Recommended name (nickname)
o Examples: Co-enzymes, Activators ▪ α-Amylase (for amylase)
o Most of the enzymatic reactions need another
regulator molecule before it undergoes enzymatic o Abbreviated name
reaction. ▪ AMS (for amylase)
CLASSIFICATION OF ENZYMES • Forward Reaction
• (1) Oxidoreductase o If lactate is more than the pyruvate, the reaction will
be forward reaction
• (2) Transferase
o Lactate → Pyruvate
• (3) Hydrolase
• (4) Lyases ▪ Lactate = substrate
• (5) Isomerase ▪ Pyruvate = product
• (6) Ligases o Substrate is always in the name of the enzyme
• The number/order corresponds to the 1st digit of the
numerical code. • Reverse Reaction
o If pyruvate is at large amounts in a test tube, lactate
MNEMONIC: Oh To Hold Lyka’s Incredible Legs dehydrogenase turns it to lactate
o Lyka = Lyase o Pyruvate → Lactate
o Legs = Ligase
▪ Pyruvate = substrate
(1) Oxidoreductase ▪ Lactate = product
Oxidoreductase Reaction
• Oxidation and reduction
o The transfer of H+ ion from one substrate to another
to form the products
▪ Oxidation – removal of H+ ion
▪ Reduction – acceptance of H+
• Example: E.C.1.1.1.27
o Systematic name: L-Lactate NAD+
Oxidoreductase
▪ NAD – nicotinamide adenine dinucleotide • Dehydrogenase – One hydrogen is removed thus
becoming pyruvate
 NAD+ – oxidized form of NAD
o The oxidized NAD will receive the removed
 NADH – reduced form of NAD; due to
hydrogen and become reduced.
acceptance of hydrogen
o Recommended name: Lactate Dehydrogenase • In every oxidation-reduction reaction, one of the
o Abbreviated name: LDH substrates is oxidized to form the pyruvate while the
other is reduced.
Anatomy of the Enzymatic Reaction Symbols o Oxidation-reduction is always a pair
▪ Substates: Lactate, oxidized NAD (NAD+)
 Lactate is oxidized since H+ ion was
removed.
 Oxidized NAD (NAD+) becomes reduced
NAD (NADH) since it received the H+ ion
• Substrate NOTE: Whenever an enzyme ends in dehydrogenase, its
o Always at the left side function is oxidation.
• Product o Lactate dehydrogenase oxidizes lactate.
o At the right side (2) Transferases

• Arrow • Has a similar function to oxidoreductase
o Indicates the direction of the reaction • Transfer of functional groups other than hydrogen from
o Substrate may be on the right if the arrow is one substrate to another
reversed • Example: E.C. 2.6.1.1.
o Double arrow o Systematic name: L- Aspartate: 2-Oxaloglutarate
▪ Indicates that the enzymatic reaction is Aminotransferase
reversible ▪ Substrates for the forward reaction:
▪ “reversible” – can go either way
▪ Lactate → Pyruvate  L-aspartate, 2-Oxaloglutarate
▪ Amino group is being transferred thus the name
o The direction of the reaction depends on the amount
aminotransferase
of either the product or the substrate
o Recommended name: Aspartate Aminotransferase
• Always put the name of the enzyme above the arrow o Abbreviated name: AST
indicating that the enzyme is not changing after the
reaction ▪ Old name: SGOT
Transferase Reaction o Adding water will break the bond and turn amylose
into:
• Example: E.C. 2.6.1.2. ▪ glucose (monosaccharide) or
o Systematic name: L-Alanine: 2-Oxaloglutarate ▪ maltose (disaccharide) or
Aminotransferase ▪ dextrins (oligosaccharides)
o Recommended name: Alanine transaminase • Amylose can break by itself by adding water but
o Abbreviated name: ALT amylase can speed up the reaction
o Old name: Serum Glutamate Pyruvate • Amylase is found in the saliva (salivary glands) and
Transaminase (SGPT) pancreatic secretions (pancreas)
▪ Contains the name of the products of the o Ex. Rice (starchy) has amylose
reaction
▪ α-1,4 glycosidic bonds will break into
• α-ketoglutarate glucose/maltose/dextrins with the aid of water
o Another name for Oxaloglutarate and amylase
o Pancreatic amylase – further degradation of
carbohydrates
(4) Lyases
• Addition of a group to a double bond to become a single

bond, or
• Removal of a group to form a double bond
• Example: Carbonic Anhydrase & Citrate Lyase
Lyase Reaction
• ALT is an aminotransferase
o The amino group from L-Alanine is transferred to α-
ketoglutarate
▪ L-Alanine becomes pyruvate
▪ α-ketoglutarate becomes L-Glutamate
 An α-ketoglutarate that gained an amino • Carbon dioxide and water becomes carbonic acid with
group the help of carbonic anhydrase
 Transfer of amino group o Lyase – adds something to a double bond (water)
and becomes a single bond
• It is a forward reaction
o Substrate is in the name of the enzyme. • Blood pH
o Carbon dioxide in the lungs/bloodstream reacts with
(3) Hydrolases water with the help of carbonic anhydrase producing
• Hydrolysis of various bonds carbonic acid which becomes bicarbonate
o Addition of H2O to a bond resulting in bond ▪ Bicarbonate acts as a buffer for blood pH
breakage (5) Isomerases
• Example: E.C. 3.2.1.1.
• Rearrange the functional groups within the molecule
o Systematic name: 1,4-D-Glucan and catalyze the conversion of one isomer into another
Glucanohydrolase
o Isomers are the same molecules (same number of
▪ D – stereoisomerism atoms) but with a different arrangement
o Recommended name: α-Amylase • Example: Phosphoglycerate mutase
▪ Only acts on α-1,4 glycosidic bonds o Mutase – an isomerase; “mutates” a molecule into
another type of molecule
Hydrolase Reaction
Isomerase Reaction
• Amylose has a glycosidic bond (α-1,4 glycosidic bond)
o Has 3 isoenzymes, which are composed of 2

• 3-Phosphoglycerate
subunits of the tertiary structure of a protein:
o Glycerate (3 carbons) but the phosphate group is in
the 3rd carbon ▪ CK-BB – B and B subunits (migrate fastest
o Phosphoglycerate mutase will transfer the towards the anode/ travels the farthest)
phosphate to the 2nd carbon and becomes 2- ▪ CK-MB – M and B subunits
Phosphoglycerate ▪ CK-MM – M and M subunits (slowest mobility)
o Same function but differ in the subunits of their
(6) Ligases proteins
• Catalyze a reaction in which C-C, C-S, C-O or C-N • Isoenzymes have different physical properties which
bond is made or broken includes electrophoretic mobility, solubility and
o Bond formation or breaking down of a bond (if resistance to activation.
reversible reaction) o If undergo electrophoresis, they have different
▪ Energy – needed to form a bond mobilities.
• Accompanied by an ATP-ADP conversion or a similar ▪ Electrophoresis – serum is added in the agarose

compound such as GTP (nucleotide triphosphate) gel,  electric current is introduced for it to flow
from cathode to anode.
Ligase Reaction
 They have different speeds at which they
travel the agarose gel.
o Some are easily activated while some are not easily
activated.
Isoenzyme vs. Isoforms
• Central Dogma of Life

𝑡𝑟𝑎𝑛𝑠𝑐𝑟𝑖𝑝𝑡𝑖𝑜𝑛 𝑡𝑟𝑎𝑛𝑠𝑙𝑎𝑡𝑖𝑜𝑛
𝐷𝑁𝐴 (𝑟𝑒𝑝𝑙𝑖𝑐𝑎𝑡𝑖𝑜𝑛) → 𝑅𝑁𝐴 → 𝑃𝑟𝑜𝑡𝑒𝑖𝑛
• Enzymes are proteins

• DNA Ligase
o Embedded in the genome in the same way that
o Whenever there is a DNA strand in the 3’ end, body produce them.
another DNA should be added. o Genome has genes that encode for an enzyme
o If the triphosphate will break (2 phosphates will be
removed) with the help of ATP → Monophosphate • Isoenzyme
will create a phosphodiester bond o Structurally, it has different enzymes because they
• DNA Polymerase arise from different genes.
o Different genes which encode for different proteins
o When dinucleotide is added, it is accompanied with
ATP-ADP conversion. • Isoforms
NOTE: No ligase in the clinically significant enzymes so far. o Same genes and same proteins after translation but
differ during the post-translational modifications in
the Golgi complex.
TERMINOLOGIES o Different in structure because they were modified by
Substrate the Golgi complex differently.
▪ Ribosomes – where proteins are translated
• Molecule acted upon by enzyme
▪ Golgi body or complex – process the protein
• Specific for every enzyme produced from translation
• E.g., Amylase
 Also called as post-translational processing
o Substrate: α-1,4-glycosidic bond between amylose
and amylopectin Cofactor
o If triglyceride is introduced to amylase, nothing will
happen since that enzyme is specific for α-1,4- • May be necessary for enzyme activity
glycosidic bond. • Non-protein molecule
o Helps in the enzyme activity
Isoenzyme
o It goes into and connects to the allosteric site
• Iso – “the same”
Two types of Cofactors
• Same enzymes with the same action but have different
forms • Activator
• E.g., Creatine kinase
o Inorganic cofactor
o Kinase – involves ATP-ADP, but these are not o E.g. Calcium, Magnesium, Manganese, Zinc, Iron
ligases.
• Coenzyme Example 3
o Non-protein
o Organic cofactor
o E.g. Adenine Triphosphate (ATP), Nicotinamide
adenine dinucleotide (NADH)
▪ The larger the molecule, the more organic it is.
o Prosthetic group
• Apoenzyme + substrate + absence of cofactor
▪ When coenzyme is bound tightly to an enzyme
▪ Already present in the enzyme, not a separate o No enzyme-substrate complex, no reaction
entity from the enzyme o Needs a cofactor
Holoenzyme
• Apoenzyme + Coenzyme
o Absence of cofactor = no reaction
• Apoenzyme + Substrate + presence of cofactor =

reaction occurs
o Cofactor: Copper
▪ Activator (Inorganic element)
Proenzyme
Apoenzyme • Also known as zymogen
• Polypeptide portion of the enzyme o Generates the actual enzyme
o Protein portion of the enzyme that already contains ▪ Zymogen = “gen” - genesis
tightly bound coenzyme
• Inactive form of enzyme
Example 1
o Example: Preproprotein, undergoes post relational
modification in the Golgi complex and forms
proprotein, to protein
▪ Preproprotein → proprotein → protein
▪ It has to be converted first into an actual enzyme
before activated
o Where is it activated?
• Nicotinamide adenine dinucleotide (NAD) ▪ Depends on the enzyme
▪ Inactive form of enzyme reaches its destination
o Considered a substrate and as a coenzyme
before activated, usually by cutting amino acid
▪ Coenzyme – Organic cofactor because lactate • It is then converted, usually by proteolysis, to the active
dehydrogenase cannot proceed with the reaction form when it has reached the site of its activity
without NAD.
Example 2 ENZYME KINETICS
• How does enzyme behave inside the body or in the test
tube (laboratory)
• Kinetics “to move”
• Adenosine triphosphate (ATP) Michaelis – Menten Theory

o Organic cofactor • Leonor Michaelis (1875-
▪ Thus, it is a coenzyme 1949) and Maud Menten
(1879-1960)
o Involved in Creatine Kinase • Hypothesize the role of
substrate concentration in the
formation of the ES Complex
• Basic tenet of enzyme kinetics Bond Specificity

• E + S → E-S → P + E
• The enzyme is specific to a particular bond.
o An enzyme-substrate complex must first be formed
for an enzymatic reaction to occur o E.g., Proteases
▪ The product can only be achieved, if the ▪ Proteases digest proteins because they are
substrate forms a complex with the enzyme. particular to a peptide bond.
▪ There must be a physical interaction between ▪ Regardless of the type of amino acid, as long as
the enzyme and the substrate they are connected by a peptide bond.
Stereoisometric Specificity
• The enzyme will only act upon a specific isomer of

substrate.
• Base on arrangement of molecules
• (1) At first, there is a Substrate [S] and an enzyme [E] o E.g., Glucose dehydrogenase
• (2) Before the Substrate [S] turns into a Product [P],
there must be a formation of ES complex [ES] ▪ Classification of enzyme: Oxidoreductase
o ES Complex [ES] - the physical binding of the  “dehydrogenase” – removal of hydrogen

substrate to the active site of the enzyme ▪ A stereoisometric specific enzyme because it
only acts upon β-D-glucose.
▪ Crucial intermediate step in the enzymatic
reaction  If the glucose is an α-D-glucose, the glucose
▪ Explains the behavior of enzyme dehydrogenase will not react only to β-D-
glucose.
Analogy on Enzymatic Reaction of the Body
• How the enzyme work?

o If there are substrate and an enzyme, the enzyme
and substrate must form a complex. Then the
enzyme will turn the substrate into a product
• The enzymatic reaction is dependent on the availability
of the active site.
o The active site must be available because the
substrate must be place on the active site before the
• Enzyme is still the same enzyme at the end of reaction product is formed.
o Enzyme does not change chemically after the
reaction
Specificity
Absolute Specificity
• The enzyme can only form a complex to a specific

substrate.
o E.g., Amylase • The graph above shows constant amount of enzymes
▪ Amylase can only react with amylose because o Increasing the amount of substrate will increase the
amylase has absolute specificity towards speed of the reaction. However, it will eventually hit
amylose. a limit (plateau) despite adding more substrate since
▪ If alanine, albumin, and triglyceride are added, the active site is limited.
amylase will not react to it. o Enzymes cannot cater adding more substrate
Group Specificity • Adding more substrate in any enzymatic reaction will
lead to a limit or maximum velocity.
• The enzyme is specific for a particular functional
group. o X axis
• Regardless of the molecule, for as long as there is a ▪ Substrate concentration
functional group. ▪ From left to right, substrate increases in
o E.g., Phosphate esterase concentration
▪ No matter what molecule, for as long as it o Y axis

contains a phosphate ester, it will act on that ▪ Velocity or speed of the reaction
molecule. ▪ As you go higher, the speed increases
Michaelis-Menten Equation • The formula of Michaelis-Menten was reciprocated to

form the Lineweaver-Burk equation:
𝟏 𝐊𝒎 𝟏 𝟏
= +
𝑽 𝑽𝒎𝒂𝒙 [𝑺] 𝑽𝒎𝒂𝒙
o Where:
1
▪ Y axis =
𝑉
1
▪ X axis =
[𝑆]
▪ Vmax is represented by the y-intercept, where
the line of enzymatic reaction crosses the y axis
▪ Km corresponds to the x-intercept
Enzyme Concentration vs Rate of Reaction
• The graph above describes the kinetics of the enzymes

as the substrate increases. • This graph shows that
the more enzymes
o Y axis – Reaction velocity or speed added, the more active
o X axis – Substrate concentration sites → the faster the
reaction
• Michaelis-Menten equation:
o More enzyme
𝑽𝒎𝒂𝒙 [𝑺] molecules can react
𝑽=
𝑲𝒎 + [𝑺] with more substrate
molecules, so the
o This equation will obtain the Michaelis-Menten reaction rate
curve. increases
• Characteristics of the curve:
o The velocity of the reaction increases in proportion • Assumes that there is a large excess of substrate
to the concentration of the substrate. o The rate of reaction will increase only if there is
o However, when there is an excessive substrate increased substrate concentration
concentration, the velocity of the reaction can no o Less substrate and more enzymes = Rate of
longer increase → reach a maximum velocity. reaction remains the same
▪ Hence, the speed of the reaction plateaus. • This graph is used as a principle in the laboratory in
• Km (Michaelis-Menten constant) assays involving enzymes because we want to know
the concentration of enzymes.
o The substrate concentration in which its velocity is
half of the Vmax (maximum velocity)
TWO THEORIES OF ENZYME-SUBSTRATE
▪ Example: amylase, amylose, lipase and
COMPLEX
triglycerides have its own Km.
Lineweaver-Burk Plot
• A mathematical transformation of the Michaelis-Menten

curve
o Problem of
Michaelis-Menten
curve:
▪ The curve is
difficult to
read, and
difficult to
discover the
Km of a given
enzyme
Lock and Key Theory (1) Substrate Concentration
First-order Reaction
• Are those which proceed at a rate exactly proportional

to the concentration of only one reactant (substrate).
o Are enzymatic reactions in which the speed of the
reaction is dependent on the concentration of the
substrate; one substrate alone.
• 𝑨→𝑷
o Rate of reaction is exactly proportional to the rate of
disappearance of Substrate (A) or the appearance
of Product (P)
• Proposed by Emil Fischer
• Enzyme serves as the key and the substrate serves as Second-order Reaction
the lock
• Problem: It assumes that enzymes are rigid when in • Are those in which the rate is proportional to the product
reality, it is flexible. of the concentration of two substrates
• 𝑨 + 𝑩 → 𝑷
Induced Fit theory
o Rate of reaction is exactly proportional to the rate of
disappearance of Substrate (A) or the appearance
of Product (P)
Zero-order Reaction
• The reactions are zero-order with respect to the

reactants (substrates)
• Rate of reaction depend not on the concentration of the
substrate, but on the concentration of the enzyme.
o Rate of reaction depends on the concentration of the
• Proposed by Robert Copeland molecular species undergoing reaction
• There is a conformational change in the active site until Michaelis-Menten Curve
the substrate is complementarily fit into the active site.
o Active site if flexible.
• Lysozyme
o An enzyme that helps kill bacteria by binding to the
polysaccharide coating of the bacteria.
• Induced fit
o Change of shape of the active site
▪ The shape of the active site changes to fit the
polysaccharide substrate
▪ By initiating the “induced fit”, the enzyme breaks
• First-order reaction
the polysaccharide, which helps in killing the
bacteria. o The speed of the reaction increases as the
concentration of the substrate increases.
o Depends on the concentration of the substrate.
FACTORS THAT INFLUENCE ENZYMATIC • Zero-order reaction:
REACTIONS
o The rate of the reaction in the plateau of the graph
• Factors that affect proteins are the same factors that does not depend on the substrate concentration.
affect enzymes
• No matter how much substrate is added, the reaction
o But there are factors that are unique to enzyme will stay the same.
alone (e.g., Substrate concentration)
o The only way to increase the maximum velocity is by
• In every substrate reaction: adding more enzyme.
o Enzyme + Substrate → Enzyme-Substrate complex o Depends on the enzyme concentration
→ Enzyme + Product NOTE: To achieve the zero-order reaction in the laboratory
to measure the enzyme concentration of a patient
sample, we must increase in excess the substrate.
(2) Enzyme Concentration
• The higher the enzyme level,

the faster the reaction will
proceed
• More enzyme molecules can
react with more substrate
molecules, 50 the reaction
rate increases
• This scenario assumes that
there is a large excess of
substrate.
(3) pH
• pH = 7.0 - 8.0
• Often a product of the enzyme-catalyzed reaction.
o Depends on the enzyme
o When a lot of products are made, the product would
• Changes in pH may denature the enzyme compete with substrate for the enzyme’s active site,
• Protein in nature thus, slowing the reaction.
o pH affects proteins because pH (amount of • The competitive inhibitor has roughly the same shape
hydrogen ions in a solution) affects how carboxylic as the substrate, so it can fit in the active site.
acid group and the amino group in amino acids o When it is there the substrate is unable to bind to
behaves the enzyme and the desired reaction does not occur.
▪ Which is to either gain or lose hydrogen.
• A competitive inhibitor may be the end product of the
(4) Temperature reaction (negative feedback), or it may be another
chemical which blocks the substrate from binding to the
• 37°C (normal) active site.
• Denaturation at 40-50°C • Adding more substrate can decrease the inhibition
• Assay temperatures:
o More chances that the substrates will go to the
o At 25°, 30°, or 37°C active site
(5) Cofactors Noncompetitive Inhibition of Enzymes (Allosteric
Inhibition)
• Non-protein entities that must bind to particular
enzymes before a reaction occurs • Noncompetitive inhibitor inhibits the allosteric site.
• Activators: metallic or nonmetallic
o Once the connect to the allosteric site of the
o Calcium, ferrous, magnesium, manganese, zinc, enzyme, they might change the form of the enzyme
potassium, bromide, chloride ions to the point that the substrate can no longer fit to the
active site.
• If they are absent and they are needed in the enzymatic
reactions then enzymatic reactions cannot proceed
Activators
• Proper substrate binding

• Linking substrate to the enzyme or coenzyme
• Undergoing oxidation or reduction
Coenzymes
• Second substrates of enzymatic reactions

• Prosthetic group
• In assays, this should be provided in excess
(6) Inhibitors
• Interfere with enzyme reactions

o a certain molecule that would inhibit a reaction
Competitive Inhibition of Enzymes
• Competitive inhibitor competes with the substrate for

the active site.
• The regulatory site is often called an allosteric site. Maximum Velocity and Michaelis-Menten Constant
• Inhibiting molecule is often the product of the enzyme-
catalyzed reaction Competitive Inhibition
o Inhibition is said to be a form of a negative feedback
because an increase in the product, forces a • Black curve – uninhibited
decrease in the enzyme reaction reaction
• When in high concentrations, the allosteric inhibitor o Vmax = 1 (unchanged)

binds to the regulatory site. o Km = 1 (unchanged)
o This changes the shape of the enzyme, thus • Purple curve –

changing the shape of the active site, and thus competitively inhibited reaction
preventing the enzyme from catalyzing its reaction. o Vmax = 1 (unchanged)
o When the concentration of inhibitor decreases, the o Km = 4.0 (increased)
inhibitor leaves the regulatory site, and the active
site then resumes its normal shape. ▪ Takes more substrate to reach the maximum
velocity
▪ The enzyme resumes catalyzing its reaction
Noncompetitive Inhibition
• Adding more substrate will not decrease the
inhibition • Black curve – uninhibited
o It will only compete with the allosteric site reaction
Types of Noncompetitive Inhibitor o Vmax = 1 (unchanged)
o Km = 1 (unchanged)
• Reversible • Purple curve – noncompetitively inhibited reaction
o Once inhibitor is removed to the allosteric site, the o Vmax = 0.5 (decreased)
active site returns to normal o Km = 1.0 (unchanged)
• Irreversible ▪ Maximum velocity will not be reached if inhibited
o Once it joins to the allosteric site, it will destroy the noncompetitively.
enzyme. Uncompetitive Inhibition
Uncompetitive Inhibition of Enzymes
• Black curve – uninhibited
reaction
o Vmax = 1 (unchanged)
• Red curve – uncompetitively inhibited reaction
o Vmax = 0.5 (decreased)
o Km = 0.5 (decreased)
• Problem:
o Changes between competitive, noncompetitive, and
uncompetitive inhibition in the Michaelis-Menten
Curves are not apparent.
• Uncompetitive inhibitor will not inhibit unless enzyme
substrate complex is formed. • Solution: Use Lineweaver-Burk transformation line in
o It will not bind to active site nor the allosteric site of analysing enzymes to better visualize inhibitions.
the enzyme, unless there is an enzyme substrate
Lineweaver-Burk Line Interpretation
complex.
• The more substrates added, the more enzyme- • Vmax – point where the line intersects the y-axis
substrate complex will form, the more the reaction is • Km – point where the line intersects the x-axis
inhibited. • Changes in the Lineweaver-Burk Line indicates what
• Adding more substrate will result to increase type of inhibition inhibits the reaction
inhibition Competitive Inhibition
o More enzyme-substrate complex, more inhibition
• Black line – uninhibited
Summary reaction
• The more substrates added: o Vmax = 1 (unchanged)

o Competitive inhibition: Decreased inhibition
o Noncompetitive inhibition: Inhibition stays the same • Red line – competitively
o Uncompetitive inhibition: Increased inhibition inhibited reaction
o Vmax = 1 (unchanged) o If the serum of the patient is added to the reagent,

o Km = 2.5 (increased) lactate becomes pyruvate and NAD+ becomes
NADH.
▪ Slope increases but Y-intercept is still the same
▪ NAD+, overtime, increases in
Noncompetitive Inhibition
absorbance/concentration.
• Black line – uninhibited reaction o The rate of the increase in concentration

corresponds to the enzyme concentration.
o Km = 1 (unchanged) • Zero-order reaction is important because lactate and
NAD+ may run out, resulting in a plateau of the rate of
• Red line – noncompetitively the reaction
inhibited reaction
o Hence, enzyme concentration cannot be measured
o Vmax = 0.5 (decreased)
o Km = 1 (unchanged) • Time over absorbance graph
▪ Vmax (y-intercept) moves up while the Km (x- o Linear portion of the graph is where the enzyme
intercept) remains the same. concentration can be measured.
o NOTE: Lineweaver-Burk line shows a reverse ▪ If there will be too much enzyme, lactate and
interpretation of the Vmax. The higher the Vmax (y- NAD+ will be immediately utilized. The graph will
intercept), the slower the reaction velocity. show a sudden increase and becomes plateau.
▪ The enzyme concentration cannot be measured
Uncompetitive Inhibition if the substrate is inadequate. Hence, excess
substrate concentration is needed.
• Black line – uninhibited reaction
Enzymatic Assays
o Km = 1 (unchanged) Coupled-Enzyme Assay
• Red line – Uncompetitively • Substances other than substrate or coenzyme are
inhibited reaction necessary and must be present in excess.
o Vmax = 0.5 (decreased) • NAD+ or NADH
o Km = 0.5 (decreased)
o Convenient when neither is a coenzyme
▪ Shows parallel lines
• Use of auxiliary enzyme and indicator enzyme
MEASUREMENT AND ASSAYS

Measurement of Enzymatic Activity
• Ways to measure the enzyme activity in the lab:

o Increase in product concentration
o Decrease in substrate concentrate
o Decrease or increase in coenzyme concentration
o An increase in the concentration of the altered
coenzyme
[𝑬𝒏𝒛𝒚𝒎𝒆]
[𝑺𝒖𝒃𝒔𝒕𝒓𝒂𝒕𝒆] → [𝑷𝒓𝒐𝒅𝒖𝒄𝒕] • Example: Lipase Test
o In measuring the lipase of the patient, we do that in
• Enzymes can be measured by measuring either its
a series of enzymatic reactions.
substrate or product
▪ Triglycerides and Glycerokinase are part of the
o NOTE: Coenzymes can be considered as
reagent.
substrates while altered coenzymes can be
considered as products o This assay is performed in sequence because there
is no spectrophotometric analysis that could directly
• How to measure the LDH of a patient? measure glycerol and fatty acids.
𝐿𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷+ ↔ 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 +
Fixed-Time Method (End-point)
o Reagent must contain the substrates lactate and
NAD+, and must be provided in excess to achieve • Reactants are combined.
the zero-order reaction. • Reaction proceeds for a designated time.
o LDH is provided by the serum of the patient, and it is • Reaction is stopped.
measured using spectrophotometry. o Usually by inactivation (weak acid)
▪ NADH can absorb light at 340 nm, • Measurement is made of the amount of reaction has
▪ NAD+ cannot absorb light at 340 nm. occurred
Continuous-Monitoring Method (Kinetic Assay)
• Multiple measurements
• Absorbance change
o Increasing in absorbance
o Decreasing in absorbance
• Time intervals or continuously
• Linearity is verified and deviation is observable
Difference between Fixed-Time Method and Continuous-
Monitoring Method
• Fixed-time Method
o When the absorbance is measured only once.
• Continuous Method
o Several measurements over time
▪ Measure the absorbance over a series of period
of time (after 1 min, 2 mins, etc.)
• In enzymatic concentration, continuous method is much
better to see how fast the enzymatic reaction is.
Common Cause of Deviation from Linearity
• Very high concentration of enzymes

o In comparison to substrate
o Too much enzyme, substrates are consumed =
absorbance becomes plateau
▪ Enzyme concentration cannot be measured
• Too low substrate
Calculation of Enzyme Activity
International Unit (IU)
• Amount of enzyme that will catalyse the reaction of 1

micromole of substrate per minute under specified
conditions
• Enzyme activity
International Unit per Liter (IU/L)
• Enzyme concentration
Katal Unite (mole/s)
• Amount of enzyme that will catalyse the reaction f 1

mole of substrate per second under specified conditions
o 1.0 IU = 16.7 nkat
[TRANS] LABORATORY UNIT 02: AMS, LIP, CK and LDH (LABORATORY)
ENZYMES
Table 1. Two Types of Isoenzymes
Review of Concepts:
S – Type P – Type
• Enzymes are proteins and it catalyzes different
biochemical reaction in the body Derived from Salivary tissue,
Derived from Pancreatic tissue
• Enzymes require substrates to yield a product(s) fallopian tube and lung
• Requires specific: S1, S2, S3 P1, P2, P3
o Environmental condition(s) to be active a.k.a. ptyalin
▪ E.g., pH - a pH above or below could denature
the proteins rendering it to be inactive • For electrophoresis:
o Cofactors to be active o S-type: Migrates more quickly than P-type.
o P-type: Migrates slower than S-type.
▪ E.g., Ca2+ in alpha-amylase
• For activity:
o Enzymes can be activated or inhibited
o S-type: Terminated in the stomach because of the
gastric pH.
Alpha-Amylase
Tissue Sources
Properties
• Two major sources of the alpha-amylase:
• IUB Nomenclature:
o Acinar cells of the pancreas
o E.C. 3.2.1.1; 1,4-α-D-glucan glucanohydrolase o Salivary glands
(AMY)
• Other sources: skeletal muscle, small intestine and
• Classification: Hydrolase fallopian tubes
• Molecular weight:
Diagnostic Significance
o 50,000 to 55,000 Da
▪ Amylase is normally occurring in the plasma • Serum and urine AMY measurements is in the
because of its small size. diagnosis of Acute pancreatitis.
▪ Since it is small, it can pass through the o Alpha-AMY - not specific for acute pancreatitis
glomeruli of the kidneys. because it can also be present in other tissues.
o NOTE: Amylase is the only plasma-enzyme found in ▪ If the other tissues are injured, there will also be
the urine. an elevation in the alpha-amylase.
• Activity: • Elevates: 5 to 8 hours after the onset of an attack
o Catalyse the breakdown of starch and glycogen • Peak: 24 hours
• Return to normal levels within 3 to 5 days.
• Substrates: • Values generally range from 250 to 1,000 Somogyi
o Amylose, amylopectin, and glycogen units per dL (2.55×ULN).
▪ Substrates are bigger molecules.

Table 2. Assay for Enzyme Activity
• Products:
o Glucose, Maltose and Dextrins Characteristics Amylase Methodologies
▪ Products are smaller molecules. Measures the disappearance of starch
Amyloclastic
• Optimum pH: 6.9 – 7.0 substrate.
• Cofactors: Calcium and Chloride Saccharogenic Measures the appearance of the product.
o For them to be active. Measures the increasing color from
Chromogenic production of product coupled with a
• Activators: Bromide, nitrate, cholate, or monohydrogen chromogenic dye.
phosphate
Continuous Coupling of several enzyme systems to
• Isoenzyme: S-type and P-type monitoring monitor amylase activity.
ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO.LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. CORTEZ. BSMLS 3 1
AQUINO. CAGAS
LABORATORY UNIT 02: AMS, LIP, CK AND LDH
Specimen Collection, Storage, and Sources of Error • Reaction Principle
• Specimen 5 𝐶𝑁𝑃𝐺3 →
α−𝐴𝑚𝑦𝑙𝑎𝑠𝑒
3 CNP + 2 CNPG2 + 3 G3 + 2 G
o Ideal: Serum, Heparinized plasma, urine
• Storage: Table 3. Contents of the Reagent
o Room temperature for 1 week or at 4°C for 2
months Reagent Solution Measurement
• Sources of Error:
MES buffer 36 mmol/l
o Elevated plasma triglycerides
CNPG3 1.6 mmol/l
▪ suppress or inhibit serum amylase activity
Calcium acetate 3.6 mmol/l
o Morphine and other opiates
Sodium chloride 37 mmol/l
▪ Numb the sensation of pain and it will lead to
falsely elevated amylase levels when Potassium thiocyanate 253 mmol/l
administered
Sodium azide 0.095%
Reference Range
• Content of Reagent
• Serum: 28 to 100 U/L (37°C) (0.5 to 1.7 µkat/L)
o The different substances stabilize the enzymes.
o NOTE: µkat/micro-Katal – unit of an enzyme’s o Specific pH range – for an enzyme to be active
catalytic activity
▪ MES buffer –stabilizes the solution and pH (6.0),
• Urine: 1 to 15 U/h an ideal pH for α-amylase
o Activators
Package Insert
▪ Calcium
▪ Chloride
• Reagent Preparation and Stability
o Reagent is stable unopened up to the stated expiry
date when stored at 2-8°C.
▪ If the reagent is stable at room temperature or
lower temperature.
▪ For enzymes or other proteins, if it is beyond or
below the required temperature, the enzymes
may be destroyed or inactivated.
o Reagent is ready for use. After opening, it is stable
for 12 weeks when stored light protected at 2-8°C
and for 4 weeks at 15-25°C.
o Contamination of the reagent solution must be
avoided.
• Specimen
o Ideal: Serum, heparinized plasma, and urine
▪ Found in Urine since α-amylase is small in size
o No loss of activity within 5 days at 4-25°C.
• Assay Parameters
• Method
o Wavelength: Hg 405 nm, (400-410 nm)
o The α-amylase liquicolor colorimetric test comprises o Optical path: 1cm
a new substrate, 2-chloro-4-nitrophenyl- o Temperature: 25°C, 37°C
maltotrioside (CNPG3). o Measurement: against H2O (increasing absorbance)
o The substrate reacts directly with α-amylase and o Warm the reagent solution and the cuvettes to the
does not require the presence of ancillary enzymes. desired temperature. Temperature must be kept
The release of 2-chloro-4-nitrophenol (CNP) from constant (±0.5°C) for the duration of the test.
the substrate and the resulting absorbance increase
per minute is directly related to the α-amylase
activity in the sample.
o The method is calibrated for the new IFCC method
to obtain the same Reference value.
AQUINO. CAGAS
Table 4. Procedure Table 6. Reference Values
Pipette into cuvettes 25°C 37°C Values
Sample 20 µl 10 µl Temperature 25OC 37OC IFCC
Serum, plasma 120 U/I 220 U/I 28-100 U/I

Reagent 1000 µl 1000 µl
Spontaneously
600 U/I 1000 U/I ≤ 460 U/I
voided urine
• Procedure 24-hr urine ** 450 U/24h 900 U/24h ≤ 410 U/I
o (1) Mix well, incubate for 1 minute at the desired
temperature. ** Calculated Values
o (2) Read the absorbance and at the same time start
the stopwatch. • Quality Control
o (3) Read the absorbance again after 1, 2, and 3 o All control sera with α-amylase values determined
minutes. by the α-AMYLASE liquicolor method may be used
o Take note of the sample volume to be used for each o We recommended to use our HumaTrol quality
incubation temperatures (25°C/37°C) and the control sera, based on animal serum, or our
amount of reagent to be added. SERODOS made of human serum
o E.g., 25°C
• Automation
▪ Sample: 20 µl
▪ Reagent: 1000 µl o Proposals to apply the reagents on analysers are
available on request. Each laboratory has to validate
• Take note of the calculations and factors to be used: the application in its own responsibility.
o For amylase, CK and LDH • Notes
o Incubation: 25°C or 37°C
o Saliva and sweat contain α-amylase. To avoid
possible contamination do not pipette by mouth and
Calculation for Alpha-Amylase avoid contact of the reagent and pipette tips with the
skin.
• From the readings determine the mean absorbance o RGT may turn yellowish during shelf life. This does
change per minute (∆𝐴/𝑚𝑖𝑛) and employ this for the not affect the test performance as long as the
calculation of the α-amylase activity in the sample. Use reagent blank is < 0.200 A. Otherwise RGT should
the following factors: no longer be used.
o RGT contains sodium azide (0.095%). Do not
Table 5. Calculation for Alpha-Amylase swallow. Avoid contact with skin and mucous
membranes.
Factors Mean Absorbance Change per minute Lipase
U/I (25°C) = ∆𝐴/ min 𝑥 9864 Properties

U/I (37°C) = ∆𝐴/ min 𝑥 24820 • IUB Nomenclature:
IFCC: U/I (37°C) = ∆𝐴/ min 𝑥 10183 o E.C. 3.1.1.3 : Triacylglycerol Acylhydrolase
• Classification: Hydrolase
• Activity:
• Conversion factors:
o Hydrolyzes the ester linkages of fats to produce
o 1 U/I = 16.67 x 10-3 µkat/I
alcohols and fatty acids
o 1 µkat/I = 60 U/I
Tissue Source
Performance Characteristics
• Pancreas
• Stomach and small intestine
• Linearity
o If the absorbance change per minute exceeds Diagnostic Significance
∆𝐴/ min = 0.300, dilute 0.1 ml sample with 0.5 ml
NaCl solution (0.9%) and repeat the assay using this • Diagnosis of acute pancreatitis
dilution. Multiply the result by 6. • Acute pancreatitis: increases 4 to 8 hours after an
o Typical performance date can be found in attack
Verification Report • Peak: 24 hours
• Decrease within 8 to 14 day
• Persist for approximately 8 days in acute pancreatitis
o The best diagnostic test to use is lipase
AQUINO. CAGAS
o Lipase arises early and persist for up to 8 days as • Optimum pH:

compared to α-amylase which only persist 2-3 days
o 9.0 (Forward reaction)
after the acute attack.
o 6.7 (Backward reaction)
Assays For Enzyme Activity
Tissue Sources
• Classic Cherry-Crandall Method
• Skeletal muscles
o Reference method • Heart muscle
o Substrate: Olive Oil • Brain tissues
o Endpoint: Measurement of the liberated fatty acids • Other sources: Bladder, placenta, GIT, thyroid, uterus,
by titration after a 24-hour incubation kidney, lung, prostate, spleen, liver, and pancreas
▪ Modifications of the Cherry-Crandall Method are
available but lacks stable and uniform substrate. NOTE: If a tissue needs ATP, it contains creatine kinase.
▪ Triolein (substrate) – pure form of triglyceride; Almost all tissues in the body have creatine kinase
used today to address the problem with modified
methods. Table 7. Creatine Kinase
• Turbidimetric Method
Isoenzymes Tissue Source Electrophoresis
o Fats in the solution create a cloudy emulsion
o As the fats are hydrolyzed by LPS, the particles CK-MM Skeletal muscle CK-3
disperse
CK-MB Heart muscle CK-2
▪ It is a turbidimetric method due to the
involvement of particles.
CK-BB Brain tissues CK-1
o The rate of clearing can be measured as an
estimation of LPS activity
o Relatively simpler and more rapid than the reference
method
• Colorimetric Method
o Based on coupled reactions with enzymes such as
peroxidase or glycerol kinase
Specimen Collection, Storage, and Sources of Error • On electrophoretic separation, CK-BB will migrate
fastest towards the anode and is therefore called CK1
• Specimen: Serum (ideal specimen) o Followed by CK-MB (CK-2)
• Storage: in activity at room temperature for 1 week or o Finally, CK-MM (CK-3)
for 3 weeks at 4°C
• Sources of error: Hemolysis should be avoided ▪ Exhibit the slowest mobility
because hemoglobin inhibits the activity of serum LPS NOTE: The MM-4 from the skeletal muscles is the major
o Hemolysis causes falsely low serum LPS values. isoenzyme in the sera of healthy people.
Reference Range Diagnostic Significance
• LPS (serum): <38 U/L (37°C) or (<0.6 µkat/L) • Used to determine if the person/patient have
Myocardial Infarction
o U/L – units per liter
o µkat/L – microkatal per liter o Rise: within 4-8 hours after the onset
o Peak: 12-24 hours
Creatine Kinase o Return to normal: within 48-72 hours
Properties • Elevated levels of the CK-MB, ≥6% of the total CK is

considered a good indicator of myocardial damage,
• IUB Nomenclature: particularly acute myocardial infarction
o E.C. 2.7.3.2 o There is obstruction in the flow of oxygenated blood
supply in the cardiac muscles itself
• ATP: Creatine N-phosphotransferase o If prolonged, cardiac cells are damaged, resulting to
• Classification: Transferase the leaking of CK enzymes inside of the cells
• Molecular weight:
• Troponins
o MW: 82,000 Da
o Non-enzyme proteins
• Activity: o More specific and elevated in the absence of CK-MB
elevations
o Catalyzes the rephosphorylation of ADP to ATP
using creatine phosphate as the phosphorylation
reservoir
AQUINO. CAGAS
Assays for Enzyme Activity Reference Range
• Tanzer-Gilvarg • Total CK:

o Forward reaction o Males: 46 to 171 U/L (37°C) (0.8 to 2.9ukat/L)
o Reaction Principle:
▪ a bit higher due to muscle mass
▪ Starts with Creatine and ends with lactate
o Females: 34 to 145 U/L (37°C) (0.6 to 2.4ukat/L)
• CK-MB: <5% total CK
Package Insert
o Enzymes involved:
▪ Creatine kinase (CK)
▪ Phosphoenolpyruvate kinase
▪ Lactate dehydrogenase
• Oliver-Rosalki
o Reverse Reaction
o Most commonly performed method
o Proceeds 2-6 times faster than the forward reaction
▪ There is less interference from the side reaction
o Reaction Principle:
▪ Starts with Creatine phosphate and ends with 6-
phosphogluconate
o Enzymes involved:
▪ Creatinine kinase
▪ Glucose-6-phosphate dehydrogenase
Specimen Collection, Storage, and Sources of Error

• Method
• Specimen:
o HumaZym CK-MB is based on an enzymatic CK
o Serum, Heparinised Plasma, EDTA Plasma
determination accompanied by an immunoinhibition
• Storage: method. An antibody is incorporated into the reagent
which will bind specifically to the M-subunit,
o <8hrs at room temperature inhibiting the enzymatic activity of that subunit.
▪ 48hrs at 4°C, and ▪ Thus only the remaining activity of the B-subunit
▪ 1 month -20 C is measured.
• Sources of error: o Due to negligible concentrations of CK-BB in the
o Moderate and severely hemolysed specimens are circulation, the remaining activity, multiplied by the
not accepted for analysis or a request for creatine factor 2, represents the activity of the CK-MB
kinase is made isoenzyme.
▪ RBCs do not contain CK; they are rich in

adenylate kinase • Reaction Principle
▪ Adenylate kinase reacts with ADP to produce 𝐶𝐾
ATP → ATP participates in the assay → causing 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐴𝐷𝑃 ↔ 𝐶𝑟𝑒𝑎𝑡𝑖𝑛𝑒 + 𝐴𝑇𝑃
a falsely elevated CK level. 𝐻𝐾
𝐴𝑇𝑃 + 𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 ↔ 𝐴𝐷𝑃 + 𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 − 6 − 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 (𝐺6𝑃)
𝐺6𝑃−𝐷𝐻
𝐺6𝑃 + 𝑁𝐴𝐷𝑃 ↔ 6 − 𝑝ℎ𝑜𝑠𝑝ℎ𝑜 − 𝐷 − 𝑔𝑙𝑢𝑐𝑜𝑛𝑜 − 𝑙𝑎𝑐𝑡𝑜𝑛𝑒 + 𝑁𝐴𝐷𝑃𝐻 + 𝐻 +
AQUINO. CAGAS
Table 8. Contents, Reagent Composition in the Test • Procedure

o Prior to determining CK-MB activity it is
Reagents and its Contents Measurement recommended to measure total CK activity using the
CK-NAC activated method (CK-NAC liquiUV, REF
[BUF] 1 x 60 ml buffer solution
12015, HumaZym M-Test, REF 12005) in order to
ensure correct diagnostic interpretation of the test
Imidazole buffer (pH 6.7) 0.10 mol/l results.
o Warm reagents and cuvettes to the desired
Glucose 20 mmol/l
temperature. Temperature must be kept constant
Mg-acetate 10 mmol/l (±0.5°C) for the duration of the test.
EDTA 2.00 mmol/l

Table 9. Macro and Micro
[ENZ] 20 x 3 ml enzyme/antibody reagent (lyoph.)
ADP 2.00 mmol/l Macro Micro
Pipette directly into the

AMP 5.00 mmol/l Pipette into cuvettes
reconstituted working reagent
Diadenosine pentaphosphate 10 mmol/l Sample/[CAL] 40 µl
Sample/[CAL] 100 µl
Working Reagent 1000 µl
NADP 2.00 mmol/l (1) Mix and incubate at the
(1) Mix and transfer the solution
desired temperature for 10
to a cuvette.
HK > 2.50 U/ml minutes.
(2) Incubate at the desired
G6P-DH > 1.50 U/ml temperature for 10 minutes. (2) Read the absorbance A1.
Read the absorbance A1.
N-Acetylcysteine 20 mmol/l (3) Exactly after 5 minutes read (3) Exactly after 5 minutes read
the absorbance A2. the absorbance A2.
Creatine phosphate polyclonal
30 mmol/l
Antibody to CK-M subunit
Additional material but not supplied with the kit
Calculation
[REF] 13611
• Calculate the absorbance change per minute (ΔA/min)
[LowHigh] 4 x 2 ml CK-MB Control Human Serum (lyophilised) according to:
[REF] 13612 o ΔA/min = A2-A1: 5.
[CAL] 2 x 1 ml CK-MB Calibrator Human Serum (lyophilised) • The CK-MB activity is calculated using the following
factors:
• Reagent Preparation and Stability Table 10. CK-MB Activity (U/I)

o (1) Reconstitute one vial ENZ with exactly 3ml BUF.
o (2) Swirl gently and incubate for 5 min. at room Semi-micro Macro
temperature prior to use.
o The reagents are stable up to the stated expiry date Hg 334 nm 8414 x ΔA/min 10032 x ΔA/min
when stored at 2…8°C
o The reconstituted working reagent is stable for 5 340 nm 8254 x ΔA/min 9842 x ΔA/min
days at 2…8°C
Hg 365 nm 14858 x ΔA/min 17716 x ΔA/min
• Specimen
o Serum • Conversion factor of traditional units (U/I) in SI-units
o Heparinised plasma or EDTA plasma (kat/l)
o Loss of activity within 1 day at 2…8°C: <10%
o 1 U/l = 16.67x10-3 µkat/l
• Assay o 1 µkat/l = 60 U/l
o Wavelength: Hg 334nm, 340 nm, Hg 365 nm Performance Characteristics
o Optical path: 1 cm
o Temperature: 25°C, 30°C or 37°C • Linearity
o Measurement: against air (increasing absorbance)
o The antibody inhibits the activity of M-subunits up to
2000 U/I of CK MM
• Reference Range, Myocardial Infarction (MI)
o The likelihood of myocardial damage is high if the
following 3 criteria are met:
AQUINO. CAGAS
Table 11. Reference Range (MI) Table 12. Lactate Dehydrogenase (LDH) Isoenzymes –
Tissue Localization and Sources of Elevation
25℃3 30℃4 37℃
Isoenzyme Tissue Disorder
Total CK
Heart Myocardial infarction
Men > 80 U/I > 130 U/I > 195 U/I LDH-1 (HHHH)
Red blood cells Hemolytic anemia
Women > 70 U/I > 110 U/I > 170 U/I
Megalobalastic anemia
Heart
LDH-2 (HHHM) Acute renal infarct
CK-MB > 10 U/I > 16 U/I > 25 U/I Red blood cells
Hemolyzed specimen
CK-MB activity ranging between 6% and 25% of the total CK Pulmonary embolism
activity Lung Extensive Pulmonary
Lymphocytes pneumonia
LDH-3 (HHMM)
Spleen Lymphocytosis
• Quality control Pancreas Acute pancreatitis
Carcinoma
o All control sera with CK-MB values determined by Hepatic injury or
this method can be employed LDH-4 (HMMM) Liver
inflammation
• Automation LDH-5 (MMMM) Skeletal muscle Skeletal muscle Injury
o This kit is designed for manual use. For use on
automates we recommend [REF] 12118. Proposals
to apply this reagents on analysers are available on
request. Each laboratory has to validate the Table 13. Lactate Dehydrogenase (LDH) Isoenzymes
application in its own responsibility. as a Percentage of Total LDH
• Note
o [BUF] contains sodium azide (< 0.095%) as Isoenzyme %
stabiliser. Avoid contact with the skin and mucous
membranes. LDH-1 14-26
Lactate Dehydrogenase LDH-2 29-39
Properties LDH-3 20-26
LDH-4 8-16
• IUB Nomenclature:
o E.C. 1.1.1.27 LDH-5 6-16
• ATP: L-Lactate: NAD+ Oxidoreductase

• Classification: Oxidoreductase • LDH-2 - The major isoenzyme infraction in the sera of
• Activity: healthy individuals
o Catalyzes the interconversion of lactic & pyruvic Diagnostic Significance
acids
• Myocardial Infarction
• Cofactor: Zinc
• Optimum pH: o Similar to CKMB, LDH is also elevated in MI
because of its wide distribution to the body.
o Forward reaction: 8.3-8.9
o Backward reaction: 7.1-7.4 ▪ However, the heart tissue and red blood cells
contains higher concentration of LDH. Thus, it is
not specific for MI and not preferred marker for
Tissue Sources diagnosing acute myocardial infraction.
• Widely distributed in the body: o Rise: within 12-24 hours after the onset
o Peak: 48-72 hours
o Heart o Remain elevated: 10 days
o Liver
o Skeletal muscle ▪ LD 1 > LD 2
o Kidney  LD 1 is the dominant isoenzyme
o Erythrocytes
o Lung  LD flipped pattern
o Smooth muscle  Suggestive AMI
o Brain
• Lactate dehydrogenase is elevated in a variety of
disorders due to its widespread activity in numerous
body tissue, with the highest levels noted in pernicious
anemia and hemolytic disorders
AQUINO. CAGAS
Assays for Enzyme Activity • Method

o “Modified method” based on the recommendations
of the SCE (Scandinavian Committee on Enzymes).
• Reaction Principle
• Forward reaction 𝐿𝐷𝐻
o Substrate: lactate 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 + ↔ 𝐿𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷 +
o Also known as _______
o Produces NADH Table 14. Contents, Reagent Composition in the Test
o Not affected by the product inhibition
o LD-1
Reagents and its Contents Measurement
• Reverse reaction
o Substrate: pyruvate [REF] 12214 12014 12024
o Also known as ________
o LD 5 [BUF] 16 x 4 ml 10 x 8 ml 8 x 40 ml
o Rate of the reverse reaction is approx. 3 times faster [SUB] 1 x 16 ml 2 x 10 ml 8 x 10 ml
but more susceptible to substrate exhaustion and
loss of linearity [BUF] Buffer / Substrate
▪ Allowing smaller samples and volumes and TRIS Buffer (pH 7.35) 62.5 mmol/l
shorter reaction times
Pyruvate 1.5 mmol/l
o Optimal pH
Sodium azide 0.095%
▪ Forward: 8.3 – 8.9
▪ Backward: 7.1 – 7.4 [SUB] Substrate
Specimen Collection, Storage, and Sources of Error NADH 0.75 mmol/l
Sodium azide 0.095%

• Specimen:
o Serum, Heparinised or EDTA plasma • Reagent Preparation
• Storage: o Procedure 1 with reagent start
o 25℃ analysed within 48hrs ▪ The reagents are ready for use
▪ The reagents are stable, even after opening, up
• Source of error: Hemolysis to the stated expiry date when stored at 2-8oC.
o Erythrocytes contains an LD concentration ▪ BUF must be kept light protected.
approximately 100 to 150 times found in the serum. ▪ Contamination of reagents must be avoided
o If there is any red coloration after centrifugation of o Procedure 2 with sample start
the serum or plasma, it is unacceptable for analysis
because it causes falsely elevation of LDH. ▪ REF 12024: Pour the entire contents of one
bottle SUB into one bottle BUF, mix thoroughly.
Reference range ▪ REF 12214: Pipette 1 mL from bottle SUB into
one bottle BUF, mix thoroughly.
• LD: 125 to 220 U/L (37℃) ▪ REF 12014: Pipette 2 mL from bottle SUB into
one bottle BUF, mix thoroughly.
Package Insert o The working reagent is stable for 3 weeks at 2…8oC
and 3 days at 15…25oC.
o The working reagent must be light protected.
• Specimen
o Serum, heparinized or EDTA plasma
o Avoid Hemolysis
o Loss of activity within 3 days 8% at + 4℃, 2% at
15…25℃
• Assay
o Wavelength: Hg 334 nm, 340 nm, Hg 365 nm
o Temperature: 25℃, 30℃ or 37℃
o Measurement: against air (decreasing absorbance)
o Warm the reagents and the cuvettes to the desired
temperature.
o Temperature must be kept at constant (±0.5℃) for
the duration of test.
AQUINO. CAGAS
Table 15. Procedure 1 Performance Characteristics
Pipette into • Linearity

25℃, 30℃ 37℃
cuvettes
o If the absorbance change per minute (∆𝐴/min )
Sample 20 µl 10 µl exceeds 0.150 at Hg 334 nm, 340 nm or 0.070 at Hg
[BUF] 1000 µl 1000 µl 365 nm.
Mix, incubate for 1-5 min. at 25℃, 30℃, or 37℃ o Dilute 0.1 mL of the sample with 0.9 mL
physiological saline (0.9%) and repeat the assay
[SUB] 250 µl using this dilution.
o Multiply the result by 10.
Mix, read the absorbance after 1 minute and at the same time start
the stop watch. Read the absorbance again exactly after 1, 2 and 3
minutes. Table 19. Reference values
Table 16. Procedure 2 Temperature 25 30 37 IFCC4
Pipette into Adults [U/I] 120-240 160-320 225-450

25℃, 30℃ 37℃
cuvettes
Men [U/I] < 243
Sample 20 µl 10 µl
Working reagent 1000 µl 1000 µl
Mix, read the absorbance after 1 minute and at the same time start Women [U/I] < 244
the stop watch. Read the absorbance again exactly after 1, 2 and 3 Children [U/I]
minutes. (up to 12 Up to 500
months)
• Semi-micro method; for macro methods double the
volumes.
• Quality Control
Calculation
o All control sera with LDH values determined by this
• Using the absorbance readings calculate the mean method can be employed.
absorbance change per minute (ΔA/min) o We recommended to use our animal serum based
• Calculate the LDH activity in the sample by multiplying HumaTrol or our human serum based SERODOS
ΔA/min using the following factors: quality control sera.
• Automation
Table 17. Calculation for Procedure 1
o Proposals to apply the reagents on analysers are
available on request.
Hg 334 nm 340 nm Hg 365 nm o Each laboratory has to validate the application in its
own responsibility.
U/I (25℃, 30℃) =
10275 10080 18675 • Note
ΔA/min. x
U/I (37℃ ) = o [BUF] and [SUB] contain sodium azide (0.095%). Do
20390 20000 37060
ΔA/min. x
not swallow. Avoid contact with skin and nucous
membranes.
Table 18. Calculation for Procedure 2
SUMMARY
• A single enzyme is not diagnostic and not specific for a
Hg 334 nm 340 nm Hg 365 nm
disease
U/I (25℃, 30℃) =
• To avoid falsely increased/falsely decreased values we
8250 8095 15000 must take note of the different sources of errors for
ΔA/min. x
U/I (37℃ ) = different enzymes
ΔA/min. x
16345 16030 29705 • Observe the proper storage of specimen if we can’t
analyse them right away
• Conversion factor of the traditional units (U/I) in SI-units

(kat/I):
o 1 U/I = 16.67 x 10-3 ukat/I
o 1 µkat/I = 60 U/I
• Factor to convert results to the new IFCC
recommended method:
o U/I (LDH SCE) x 0.4796 = U/I (LDH IFCC)
AQUINO. CAGAS
[TRANS] LABORATORY UNIT 03: TRANSAMINASES AND PHOSPHATASES (LABORATORY)
TRANSFERASES • Reaction with diazonium salts couples the salt with the
keto acid product and forms a color
• Aspartate aminotransferase
o Salt reacts with oxaloacetate and color change is
• Alanine aminotransferase
observed
Aspartate aminotransferase (AST) Sources of Assay Error
Assays for Enzyme Activity • Hemolysis should be avoided
• Karmen Method o Even though erythrocytes are in smaller

concentrations, it should still be observed as it can
• Reitman and Frankel Method
be a source AST.
(1) Karmen Method o Hemolysed samples can cause falsely increase
AST levels.
• Common method employed in measuring the AST level
• A coupled enzymatic reaction • Stable in serum for 3 – 4 days at ref temp
• Composed of two enzymatic reactions
• Optimum pH: 7.3 - 7.8 NOTE:
• Serum • In all tests, hemolysed samples are not allowed
o Assumed sample • Practice good phlebotomy technique
• 1st reaction: AST Package Insert
o Aspartate aminotransferase (AST) – enzyme

▪ Aside from the enzyme, vitamin B6 is assumed
to be in the serum
• 2nd reaction:
o Seen in Krebs cycle

o Kinetic method
▪ Change in absorbance is monitored continuously
o Malate dehydrogenase (MD) - Indicator enzyme
▪ Causes a color change as malate forms
▪ produces a reaction
o Products:
▪ Malate and NAD+ (oxidized form of NAD)
• After the enzymatic reactions, change in absorbance at

340 nm is monitored continuously.
• Absorbance of NAD+ is specifically measured
o 340 nm – subject to change, depends on the
reagents used
(2) Reitman and Frankel Method
• Reaction with dinitrophenylhydrazine couples color

reagent with keto acid product
o The color reagent, dinitrophenylhydrazine reacts
with the keto acid product, oxaloacetate
ANTOYAN. IBONES. GABAESIN. LATONIO. MACADANGDANG. MERCADO. NARAGA. OLIVA. RELATOR. VILLENA. BSMLS 3 1
CATAPANG. NAVARRO. CAGAS
LABORATORY UNIT 03: TRANSAMINASES AND PHOSPHATES (LABORATORY)
• Method • Specimen
o Kinetic method for the determination of ASAT o Serum (preferred), heparinised plasma or EDTA
activity according to the recommendations of the plasma
Expert Panel of the IFCC (International Federation o Avoid hemolysis
of Clinical Chemistry). o Loss of activity within 3 days
o Without pyridoxalphosphate activation.
▪ At + 4ºC: ~ 8% (Falsely decreased = loss of
• Reaction Principle activity)
▪ At 20-25ºC: ~ 10%
o Karmen Method
▪ Modified in this method is the involvement of
pyridoxalphosphate o Wavelength: Hg 365nm, 340 nm (common) or Hg
334 nm
 No longer needed to determine AST level
o Optical path: 1cm
o Temperature: 25ºC, 30ºC or 37ºC
𝐺𝑂𝑇
2 − 𝑜𝑥𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 + 𝐿 − 𝑎𝑠𝑝𝑎𝑟𝑡𝑎𝑡𝑒 ↔ 𝐿 − 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 + 𝑜𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 ▪ Desired: 25ºC = easier to maintain
𝑀𝐷𝐻 o Measurement: against air (decreasing absorbance)
𝑂𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 + ↔ 𝐿 − 𝑚𝑎𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷 +
▪ Autozero is performed
Table 1. Contents of the Reagent temperature. Temperature must be kept constant (±
0.5oC) for the duration of the test.
Reagent Solution Measurement

Table 2. Procedure 1
[BUF] Buffer / Enzyme reagent
TRIS buffer (pH 7.8) 100 mmol/L Pipette into cuvettes 25ºC, 30ºC 37ºC
L-Aspartate 300 mmol/L Sample 200 uL 100 uL

LDH ≥0.9 kU/L Buffer 1000 uL 1000 uL
MDH ≥0.6 kU/L Mix, incubate for 5 min. at the desired temperature
[SUB] Substrate Substrate 250 uL 250 uL
2-oxoglutarate 60 mmol/L *Semi micro method; for macro methods, double the volumes
NADH 0.9 mmol/L
• Procedure 1
o (1) Mix, read the absorbance after 1 minute
• Reagent Preparation and Stability
▪ At the same time, start the stop watch
o Procedure 1 with Substrate Start (Reagent Start)
o (2) Read the absorbance again exactly after 1, 2
▪ The reagents are ready for use and 3 minutes
▪ The reagents are stable, even after opening up o Buffer is transferred first to the test tube
to the stated expiry date when stored protected
from light at 2-8ºC ▪ Greater volume
▪ Contamination of reagent should be avoided o 4 absorbance readings
o Procedure 2 with Sample Start
▪ Problem: Table 3. Procedure 2
 Requires a working reagent before doing the
actual procedure Pipette into cuvettes 25ºC, 30ºC 37ºC
 Stability is short
Sample 200 uL 100 uL
▪ REF 12031 and 12021: Pour the entire contents
of one bottle [SUB] into one bottle [BUF], mix Working Reagent 1000 uL 1000 uL
thoroughly *Semi micro method; for macro methods, double the volumes
▪ REF 12211: Pipette 1 mL from the bottle [SUB]
into one bottle [BUF], mix thoroughly • Procedure 2
▪ REF 12011: Pipette 2 mL from bottle [SUB] into
one bottle [BUF], mix thoroughly o (1) Mix, read the absorbance after 1 minute
o The working reagent is stable for 4 weeks at 2-8 ºC ▪ At the same time, start the stop watch
(refrigerated); and 5 days at 15-25ºC (room temp) o (2) Read the absorbance again exactly after 1, 2
and 3 minutes
o 4 absorbance readings Performance Characteristics
Calculation for Aspartate Aminotransferase • Linearity

o If the absorbance change per minute (∆𝐴/min)
• For ΔA/min within 0.06-0.08 (Hg 365 nm) or 0.12-0.16 exceed
(Hg 334 nm, 340 nm) (procedure 1+2) use
measurements from the first two minutes for calculation Table 5. Linearity Reference
(1 min. incubation, 2 min. measurements).
Wavelength 25C, 30C

Table 4. Calculation for Aspartate Aminotransferase ΔA/min 37C (U/L)
(nm) (U/L)
Hg 365 0.080 170 320

U/L = ΔA/min x Sample Start Reagent Start
Hg 334/340 0.160 190 350
25C, 25C,
Wavelength 37C 37C
30C 30C
Hg 334 nm 971 1780 1173 2184 o Dilute 0.1 mL of the sample with 0.9 mL
physiological saline (0.9%) and repeat the assay
340 nm 952 1745 1151 2143 using this dilution.
Hg 365 nm 1765 3235 2132 3971
▪ Dilution procedure may be procedure 1 or 2
▪ Ex. procedure 1: Dilute with 1000 L buffer, get
the 4 absorbance readings, perform calculations,
• Conversion factor from traditional units (U/L) in SI-units then multiply by 10 to get the final answer
(kat/L):
o In sera with very high activities, the initial
o 1 U/L = 16.67 x 10-3 kat/L absorbance may be very low as most of the NADH
o 1 kat/L = 60 U/L may have been consumed before the first reading.
• Example: ▪ In this case, rerun the sample after dilution as
described above.
o (1) Take the 4 absorbance readings (ex:)
▪ A1= 0.03; A2= 0.04; A3= 0.05; A4= 0.06 Table 6. Reference Values
o (2) Determine the difference between absorbances
▪ A1 – A2 = 0.01 Temperature 25C 30C 37C IFCC*
▪ A2 – A3 = 0.01
▪ A3 – A4 = 0.01 Men up to 18 U/L 25 U/L 37 U/L 35
o (3) Calculate the mean of the differences/change in Women up to 15 U/L 21 U/L 31 U/L 31
absorbance per minute
* with pyridoxalphosphate activation
(0.01 + 0.01 + 0.01)
= 0.01
3 • Quality Control
o (4) Multiply the mean/change in absorbance per o All control sera with GOT values determined by this
minute to the conversion factor method can be employed.
o We recommend to use our quality control serum
▪ Consider the type of procedure being employed HumaTrol based on animal serum or our SERODOS
 Ex: Reagent start (25°C,30°C, 340nm) = 1151 quality control serum based on human serum
▪ 0.01 × 1151 = 11.51 U/L • Automation
o (5) Refer to reference values o Proposals to apply the reagents on analysers are
▪ Patient: Male; Temp: 25C = 11.51 U/L is within
o Each laboratory has to validate the application in its
the normal range of 0-18 U/L
own responsibility.
o (6) Conversion to kat/L (optional)
• Note
▪ 1 U/L = 16.67 x 10-3 kat/L o [BUF] and [SUB] contain sodium azide (0.095%).
▪ 1 kat/L = 60 U/L
▪ Do not swallow.
▪ Avoid contact with skin and mucous membranes
because it could cause irritation.
Alanine aminotransferase (ALT)
Assays for Enzyme Activity
(1) Wrobleuski La Due Method
• Coupled enzymatic reaction

• Difference with Karmen Method:
o Amino acid: Aspartate → Alanine
o Product: Oxaloacetate → Pyruvate
o Enzyme: ALT → AST
o Enzyme indicator: Lactate Dehydrogenase
• Employed in LD determination
• 1st reaction:
• Method
• 2nd reaction: o Kinetic method for the determination of ALAT activity
according to the recommendations of the Expert
Panel of the IFCC (International Federation of
Clinical Chemistry). Without pyridoxalphosphate
o There is still oxidation of NADH to NAD+ activation.
• Reaction Principle
• Absorbance is still measured at 340 nm 𝐺𝑃𝑇
2 − 𝑜𝑥𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 + 𝐿 − 𝑎𝑙𝑎𝑛𝑖𝑛𝑒 ↔ 𝐿 − 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 + 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒
o Wavelength depends on the reagent used
𝐿𝐷𝐻
• Optimal pH is 7.3-7.8 (same with AST) 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 + ↔ 𝐿 − 𝑙𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷 +
• May employ Reitman and Frankel Method
o Uses a coloring reagent called
Table 7. Contents of the Reagent
dinitrophenylhydrazine
▪ In ALT, it will react to pyruvate
Sources of Assay Error
[BUF] Buffer / Enzyme reagent
• ALT is stable for 3 – 4 days at 4ºC
TRIS buffer (pH 7.5) 150 mmol/L
• Relatively unaffected by hemolysis
L-alanine 750 mmol/L
ALT Package Insert LDH ≥1.2 kU/L
[SUB] Substrate
2-oxoglutarate 90 mmol/L
NADH 0.9 mmol/L
• Reagent Preparation and stability

o Procedure 1 with Substrate Start (Reagent Start)
▪ The reagents are ready to use
▪ The reagents are stable, even after opening up
to the stated expiry date
▪ When stored must be protected from light at 2-
8°C
▪ Contamination of the reagent must be avoided
of one bottle [SUB] into one bottle [BUF], mix
thoroughly.
one bottle [BUF], mix thoroughly

our bottle [BUF], mix thoroughly • Procedure 2
▪ Procedure 2 is not preferred it is a waste of
reagent (uses more reagent)
o The working reagent is stable for 4 weeks at 2-8°C ▪ At the same time, start the stop watch
and 5 days at 15-25°C o (2) Read the absorbance again exactly after 1, 2
and 3 minutes
• Specimen
o Serum, heparinized plasma or EDTA plasma ▪ Both procedures are for
o Avoid hemolysis o 4 absorbance readings
o Loss of activity within 3 days
▪ at +4°C: ~ 10% Calculation for Alanine Aminotransferase
▪ at 20-25°C: ~ 17%
• Assay • For ΔA/min within 0.06-0.08 (Hg 365 nm) or 0.12-0.16
(Hg 334 nm 340 nm) (procedure 1+2) use only
o Wavelength: Hg 365 nm, 340 nm or Hg 334 nm measurements from the first 2 minutes for calculation (1
▪ Common: 340 nm min. Incubation, 2 min. Measurements).
o Temperature: 25°C, 30°C or 37°C Table 10. Calculation for Alanine Aminotransferase
▪ Desired: 25ºC
o Measurement: against air (decreasing absorbance) U/L = ΔA/min x Sample Start Reagent Start
temperature. 25C, 25C,
Wavelength 37C 37C
o Temperature must be kept at constant (±0.5°C) for 30C 30C
the duration of test. Hg 334 nm 971 1780 1173 2184
340 nm 952 1745 1151 2143

Table 8. Procedure 1
Hg 365 nm 1765 3235 2132 3971
Pipette into cuvettes 25ºC, 30ºC 37ºC
Sample 200 uL 100 uL • Conversion factor from traditional units (U/l) in SI-units
(kat/l):
Buffer 1000 uL 1000 uL o 1 U/l = 16.67 x 10-3 µkat/l
o 1 µkat/l = 60 U/l
Mix, incubate for 5 min. at the desired temperature
Substrate 250 uL 250 uL
*Semi micro method; for macro methods, double the volumes • Linearity
o If the absorbance change per minute (ΔA/min) or the
• Procedure 1 activity exceed

Table 11. Linearity Reference
▪ At the same time, start the stop watch
o (2) Read the absorbance again exactly after 1, 2 Wavelength
ΔA/min
25C, 30C
37C (U/L)
and 3 minutes (nm) (U/L)
o Buffer is transferred first to the test tube
Hg 365 0.080 170 320
▪ Greater volume
Hg 334/340 0.160 190 350
o 4 absorbance readings
o Dilute 0.1 mL of the sample with 0.9 mL
Table 9. Procedure 2 physiological saline (0.9%) and repeat the assay
using the dilution.
Pipette into cuvettes 25ºC, 30ºC 37ºC o In sera with very high activities, the initial
absorbance may be very low as most of the NADH
Sample 200 uL 100 uL may have been consumed before the first reading
Working Reagent 1000 uL 1000 uL ▪ In this case rerun the sample after dilution as
described above.
*Semi micro method; for macro methods, double the volumes
Table 12. Reference values • pH: 10.2 (Bishop)

o must be alkaline; should be near the optimal pH of
Temperature 25C 30C 37C IFCC* ALP which is pH 9 to 10
o 10.15; 10.3 (Tietz)
Men up to 18 U/L 25 U/L 37 U/L 35
• Products:
Women up to 15 U/L 21 U/L 31 U/L 31 o p-Nitrophenol
*with pyridoxalphosphate activation ▪ p-Nitrophenoxide ion is also another name for p-
• Quality control Nitrophenol (Tietz)
▪ p-Nitrophenol can be seen by the naked eye –
o All control sera with GPT values determined by this the mixture becomes yellow
method can be employed.
o We recommend to use our animal serum based o Phosphate ion
HUMATROL, or our human serum based • Substrate → hydrolyzed by ALP with an alkaline pH →
SERODOS quality control sera. produces p-Nitrophenol and Phosphate ion → Mixture
• Automation of reagent and serum sample becomes yellow due to p-
Nitrophenol
o Proposals to apply the reagents on analyses are • Enzyme activity is measure at 405 nanometers
o Each laboratory has to validate the application in its
own responsibility. Table 13. Summary of Other Methods of ALP Assays
• Note
o SUB and BUF contain sodium azide (0.095%). Method Substrate End Products
▪ Do not swallow. beta- Inorganic phosphate +

Bodansky
▪ Avoid contact with skin and mucous membrane glycerolphosphate glycerol
beta- Inorganic phosphate +

PHOSPHATASES Shinowara
glycerolphosphate glycerol
• Alkaline Phosphatase
beta- Inorganic phosphate +
• Acid Phosphatase Jones
Alkaline phosphatase (ALP) beta- Inorganic phosphate +
Reinhart
King and Phenol + phosphate
phenylphosphate
Continuous-Monitoring technique (Bowers and McComb) Armstrong ion
Huggins and Phenolphthalein Phenolphthalein red +

• Calculation of ALP activity based on the molar Talalay diphosphate phosphate ion
absorptivity of p-nitrophenol
• Bowers and McComb method is the established and Moss
Alpha-naphthol Alpha-naphthol +
most commonly used phosphate phosphate ion
• Bessy-Lowry-Brock Method
Buffered
Klein, Babson Free phenolphthalein
o Substrate and products are the same for both and Read
phenolphthalein
+ phosphate ion
Bowers and McComb method and Bessy-Lowry- phosphate
Brock Method
• Reaction Principle: Sources of Assay Error
• Hemolysis can cause slight elevations

o Causes falsely increased ALP results
o ALP being 6 times more concentrated in the red
blood cells compared to in the serum.
• Citrate and EDTA shouldn’t be used as anticoagulants
o They both will bind to cations like magnesium and
zinc
o Magnesium and zinc are activators thus interfere
with ALP testing
o Can lead to falsely decreased ALP results
• Should be run ASAP
• Substrate: p-Nitrophenyl-phosphate o Activity increases 3 – 10% on standing at RT or
o New name: 4-Nitrophenyl phosphate 4oC for several hours
o If days have passed, the activity of the enzyme will Table 14. Contents of the Reagent
decrease
• Diet may induce elevations “B” and “O” individuals who Reagent
are secretors
[REF] 12217 12017 12027 12037
o Can lead to falsely increased ALP results
[BUF] 16 × 4 ml 10 × 8 ml 8 × 40 ml 4 × 200 ml
o 25% higher following fat meal
o Fasting is preferred for patients before submitting [SUB] 1 × 16 ml 2 × 10 ml 8 × 10 ml 4 × 50 ml
themselves for ALP testing Buffer
NOTE: Diethanolamine buffer (pH 10.35 ± 0.2) 1.25 mol/l
[BUF]
• The activity of the enzyme is tested since the enzyme 0.625
itself cannot be measured Magnesium chloride
mmol/l
• The amount of enzymes present in the body is then
Substrate
correlated with the result of the activity of the enzyme [SUB]
acquired from the assays p-Nitrophenyl phosphate 55 mmol/l
p-Nitrophenyl phosphate 55 mmol/L
ALP Package Insert
• Buffer
o Magnesium chloride (activator)
o Diethanolamine buffer
▪ ensures that the environment of the enzyme is
alkaline
• Reagent Preparation
o Procedure 1 with Reagent Start
▪ Commonly used
▪ The reagents are ready for use
▪ The reagents are stable even after opening up to
the stated expiry date when stored at 2-8°C
▪ Contamination of the reagents must be avoided
▪ Working reagent is still prepared and there could
be a lot of excess waste
of one bottle SUB into one bottle BUF, mix
thoroughly
▪ REF 12217: Pipette 1 ml from the bottle SUB
into one bottle BUF, mix thoroughly
▪ REF 12017: Pipette 2ml from bottle SUB into
one bottle BUF, mix thoroughly
▪ The working agent is stable for 4 weeks at 2-
8°C, for 5 days at 15-25°C. The working agent
must be kept light protected
• Specimen
o Serum or heparinised plasma
▪ Do not use EDTA or Citrate (bind to activators)
▪ Falsely decreased result
o Avoid hemolysis
▪ RBC source of ALP
• Method
o Loss of activity within 7 days; 0% at 4°C, 10% at 20-
o Optimised standard method according to the 25°C
recommendations of the German Clinical Chemistry
Association (Deutsche Gesellschaft fur Klinische ▪ Loss of activity – days are considered
Chemie) ▪ Organic substances may deteriorate when
outside the body
• Reaction Principle ▪ Refrigerated (4°C) – 0% after one week
𝐴𝐿𝑃 ▪ Room temperature (20-25°C) – falsely
𝑝 − 𝑁𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑦𝑙𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐻20 ↔ 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝑝 − 𝑛𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑜𝑙 decreased result after 7 days
• Assay Performance Characteristics

o Wavelength: Hg 405 nm (400-420 nm)
o Optical path: 1cm • Linearity
o Temperature: 25°C, 30°C or 37°C o If the absorbance change per minute (Δ A/min.)
o Measurement: Read against air (increasing exceeds 0.250 dilute 0.1 ml of the sample with 0.5
absorbance); empty cuvette ml physiological saline (0.9 %) and repeat the assay
o Warm the reagent and the cuvette to the desired using this dilution. Multiply the result by 6.
temperature. Temperature must be kept constant (± o After diluting the serum sample, either of the
0.5°C) for the duration of the test procedures 1 or 2 should be performed.
Table 15. Procedure 1 with Reagent Start
Table 17. Reference Values
Pipette into cuvettes 25ºC, 30ºC, 37ºC
Temperature 25ºC U/I 30ºC U/I 37ºC U/I
Sample 20 µl
[BUF] 1000 µl
Women2 40-90 49-232 64-306
Mix, incubate for 1 min. at the desired temperature
Men2 50-190 61-232 80-306
[SUB] 250 µl
Children up to 15
Up to 400 Up to 488 Up to 644
years2
• Procedure 1 Children up to 17
Up to 300 Up to 366 Up to 483
o (1) Mix, read the absorbance after 1 minute and at years2
the same time start the stop watch.
o (2) Read the absorbance again exactly after 1, 2 • Reference values
and 3 minutes.
o If the patient in terms of age is not older than 17
• One test tube is needed years, their sex will not be considered
• Add first the buffer (greater volume) o If the patient is above the age of 17, their sex will be
• Should acquire four (4) absorbance readings considered
o Note: Age, sex and desired temperature (during the
procedure) are the ones that should be identified
Table 16. Procedure 2 with Sample Start first
• Quality Control
Pipette into cuvettes 25ºC, 30ºC, 37ºC
o All control sera with alkaline phosphatase values
Sample 20 µl determined by this method can be employed.
o We recommend to use our animal serum based
Working Reagent 1000 µl HumaTrol quality control sera or our human serum
*Semi-micro method; for macro methods multiply volumes by 2 (only based SERODOS.
differ in volume)
• Automation
• Procedure 2 o Proposals to apply the reagents on analysers are
o (1) Mix, read the absorbance after 1 minute and at available on request. Each laboratory has to validate
the same time start the stop watch. the application in its own responsibility
o (2) Read the absorbance again exactly after 1, 2 • Notes
and 3 minutes.
o BUF and SUB contain sodium azide (0.095%) as
• Should acquire four (4) absorbance readings preservative. Do not swallow. Avoid contact with
Calculation skin and mucous membranes!
o During the reaction p-nitrophenol is produced.
• From the readings calculate the mean absorbance This substance is poisonous when inhaled,
change per minute (ΔA/min) swallowed or when absorbed through the skin. If
• Calculate the alkaline phosphatase activity using the the reaction mixture comes into contact with skin or
following factor: mucous membranes wash copiously with water and
consult a doctor.
o U/I = ΔA/min. x 3433 (procedure 1 with reagent
start) OR
o U/I = ΔA/min. x 2757 (procedure 2 with sample Acid phosphatase (ACP)
start)
• Conversion factor from traditional units (U/l) in SI units
(kat/I): • Shinowara Method
o 1 U/l = 16.67 x 10-3 µkat/l • Immunochemical Techniques
(1) Shinowara Method • Consider the bilirubin level of the patients when
measuring the:
𝐴𝐶𝑃
𝑝-𝑁𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑜𝑙𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 ↔ 𝑝-𝑁𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑜𝑙 + 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 𝑖𝑜𝑛 o Tartrate-Resistant Acid Phosphatase (TRAP) to
𝑝𝐻 5 diagnose Hairy Cell Leukemia
• Substrate: ▪ Increased bilirubin → falsely low tartrate-

resistant ACP
o p-Nitrophenolphosphate
• End Products: ACP Package Insert
o p-Nitrophenol
o phosphate ion
• Almost the same with ALP
• Difference: enzyme and pH level
(2) Immunochemical Techniques
• RIA
o Nonacidified serum samples are needed for the
measurement of prostatic ACP
▪ activity is stable for 2 days at 4 degree C
• Counterimmunoelectrophoresis
• Immunoprecipitation
• Immunoenzymatic assay (Tandem E)
o Similar to the reaction of Shinowara method
o (1) Incubation with an antibody to prostatic ACP
o (2) Wash and incubate with p-NPP
▪ End product: p-Nitrophenol
▪ proportional to the prostatic ACP in the sample
Sources of Assay Error
• Serum should be separated from the red cells ASAP to

prevent leakage of RBC and platelet ACP to the serum
o To avoid falsely increased ACP level
o When RBC is separated from the serum, add 50 μL
of acetic acid/mL of serum to stabilize the serum
▪ to lower the pH to below 6.5 • Method
• Activity of the enzyme decreases within 1-2 hours if the o 1-Naphthyl phosphate is hydrolysed by acid
sample is left at RT w/o the addition of a preservative phosphatase (Ac. P.) to phosphate and naphthol,
which is converted with FRTR-salt to an azo dye.
o Decreases due to the loss of CO2 from the serum The increase of absorbance at 405 nm is
o Loss of CO2 → increase the blood pH proportional to the total acid phosphatase activity in
▪ Alkaline – not suitable for ACP activity the sample.
▪ Could interfere the ACP assay • Reaction Principle
▪ Resulted to falsely decrease
𝐴𝐶 𝑃.
o Optimal pH of ACP: acidic 1-𝑁𝑎𝑝ℎ𝑡ℎ𝑦𝑙 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐻2𝑂 → 𝑃ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 1-𝑁𝑎𝑝ℎ𝑡ℎ𝑜𝑙
o If not assayed immediately, serum:
1-𝑁𝑎𝑝ℎ𝑡ℎ𝑜𝑙 + 𝐹𝑅𝑇𝑅 𝑠𝑎𝑙𝑡 → 𝑎𝑧𝑜 𝑑𝑦𝑒
▪ Should be frozen
▪ Acidified to a pH lower than 6.5 o The substrate 1-Naphthyl phosphate is also called
as α-Naphthyl phosphate by other references
 by addition of acetic acid (50 μL/mL of serum)
o (1) The substrate will be hydrolyzed by Ac P. in the
• With acidification: presence of water and the product will be phosphate
and 1-naphthol
o ACP is stable for 2 days at room temperature
o (2) 1-naphthol will react to FRTR salt and then there
• Avoid hemolysis to avoid contamination of the RBC will be an azo dye that will be produced
ACP o Babson, Read and Philipps method
• RIA procedures for measurement of prostatic ACP ▪ According to a reference, this method is utilized
require nonacidified serum samples if the substrate is 1-Naphthyl phosphate and the
o Activity is stable for 2 days at 4C product is phosphate + 1-naphtol
Table 18. Contents of the Reagent • Sample Preparation

o Stabilize samples with acetic acid
Reagent o AUTOCAL and controls by addition of 1 drop of
acetic acid (stabilizer) to 1 ml sample immediately
[BUF] 1 × 32 ml Buffer Solution 100 after separation / reconstitution
Citrate Buffer (pH 5.2) mmol/l
[SUB] 16 × 2ml Substrate (powder) ▪ Stabilizers must be mixed in the serum sample
1-Maphtyhyl phosphate 19 µmol before proceeding to the procedure
FRTR-salt 2 µmol ▪ Should be done immediately after separating
(4-choloro-2methylphenyl diazonium serum form the RBCs
salt)
[STAB] 1 × 2ml Stabilizer o Thus, samples remain stable for 3 days at 2-8ºC
Acetic Acid 0.7 mol/l and 24 hours at 15-25°C
Additional material recommended but not supplied with the test kit • Assay Parameters
o Wavelength: Hg 405 nm
[REF] 13160
[CAL] 4x AUTOCAL o Temperature: 25°C, 30°C or 37°C
For 5ml lyophilized calibrator for HUMAN Clinical
o Measurement: against air (increasing absorbance)
Chemistry Reagents • Procedure
[REF] 13511 13512 13951 13151
o Warm working reagent and cuvettes up to the
[CONT HumaTrol HumaTrol SERODOS SERODOS desired temperature (25oC, 30oC or 37oC).
ROL] N P plus Temperature must be kept constant (±0.5oC) for the
6x for 5ml 6x for 5ml 6x for 5ml 6x for 5ml duration of the test.
Control Control Control Control
serum serum serum serum
normal abnormal normal abnormal Table 19. Procedures for Macro and Semi Micro
Lyophilized control serum for HUMAN Clinical
Chemistry Reagents Pipette into
Macro Semi Micro
cuvettes
Pipette directly into
• Reagent Preparation Pipetting the bottle containing Pipette into cuvette
o Before doing the procedure, a working reagent the working reagent
should be done first Sample 200 µL 100 µL
o Working reagent (total acid phosphatase Working reagent - 1000 µL
determination)
Mix and transfer the
▪ Before doing the working reagent, make sure solutions into cuvettes
that the reagents are being brought to the Mix, read the absorbance A1 after 5 minutes and
desired temperature start the stop-watch at the same time.
▪ (1) Dissolve the contents of the bottle SUB with Read the absorbance A2 exactly after 3 min. at
30ºC and 37ºC or after 5 min. at 25ºC.
exactly 2ml of BUF.
A2 – A1= ∆A
▪ (2) Mark label with date of preparation.
• Reagent Stability • Procedure (example)
o The reagents are stable up to the stated expiry date o (1) Get 200 µL of sample with stabilizer
when sealed and stored at 2…8ºC o (2) Mix it with the working reagent
o After reconstitution, the working reagent is stable for o (3) Read the absorbance
5 days at 2…8ºC and for 24 hours at 15…25ºC
protected from light ▪ Only after 5 minutes = That absorbance is A1
▪ After another 3 minutes = The absorbance is A2
• Specimen o (4) Get the change in the absorbance
o Strictly serum
▪ A2 – A1= ∆A
▪ No plasma ▪ The difference will be multiplied with conversion
factors
o Avoid hemolytic and icteric sera
o Do not use the whole bottle of working reagent in
▪ Hemolytic when erythrocyte burst and releases semi micro to save the volume of the working
erythrocyte ACP will falsely increase ACP level reagent
▪ Icteric sera will interfere in diagnosing hairy cell o E.g., for saving the working reagent
leukemia.
▪ Prepare another test tube
 Icteric means high bilirubin levels and it will
▪ Add 1000 µL of working reagent together with
interfere with the measurement of TRAP
100 µL sample
 TRAP is part of Total ACP level ▪ Disadvantage: addition of test tube
Calculation for Acid Phosphatase
• Calculate the total acid phosphate activity in the sample

using the following factors:
Table 20. Calculation for Acid Phosphatase
U/l 25°C 30°C / 37°C
Total acid
149 248
phosphatase (∆A) x
• Conversion factors:
o 1 U/l = 16.67 x10-3 µkat/l
• Linearity
o ∆A > 0.3 (30°C or 37°C) or ∆A > 0.5 (25ºC) or
activity >63 U/l, dilute 0.1 ml of sample (with
stabilizer) with 0.2 ml physiological saline (0.9%)
▪ Read the absorbance for 5 minutes and read it
again for another 3 minutes
o Repeat the assay, multiply the result by 3
o Typical performance date can be found in
Verification Report
Table 21. Reference Values
Total Acid Phosphatase
Assay temperature 25°C 30°C 37°C

Men up to (U/l) 3.6 5.0 6.5
Women up to (U/l) 3.0 4.2 5.5
• Quality Control
o Use control material recommended in “Additional
material recommended but not supplied with the kit”
or other suitable control material
o The control intervals and limits must be adapted to
the individual laboratory requirements.
o Values obtained should fall within established limits
o Each laboratory should establish corrective
measures to be taken if values fall outside the limits
• Automation
o This kit is designed for manual use. For use on
automates we recommend REF 10660.
[TRANS] LABORATORY UNIT 4: INTRO TO ELECTROLYTES, WATER DISTRIBUTION & OSMOLAL GAP
WATER Endogenous sources

• Universal solvent • Metabolic water
o Capable of dissolving variety of different substances o Produced during metabolism oxidation of food
▪ Good solvent = due to the chemical composition substances
and physical attributes of water o Water produced as an end product of the oxidation
of energy containing molecules such as
• Water molecule has polar arrangement of oxygen (-) carbohydrates, fats, and proteins.
and hydrogen (+) atoms
Distribution of Water
o Bent shape
▪ Oxygen has two lone
electron pairs that repel
each other, and those
electrons bonded to the
hydrogen atoms
• Can be heavily attracted to different molecules
o E.g., NaCl
▪ Water can disrupt the attractive forces that hold
the sodium and chloride in the salt molecule
together. Thus, it will dissolve in water
• Water is the chief
constituent of human body • Total body of water (TBW) - 60% of body weight (42 L)
o In some organisms, o Intracellular fluid
90% of their body
weight is water. ▪ 65% (28 L)
o In humans, 60% of the ▪ The largest
adult human body is ▪ Representing 2/3 of body water
water o Extracellular fluid
• Chief solvent of the human body ▪ 35% (14 L)
• Comprises 60-70% of total body weight ▪ Representing only 1/3 of body water
• Human body cannot exist without water ▪ Comprises:
 Plasma fluid – 7% (3 L)
Sources of Water
 Interstitial fluid – 28% (11 L)
Exogenous sources ▪ Both have similar electrolyte composition
 Most abundant ion – sodium and chloride
• Drinking water, Beverages
• Water from cooked food Percentage of Total Body Weight
o Water intake through mouth is highly variable 1-5
liters depends on:
▪ Social habits
▪ Climatic condition
 In many areas, there is an increase in water
temperature causing eutrophication
o Eutrophication - increase in phytoplankton
productivity and excess algae growth which will
reduce the drinking water quality \
ASOY. BAGAMANO. LAMOSTE. SARGADO. VILLAREAL. CORTEZ. CAGAS BSMLS 3 1

LABORATORY UNIT 04: INTRO TO ELECTROLYTES, WATER DISTRIBUTION & OSMOLAL GAP
Table 1. Total body weight water percentage • (2) Increases blood osmolality
o As the blood becomes more concentrated, the thirst
Percentage of total body weight response, a sequence of physiological processes,
is triggered
Newborn infant 80% • (3) Osmoreceptors in the hypothalamus
Adult male 60% o Sensory receptors in the thirst center in the
hypothalamus that monitor the concentration of
Adult female 50% solutes (osmolality) of the blood.
• (4) If blood osmolality is increased, the hypothalamus
transmits signals that result in a conscious awareness
• Why are adult male and female different? of thirst.
o Adult females normally have more fatty tissues than o The person should (and normally does) respond by
men drinking water.
• (5) Hypothalamus
Regulation of Water Intake
o Releases Antidiuretic Hormone (ADH) through the
posterior pituitary gland.
▪ ADH signals the kidneys to recover water from
urine, effectively diluting the blood plasma.
o Sends signals via the sympathetic nervous system
to the salivary glands in the mouth.
▪ Result in a decrease in watery, serous output
(and an increase in stickier, thicker mucus
output).
▪ Changes in secretions result in a “dry mouth”
and the sensation of thirst.
Functions of Water
• Involved in biochemical reactions

o Water act as reactant in many hydration
o Hydrolytic reactions of metabolic pathways.
▪ It is directly involved in many chemical reactions
to build and breakdown important cell
components
▪ Ex. Photosynthesis - process of plants to create
sugar and requires water
• Transporting media of body
o Transportation of nutrients and waste metabolites
through aqueous media of blood and tissue fluids
▪ Water is absorbed by the intestine and circulated
throughout the body in the form of body fluids,
which is in the blood.
• Regulated body temperature
o Blood circulation to the skin and sweating increases
heat dissipation
o It helps keep the body at constant temperature
• Transports Hormones, enzymes, blood platelets,
and red and white blood cells
• (1) Insufficient H2O in body (dehydrated) • Act as a solvent for Electrolytes and Non
o The water that leaks in the body as sweat, urine, or electrolytes
exhaled air is ultimately extracted from our blood
• Facilitates Digestion and promoting elimination of
plasma ingested food
▪ Blood is more concentrated when dehydrated • Serves as a tissue lubricant
▪ A net loss of water that results in insufficient
water in blood and other tissues. o Acts as tissue lubricant in Spinal Cord
o Acts as a cushion in Joints

Water Input and Output o Child with 12kg

▪ 𝐹𝑖𝑟𝑠𝑡 10𝑘𝑔 = 10 𝑥 100 = 1000𝐿
▪ 𝑁𝑒𝑥𝑡 2𝑘𝑔 = 2 𝑥 50 = 100𝐿
▪ 1000𝐿 + 100𝐿 = 1,100𝐿 𝑀𝐹𝑅
BODY ELECTROLYTES
• Electrolytes
o Substances when dissolved in solution dissociates
into ions. These ions are able to carry and electrical
current
o It develops an electrical charge when dissolved in
water
• Charged ions Na+ and Cl- called as electrolytes
Table 2. Water daily average intake and output • The concentration of these electrolytes is expressed in
mEq/L
Average Daily Intake of Average Daily Output of Water • Types of electrolytes
Water (a) (b) o Cation – Positively charged electrolyte
o Anion – Negatively charged electrolyte
Water of 250 mL or Water lost in 150 mL or
metabolism 10% sweat 6%
Water in 750 mL or Water lost in 150 mL or Distribution of Body Electrolytes
moist food 30% feces 6%
Water lost
• Electrolytes in body fluid compartments
Water in 1500 mL or 700 mL or
through skin
beverages 60% 28%
and lungs Table 4. Intracellular and Extracellular Electrolytes
Water lost in 1500 mL or
urine 60%
Intracellular Electrolytes Extracellular Electrolytes
Total Intake 2500 mL Total Output 2500 mL
Potassium Sodium
Magnesium Chloride
Daily Maintenance Fluid Requirement
Phosphorus Bicarbonate
Table 3. Holliday Segar Method Constant Values
Fluid
Body Weight Fluid Volume
Volume
(kg) (ml/kg/day)
(ml/kg/hr)
First 10 kg 4 100 A
Next 10-20 kg 2 50 B
20
(for elderly patients or
Each kg
1 patients with cardiac C
>20 kg
disease, this amount should
be reduced to 15 mL/kg/day)
Maintenance Fluid Requirement = A + B + C

• Blood plasma, Interstitial fluid, and Intracellular fluid has
electrolytes
• Computation Examples:
o Ex. Sodium
o 50kg
▪ Blood plasma – 142
▪ 𝐹𝑖𝑟𝑠𝑡 10𝑘𝑔 = 10 𝑥 100 = 1000𝐿 ▪ Interstitial fluid – 145
▪ 𝑁𝑒𝑥𝑡 10𝑘𝑔 = 10 𝑥 50 = 500𝐿 ▪ Intracellular fluid – 10
▪ 𝐸𝑥𝑐𝑒𝑠𝑠 𝑘𝑔 = 30 𝑥 20 = 600𝐿
▪ 1000𝐿 + 500𝐿 + 600𝐿 = 2,100𝐿 𝑀𝐹𝑅 Functions of Electrolytes
o 24kg
• Electrolytes are well distributed in the body
▪ 𝐹𝑖𝑟𝑠𝑡 10𝑘𝑔 = 10 𝑥 100 = 1000𝐿 compartments
▪ 𝑁𝑒𝑥𝑡 10𝑘𝑔 = 10 𝑥 50 = 500𝐿 • Electrolytes in the medium/compartments produce
▪ 𝐸𝑥𝑐𝑒𝑠𝑠 𝑘𝑔 = 4 𝑥 20 = 80𝐿 osmotic pressure, which helps in maintaining water
▪ 1000𝐿 + 500𝐿 + 80 = 1,580𝐿 𝑀𝐹𝑅 balance.

Significant Body Electrolytes
• Sodium (Na+):
o Most abundant electrolyte in the Extracellular Fluid
(ECF)
• Potassium (K+)
o Essential for normal membrane excitability for nerve
impulse
• Diffusion
• Chloride (Cl-)
o Solute molecules move from high to low
o Regulates osmotic pressure and assists in
concentration
regulating acid-base balance
• Osmosis
• Calcium (Ca2+)
o Solvent molecules move from low to high solute
o Promotes nerve impulse and muscle contraction/
concentration
relaxation
• Magnesium (Mg2+) Active Transport
o Plays role in carbohydrate and protein metabolism, • Movement of solutes

storage and use of intracellular energy and neural across membranes
transmission.
• Requires transporters and
o Important in the functioning of the heart, nerves, and
expenditure of energy
muscles.
Movement of Water and Electrolytes • Glucose absorption
o (1) The remaining glucose is absorbed by active
Diffusion transport by sodium ions.
o (2) Sodium ions are actively transported out of the
• The random movement of particles in all directions
small intestine epithelial cells and into the
through a solution
bloodstream by the sodium potassium pump.
• Best example of diffusion:
▪ This creates a concentration gradient as there is
o Products of digestion dissolve in water can pass
a higher concentration of sodium in the small
across the walls of small intestine by diffusion.
intestine lumen than in the epithelial cells.
▪ Their concentration is higher in the small o (3) Then, this causes sodium ion to diffuse from the
intestine than in the blood. small intestine lumen into the cell down their
▪ There is a concentration gradient from the concentration gradient via sodium potassium co-
intestine to the blood. transporter protein which brings glucose in to the
cell at the same time.
▪ This causes the glucose concentration in the cell
to increase
o (4) Glucose diffuses out of the cell and into the
blood through a protein channel.
Filtration
• Transfer of water and solutes through a membrane from

a region of high pressure to a region of low pressure
• Simple Diffusion
• Example:
o Diffusion without a helper protein
o Kidney
• Facilitated Diffusion
▪ Uses water to filter toxins out of the body
o Diffusion using a helper protein ▪ When the kidney uses as much as it needs
water then it gets rid of the rest through urine.
Osmosis
Osmolarity
• Movement of water across a membrane from a less
concentrated solution to a more concentrated solution • The number of moles per liter of solution
• Best example of Osmosis: Kidney dialysis • How does the body maintain osmolarity?
o The dialyzer removes the waste products in the o It is achieved by balancing the intake and excretion
patient’s blood through a dialyzing membrane. of sodium with that of water
o Dialyzing membrane – acts as the semi-permeable o Sodium is the major solute in the extracellular fluid,
membrane and it passes into a dialysis solution. so it determines the osmolarity of extracellular fluids.

Osmolality Table 5. Reference Ranges for Osmolality
• The number of moles per Kg

Range
of Solvent
• Concentration of a solution
determined by the number of Serum 275 – 295 mOsm/kg
dissolved particles per Urine (24 h) 300 – 900 mOsm/kg
kilogram of water
• Controls water movement and Urine/serum ratio 1.0 – 3.0
distribution in body fluid
compartments Random urine 50 – 1,200 mOsm/kg
Osmolal gap 5 – 10 mOsm/kg

Osmolality Clinical Significance
• Normal plasma osmolality

Calculation of Osmolal Gap
o 275 – 295 mOsm/kg of plasma H2O
o Osmoreceptors respond to small changes regulated 𝑶𝒔𝒎𝒐𝒍𝒂𝒓 𝒈𝒂𝒑 (𝑶𝒚 )
by AVP and thirst = 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑜𝑠𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 (𝑂𝑚 ) – 𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 𝑜𝑠𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 (𝑂𝑐 )
▪ AVP – Arginine vasopressin hormone (anti-
diuretic hormone)
𝑪𝒂𝒍𝒄𝒖𝒍𝒂𝒕𝒆𝒅 𝒐𝒔𝒎𝒐𝒍𝒂𝒍𝒊𝒕𝒚 (𝑶𝒄 )
Determination of Osmolality 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 [𝑚𝑔 𝑝𝑒𝑟 𝑑𝐿] 𝐵𝑈𝑁 [𝑚𝑔 𝑝𝑒𝑟 𝑑𝐿]
= 2 [𝑁𝑎+ (𝑚𝐸𝑞 𝑝𝑒𝑟 𝐿)] + ( )+( )
18 2.8
• Urine or serum
o Sodium, chloride, and bicarbonate • Illustrative case:
▪ Major electrolyte concentration o Om = 314 (Table 1)

103 𝑚𝑔 𝑝𝑒𝑟 𝑑𝐿 8 𝑚𝑔 𝑝𝑒𝑟 𝑑𝐿
o Oc = 2(141 𝑚𝐸𝑞 𝑝𝑒𝑟 𝐿) + ( )+( )
• Plasma is NOT recommended 18 2.8
o Oc = 282 + 5.7 + 2.9 = 290.6
o Osmotically active substances may be introduced in o Oy = 314 – 290.6 = 23.4 mOsm per Kg of water
the specimen from the anticoagulant
• Na+ = serum sodium level; BUN = blood urea nitrogen
• Based on colligative properties of solution
o Change in freezing point and vapor pressure
• Turbid samples must be centrifuged to remove
extraneous particles.
Calculation of Osmolality
• Either estimate of true osmolality or to determine

osmolal gap
• Osmolal gap
o Difference between measured osmolality and
calculated osmolality
▪ Measured osmolality from the osmometer
o Indirectly indicates presence of osmotically active
substances other than Na+, Urea, or Glucose
▪ Ethanol, methanol, ethylene glycol, lactate or β-
hydroxybutyrate
• Formulas for calculating osmolality:
𝒈𝒍𝒖𝒄𝒐𝒔𝒆 (𝐦𝐠⁄𝒅𝑳) 𝑩𝑼𝑵 (𝐦𝐠⁄𝒅𝑳)

𝟐 𝑵𝒂 + +
𝟐𝟎 𝟑
𝒈𝒍𝒖𝒄𝒐𝒔𝒆 𝑩𝑼𝑵
𝟏. 𝟖𝟔 𝑵𝒂 + + +𝟗
𝟏𝟖 𝟐. 𝟖

[TRANS] LABORATORY UNIT 5: SODIUM (LABORATORY METHODS AND PROCESSING)
SODIUM ION DETERMINATION Ion Selective Electrode (ISE/Glass Ion Exchange

membrane)
Specimens Used for Assays
• Serum
o Reference range: 136-145 mmol/L
• Urine (24-hour)
o Reference range: 40-220 mmol/day (varies with diet)
• CSF
o Reference range: 136-150 mmol/L
• Plasma
o Anticoagulant used:
▪ Lithium heparin • Reference electrode
▪ Ammonium heparin
o Gives a constant potential
▪ Lithium oxalate
• Whole blood • Difference between the potential of the reference and
the measuring electrode:
o depends on the analyzer available
o Reflective of the activity of the ion (similar with
• Sweat enzymes)
▪ The greater the activity exhibited, the higher the
Processing Samples for Testing concentration of the ion in the sample
• Na+ glass
• Theoretically, minimal hemolysis is acceptable in serum
and plasma o Membrane usually seen in ISE for sodium
o Change is not significant since RBCs contain only • Valinomycin
1/10 of the sodium in the plasma
o Membrane usually seen in ISE for potassium
o However, in reality, hemolyzed sample is rejected
Types of ISE
• Pseudohyponatremia (marked hemolysis)
o Could lead to dilutional effect of hemoglobin • Direct
o The sample introduced to the ISE is undiluted
Main Methods in Measuring Sodium Ions
• Indirect
• Chemical Method o Utilizes a diluted sample
o Outdated due to large sample requirements and lack ISE System for the Potentiometric Slide on the Vitros
of precision
• Flame Emission Spectrophotometry (FES)
o Dilute the serum into 1:200
o Solution should be subjected to the flame (orange
yellow)
• Atomic Absorption Spectrophotometry (AAS)
• Ion Selective Electrode (ISE/Glass Ion Exchange

membrane)
o Commonly used method
• Dry slide technology-based analyzer
ANTOYAN. GABAESIN. IBONES. LATONIO. NARAGA. NAVARRO. CAGAS BSMLS 3 1

LABORATORY UNIT 5: SODIUM (LABORATORY METHODS AND PROCESSING)
Chemical Method Table 1. Contents of the Reagent
• Albanese Lein
• Magnesium-Uranyl Method
(1) Albanese Lein Method [PREC] 60 mL Precipitating Solution
Uranyl acetate 19 mmol/L
• Principle:
Magnesium acetate 140 mmol/L
o Sodium is precipitated as sodium uranyl zinc acetate
which is then dissolved in water and determined [RGT] 60 mL Colour Reagent
photometrically by its yellow color. Ammonium thioglycolate 550 mmol/L
• Precipitant: sodium uranyl acetate Ammonia 550 mmol/L
(2) Magnesium-Uranyl Method [STD] 2 mL Standard
Sodium (Na) 150 mmol/L
• Used in package insert
*For 20 macro- / 60 semi-micro determinants
• Principle:
o Sodium is precipitated with magnesium-uranyl • Stability
acetate (protein precipitant)
o Stored unopened at room temperature (15-25ºC)
▪ The uranyl ions remaining in suspension form a and in the dark, the contents are usable until the
yellow-brown complex with thioglycolic expiry date printed on the label.
o The difference between reagent blank and analysis • Specimen
is proportional to the sodium concentration
o Serum
• Protein precipitant:
• Assay
o Uranyl acetate, magnesium acetate
o Wavelength: Hg 365 nm, Hg 405 nm, 410 nm
Package Insert o Optical path: 1 cm
o Temperature: 20-25ºC (Room Temperature)
o Measurement: against reagent blank. Only one
reagent blank per series is required.
Table 2. Procedure (Part 1)
Reagent
Macro Semi-micro
Solution
RB STD Sample RB STD Sample
[μL] [μL] [μL] [μL] [μL] [μL]
[STD] --- 50 --- --- 20 ---
Serum --- --- 50 --- --- 20
[PREC] --- 3000 3000 --- 1000 1000
• Procedure: Part 1
o (1) Prepare 3 tubes:
▪ Reagent blank
▪ Standard
▪ Sample
o (2) For the reagent blank, you will do nothing in the
first part of the procedure.
▪ STD and sample are only utilized in the first part
• Method o (3) For the Standard test tube, put 1000 μL
o Sodium is precipitated with magnesium-uranyl precipitating agent
acetate (protein precipitant); the uranyl ions ▪ Larger volumes are put first
remaining in suspension form a yellow-brown ▪ (4) To be followed by the addition of 20 μL
complex with thioglycolic Standard (STD)
o The difference between reagent blank and analysis
is proportional to the sodium concentration o (5) For the Sample tube, mix 20 μL sample instead
of 1000 μL Standard (STD) precipitating agent.

LABORATORY UNIT 5: SODIUM (LABORATORY METHODS AND PROCESSING)
o (6) Close the tube and mix well. o Calculate the result and multiply by 2.
o Multiplying the result by 2 is ONLY APPLICABLE if
▪ When mixing the tube, make sure the thumb is the dilution is 1:2 (1-part sample and 1-part diluent)
not used in covering the tubes because that
could cause a falsely increase in result ▪ Example: 100 uL sample diluted to 100 uL
 Sodium from the sweat of the fingers will be diluent)
the interference ▪ If the dilution is 1:4, then the result of the
machine will be multiplied to 4 before it will be
 Gloves is also discouraged encoded in the result form.
▪ Just swirl it clockwise and counter-clockwise o The 2 in the package insert is NOT CONSTANT.
o (7) Allow to stand for 5 minutes. ▪ The dilution factor presented under section
o (8) Shake intensively for at least 30 seconds. “linearity” is NOT constant.
o (9) Allow to stand for 30 minutes. ▪ Multiply to the diluted result the DILUTION
o (10) Centrifuge at high speed for 5-10 minutes. FACTOR used by the medical technologist.
 Case-to-case basis.
Table 3. Procedure (Part 2)
• Normal Range
Reagent
Macro Semi-micro
o Serum: 135-155 mmol/L
Solution
• Quality Control
RB STD Sample RB STD Sample
[μL] [μL] [μL] [μL] [μL] [μL] o All control sera with sodium values determined by
this method can be employed.
[PREC] 50 --- --- 20 ---- ----
o It is recommended to use HumaTrol quality control
Clear sera based on animal serum or SERODOS based
--- 50 50 --- 20 20
supernatant on human serum.
[RGT] 3000 3000 3000 1000 1000 1000
• Notes:
• Procedure Part 2 o Use the semi-micro procedure only if a very efficient
o (1) Get the supernatant centrifuge (8,000-10,000 RPM) is available.
o (2) For Reagent Blank (RB), you only need is the ▪ Otherwise, use the Macro procedure to obtain
precipitant the reliable results.
o (3) For reagent blank, standard, and sample,
prepare another set of three test tubes o [PREC] becomes discoloured when exposed to light.
Store protected from light.
▪ Each tube should contain 1000 uL of reagent.
▪ A slight turbidity does NOT affect the
o (4) Add 20 uL of clear supernatant in the test tubes determination.
labelled as sample and standard.
o (5) Add 20 uL of [PREC] in the test tube for reagent o Detergents usually contain high sodium
blank. concentrations. The equipment – test tubes pipettes,
o (6) Mix well. stoppers, cuvettes – must therefore be rinsed
o (7) After 5-30 minutes, measure absorbance of RB carefully with distilled water.
(𝚫𝑨𝑹𝑩 ), the standard (𝚫𝑨𝑺𝑻𝑫 ) and the sample
▪ Avoid contamination by traces of sodium
(𝚫𝑨𝒔𝒂𝒎𝒑𝒍𝒆 ) against distilled water at 360-410 nm (Hg
(sweat).
366 or Hg 405).
o Disposable plastic tubes are recommended for the
Calculation determination. Use Parafilm or plastic stoppers to
close the tubes.
(𝚫𝑨𝑹𝑩 − 𝚫𝑨𝒔𝒂𝒎𝒑𝒍𝒆 ) 𝒎𝒎𝒐𝒍 o [PREC] 𝑋𝑛 ; F; R: 11-20/22-33; S: 1/2-7-16-20/21-45
𝑪 = 𝟏𝟓𝟎 𝒙 [ ] o [RGT] T; R: 25-36/37/38-43; S: ½-9-16-25-26-27-28-
(𝚫𝑨𝑹𝑩 − 𝚫𝑨[𝑺𝑻𝑫] ) 𝑳
36/37/39-45-61
• Mval/L = mmol/L
o Mval – millivalence
▪ The same as milliEquivalents/liter
▪ Sodium: 1 mval/L = 1 mmol/L
• Linearity
o With sodium concentrations exceeding 300 mmol/L
(or when the result in the analyzer cannot be read),
the serum must be prediluted 1+1 with distilled
water.
o Then, run it again using the previous procedure.

[TRANS] LABORATORY UNIT 06: POTASSIUM AND MAGNESIUM (LABORATORY)
POTASSIUM • (2) NHE (Sodium hydrogen exchanger)

• Major intracellular cation o Antiport
o 20x greater concentration inside the cells than ▪ For every molecule of sodium, the channels
outside exchanges in molecule of hydrogen
o “Housed” within the cell
o Stimulated by high concentration or high
• Many cellular functions require that the body maintain a extracellular pH, catecholamines, or insulin
low extracellular fluid concentration of K⁺ ions
• (3) Barium inhibits the K⁺ channel
o As a result, only 2% of the body’s total K⁺ circulates
in the plasma o One activator - increase intracellular calcium
o E.g., regulation of neuromuscular excitability and
Exchange of Intracellular Ions
heart contraction
▪ Example: 10 potassium total in the body
▪ To get the 2%, only 0.2% of extracellular fluid
(extracellular component)
• Concentrations of cations and anions in mEq/L
o Intracellular ions
▪ Potassium
▪ Magnesium
▪ Phosphorus
▪ Protein • Na+ – K+ ATPase/pump
o Extracellular ions o Uses ATP
▪ Potassium (2%) o Since it uses ATP, it is classified under active
▪ Calcium transport.
▪ Chloride
• K+–Na+ “leak” channels
▪ Bicarbonate
o No ATP is required
Regulation of Potassium in a Cellular Level o Since no ATP is required, it is classified under
passive transport.
• 3 Na+ OUT = 2 K+ IN
o For every 3 molecules of sodium that go out of the
cell, 2 molecules of potassium enter.
Resting Membrane Potential (RMP)
• Potassium has a major effect on the contraction of

skeletal and cardiac muscle.
• (1) Potassium enters the cell through Na⁺-K⁺-ATPase or

through NKCC
o Na⁺-K⁺-ATPase
▪ Stimulated by insulin or beta-adrenergic agents
such as catecholamine, adrenaline/epinephrine
o NKCC (Na-K-Cl cotransporter)
▪ Stimulated by insulin and catecholamines
ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO. LAMOSTE. MAHINAY. PAÑA. SARGADO. VILLAREAL. AQUINO. BSMLS 3 1
CATAPANG. CAGAS
LABORATORY UNIT 06: POTASSIUM AND MAGNESIUM (LABORATORY)
• RMP in Resting State: -90mV • Insulin

o For skeletal muscle. o Promotes K⁺ entry to the cell
o Potassium is inside.
▪ Specifically, to Skeletal muscle, Liver
• RMP in Activated State: -90 to +35mV
o Insulin is very important in cases of Diabetes
o Due to the entry of sodium in the cell. mellitus.
• RPM in Inactivated State: +35 to -90mV ▪ This enzyme is deficient in those patients.
▪ If this is deficit, there is no insulin that can
o Due to the 3 sodium that have been pumped out in promote K+ entry to the cell which can result to
exchange for the 2 potassium that have been hyperkalemia.
pumped in.
• Catecholamines (epinephrine)
▪ This creates a negative potential because of the
deposit of positive ions. o Promotes K⁺ entry to the cell
o In extreme stress, this enzyme is elevated.
o For every 3 sodium molecules that go out, only 2
potassium go in. Functions
o The ratio of molecules that goes out and in is not
equal. • Regulation of neuromuscular excitability
• Contraction of the heart
Regulation of Potassium • ICF volume
• (1) Initially PCT reabsorb nearly all the potassium • Hydrogen ion concentration
• (2) Secreted into the urine in exchange for sodium in Regulation of neuromuscular excitability
both the DCT and the collecting ducts
• (3) Under the influence of aldosterone • Hyperkalemia = lack of muscle excitability
o K⁺ is concentrated outside the cell
▪ More K⁺ goes out that causes muscle weakness
by causing inactivation of the Na channels via
Chronic depolarization
▪ Membrane is always activated without rest.
 Thus, the concentration of the ions is not
balanced producing lack of muscle excitability.
• Hypokalemia = arrhythmia or paralysis
o Arrhythmia
▪ Absence of normal rhythm of the heart
o Paralysis
▪ In K⁺ medium channels, if no K⁺ goes inside the
cell, the intracellular environment becomes more
negative
 Lower than -90mV → normal RMP (resting
membrane potential) of the muscle
•  Blood K⁺ level rises and  Blood Na⁺ level falls
▪ If more negative, it is not easy to activate to drive
o The renal gland specifically the glomerulosa layer the cell into a more positive state which is
secretes aldosterone. +35mV for the muscle to be activated
o It tells the kidney to secrete potassium into the urine
in exchange for the conservation of sodium. Hydrogen ion concentration
▪ This happens in the DCT and collecting duct.
• Alkalosis: Hypokalemia
o These segments of the nephron answers to
o Low serum/ plasma K⁺
aldosterone whenever the hormone is secreted by
o As K⁺ is lost from the body, Na⁺ and H⁻ ions move
the adrenal gland.
into the cell to replace the positive ions that is lost →
leads to Alkalosis
Factors that Influence the Distribution of Potassium
▪ H⁻ ions that acidify the blood goes inside the cell
• Hypoxia, hypomagnesemia, digoxin overdose = K⁺
loss • Acidosis: Hyperkalemia
o Digoxin has narrow therapeutic index. o Excess H⁻ ions move inside the cell
▪ The slight changes in doses can lead to ▪ To maintain electroneutrality, K⁺ leaves the cell
overdose which can cause potassium loss causing Hyperkalemia
resulting to hypokalemia.
CATAPANG. CAGAS
Exercise Diuretics—thiazides, mineralocorticoids
• K⁺ is released form cells during exercise Nephritis
o Increases K⁺ by 0.3 – 1.2 mmol/L Renal tubular acidosis

Renal Loss Hyperaldosteronism
• Reversed after several minutes of rest
o Important to ask if the patient is well-rested before Cushing’s syndrome
collecting the specimen Hypomagnesemia
• Forearm exercise during venipuncture Acute leukemia
o can cause erroneous high plasma K⁺ concentrations Alkalosis
Cellular Shift
• Prolonged tourniquet Insulin overdose
o has a squeezing-like action to the muscle tissues Decreased Intake
that can erroneously elevate K⁺
Cellular Breakdown
Table 2. Causes of Hypokalemia (from Henry’s)
• Conditions that release K⁺ into the ECF:
o Severe trauma Mechanism Presentations
▪ Cells have 20x more potassium concentration
Alkalosis
inside the cells compared to ECF.
▪ The slightest trauma could affect the potassium Hypokalemic periodic paralysis
concentration of the patient.
Beta-2-adrenergic agonists
o Tumor lysis syndrome
Barium poisoning
▪ Due to the drugs used for cancer management Intracellular Shift Chloroquine and hydroxychloroquine
▪ The cancer cells being destroyed also have high poisoning
intracellular potassium levels released into the Insulin
ECF.
Administration of carbohydrates
o Massive blood transfusions
Nutritional recovery state
▪ Hemolysis of RBCs
▪ Prolonged storage of blood component Poor Intake
Vomiting
Clinical Significance Diarrhea
Gastrointestinal
Loss Intestinal drainage
Hypokalemia
Laxative abuse
• Three main mechanisms that causes hypokalemia
o (1) Intracellular shift Primary aldosteronism
o (2) Reduce intake • adrenal adenoma or hyperplasia);
o (3) Increased loss: Gastrointestinal tract and Renal PRA is suppressed
loss
Secondary aldosteronism
• Needs to request for magnesium levels and correct if • increase in aldosterone is secondary
there are any deficiencies of K⁺ to increase in renin
o Malignant hypertension
Excessive Renal
Table 1. Causes of Hypokalemia (from Bishop’s) o Renal artery stenosis
Loss
o Reninoma
o Diuretics
Mechanism Presentations o Bartter’s syndrome
o Gitelman’s syndrome
Vomiting
Excess mineralocorticoids other than
Diarrhea aldosterone
Gastric suction • e.g. Cushing’s syndrome, ACTH-
Gastrointestinal producing tumor, Iicorice, 11-
Intestinal tumor
Loss deoxycorticosterone
Malabsorption
Cancer therapy—chemotherapy, radiation
therapy
Large doses of laxatives
CATAPANG. CAGAS
(1) Intracellular Shift
• Alkalosis, insulin, and beta-2-adrenergic agonists

o Causes hypokalemia by stimulating the Na⁺-K⁺-
ATPase channel
o Alkalosis
▪ Leads to concomitant hypokalemia
 Due to the movement of H⁻ ions in the cell
 To replace the loss of positive ion (K⁺)
o Beta-2-adrenergic agonists (catecholamines)
▪ E.g., epinephrine, norepinephrine • Diuretics promoting potassium excretion
• Barium poisoning o Carbonic anhydrase inhibitors (CAI)
o Barium – used in upper gastrointestinal (UGI)
▪ Acts on the proximal convoluted tubule (PCT)
studies
o Loop Diuretics (most common)
• Chloroquine and hydroxychloroquine poisoning
▪ High ceiling – induces diuresis more effectively
o Anti-malarial agents than other diuretics
o Inhibits the potassium channel ▪ Acts on the thick ascending limb of Henle
o Thiazides (most common)
(2) Poor Intake
▪ Acts on the distal convoluted tubule (DCT)
• Rare cause of hypokalemia • Potassium (K⁺) sparing diuretics
o Spares the potassium in expense to excreting
(3) Gastrointestinal Loss magnesium
o Specifically used for patients with existing
• Vomiting hypokalemia (to prevent K⁺ loss)
o In patients with acute gastroenteritis (AGE), there is (5) Hypomagnesemia
a urine loss.
o It is a state of metabolic alkalosis • Promote urinary loss of K⁺
• Diarrhea • Diminish activity Na⁺-K⁺-ATPase
• Enhances secretion of aldosterone
o In patients with acute gastroenteritis (AGE), diarrhea
causes a direct loss in the stool. o Aldosterone is linked to conversion of sodium in
expense to excretion of K⁺
o Inc. aldosterone = Inc. Na⁺ = Excrete K⁺
(4) Excessive Renal Loss
Symptoms of Hypokalemia
• Most common cause of hypokalemia
• Asymptomatic
o Primarily due to increased aldosterone
o Aldosterone causes potassium excretion to retain o Value: 3.0 – 3.4 mmol/L
sodium
• Common complains
• Diuretics: Secondary aldosteronism o Weakness
o Promote urinary excretion of fluids or diuresis o Fatigue
o In general, it is used to lower intravascular volume in o Constipation
cases of: o Paralysis of respiratory muscles
o Arrhythmia
▪ Hypertension
▪ Pulmonary edema • Plasma K⁺ decreases below 3 mmol/L
o “Where water goes, salt follows”
Treatment of Hypokalemia
▪ Inducing diuresis to patient depletes the
intravascular volume.
▪ Electrolytes will also be excreted in urine. • Oral / IV K⁺ replacement
o IV Method - to correct the K⁺ in patient that are
about to go in the operating room
• Diet rich in K⁺
o E.g., bananas
CATAPANG. CAGAS
Hyperkalemia Due to excessive

Rare id renal excretion of K is normal
ingestion
• Three main mechanisms that causes hyperkalemia Hypoaldosteronism: Addison’s disease;
selective hypoaldosteronism
o (1) Extracellular shift (hyporeninemic hypoaldosteronism,
o (2) Increase intake heparin, congenital adrenal
o (3) Reduce renal potassium excretion enzyme deficiencies, angiotensin-
converting enzyme inhibitors)
Tubular unresponsiveness to
aldosterone (pseudohypoaldosteronism
Table 3. Causes of Hyperkalemia (from Bishop’s) types I and II): congenital and acquired
Decreased renal
salt-losing nephropathy
excretion
Potassium-sparing diuretics:
Mechanism Presentations
spironolactone, amiloride, triamterene
Acute or chronic renal failure (GFR <20 Antibiotics with ENaC-blocking effects:
mL/min) pentamidine, trimethoprim
Decreased Renal Hypoaldosteronism Antirejection medications: cyclosporine,
Excretion tacrolimus
Addison’s disease
Severe dehydration
Diuretics
Acidosis
• Pseudohyperkalemia
Muscle/cellular injury
o Falsely elevated K+
Cellular Shift Chemotherapy o Found in patients with malignancies of the blood
Leukemia
▪ E.g., leukemia, infection (increased WBC count),
Hemolysis prolonged tourniquet with fist exercise, in vitro
hemolysis
Oral or intravenous potassium
Increased Intake
replacement therapy • True Hyperkalemia
Sample Hemolysis o Extracellular shift
Artifactual Thrombocytosis ▪ K+ go out of the cell

▪ Acute acidosis
Prolonged tourniquet use or excessive
 K+ goes out of the cell to neutralize the
fist clenching
extracellular environment
▪ Digitalis
Table 4. Causes of Hyperkalemia (from Henry’s)  Inhibits Na⁺-K⁺-ATPase activity in excessive
amounts
Mechanism Presentations o Excessive ingestion

▪ Rare, only occurs when renal excretion is not
Thrombosis normal
Severe leucocytosis ▪ K+ accumulates, if the kidney is not able to
Pseudohyperkalemia regulate the concentration or excrete the K in the
Use of tourniquet with fist exercise urine
In vitro hemolysis o Decreased Renal Excretion
True Hyperkalemia ▪ Hypoaldosteronism
Acute Acidosis ▪ Tubular unresponsiveness to aldosterone
(More with inorganic acidosis) (pseudohypoaldosteronism types I and II)
Catabolic states  if there is no aldosterone, sodium is not
Periodic paralysis conserved.
Succinylcholine ▪ Antirejection medications: cyclosporine,
(Especially in those with muscular tacrolimus
Due to extracellular dystrophy)
shift Cationic amino acids (epsilon amino  In patients with tissue grafts
caproic acid) ▪ Severe dehydration
Vigorous exercise  The intravascular volume is depleted.
Digitalis intoxication, digitalis-like
chemicals  The intravascular volume which is primarily
(Herb chansu, bufotoxins) water gets depleted. Then, the electrolytes
are elevated regardless of the plasma
Fluoride poisoning
concentration.
CATAPANG. CAGAS
3 Main Mechanisms of Diminished Renal K+ Excretion Symptoms of Hyperkalemia
• Reduce aldosterone or aldosterone responsiveness • Muscle weakness

o In hypoaldosteronism, there is no hormone that • Tingling
would signal the DCT and the collecting tubules. • Numbness
• Mental confusions
▪ If the sodium is not conserved, the potassium will
be elevated. o Alter muscular conduction
o Hyporeninemic and hypoaldosteronism • Cardiac arrhythmias and cardiac arrest
▪ Most common cause of all aldosterone Potassium value and expected findings:
deficiencies states according to Henry’s.
▪ Common cause of chronic hyperkalemia among • Muscle weakness: 8 mmol/L
non-dialysis patients. • ECG (electrocardiogram) changes: 6-7 mmol/L
• Renal failure o Potassium is important in cardiac muscle conduction
• Reduce distal delivery of sodium
• Cardiac arrest: > 10 mmol/L
Drugs that Cause Hyperkalemia o Monitor and careful in high potassium level
• Captopril
Treatment of Hyperkalemia
o Inhibits ACE
o Protype of the angiotensin converting enzyme • Calcium
inhibitor
o Used to treat hypertensive patients o If the calcium levels reach to 6 – 6.5 mmol/L or there
o It has a coughing adverse reaction is ECG findings already
▪ Today, it is not allowed to be used due to the ▪ Indicates that the potassium is high, administer
pandemic. calcium to reduce the threshold potential of the
▪ If this agent is given as a side effect, it will trigger myocardial cells to avoid cardiac arrhythmias or
coughing to the patient and might be suspected cardiac arrest.
with Covid-19 infection. • Sodium bicarbonate
• Nonsteroidal anti-inflammatory agents: • Glucose
• Insulin
o Inhibit aldosterone, will not conserve sodium
o Potassium concentration in the blood will increase o To ship the potassium intracellularly
leading to hyperkalemia
• Diuretics
• Spironolactone o To secrete the potassium in the urine
o K+-sparing diuretic
o Spare potassium, increases in the extracellular fluid
Collection of Specimens & Specimen handling
• Digoxin
• Collection: Avoid prolonged application of tourniquet
o Digitalis and avoid forearm exercise or clenching of fist
o Inhibits Na+/K+ pump
o Used to treat congestive heart failure o Can lead to erroneously high potassium levels
• Cyclosporine • Sample: Heparinised plasma
o Inhibits renal response to aldosterone o To avoid Pseudohyperkalemia because clotting

o Immunosuppressant medication increases the potassium concentration
o Used to prevent organ rejection with people who o The coagulation process from the platelets may
received heterologous tissues like liver, kidney, elevate the potassium at 0.1 to 0.5 mmol/L higher
heart. than plasma.
o Not a direct inhibitor of the aldosterone itself • Storage: Room Temperature
• Heparin therapy • Specimen handling: Hemolysis should be avoided
o Inhibits aldosterone secretion
o Heparin is used as an anticoagulant Colorimetric Methods
▪ Decrease the clotting of the blood and helpful to Lockhead and Purcell Method
prevent harmful clots from forming in the blood
vessels like patients with stroke or acute MI • Potassium is reacted with sodium cobaltinitrite to
produce sodium potassium cobaltinitrite
o The pathophysiology of stroke or cerebrovascular • With the addition of phenol (color developer), a blue
accident as well as myocardial infarction is clot color is produced and determined using
formation and occlusion of the important vessels in spectrophotometer
the heart and brain.
CATAPANG. CAGAS
ISE (Ion Selective Electrode) • Method

o Potassium ions in a protein-free alkaline medium
• Current method of choice react with sodium tetraphenylboron to produce a
• Uses valinomycin membrane and KCl as inner finely dispersed turbid suspension of potassium
electrolyte solution tetraphenylboron.
o Valinomycin is used because it selectively binds the
potassium
Table 6. Contents of the Reagent
• The change in impedance correlates with the potassium
concentration
Precipitant (White cap)

Table 5. Reference Ranges for Potassium 50 ml 0.3 mol/l
Trichloroacetic acid (TCA)
TPB-Na-Reagent (black cap)
50 ml 0.2 mol/l
Sodium tetraphenylboron
Potassium
NaOH Reagent (red cap)
50 ml 2.0 mol/l
Serum 3.5 – 5.1 mmol/L Sodium hydroxide
Standard
Males: 3.5 – 4.5 mmol/L 5 ml
Potassium
5.0 mmol/l
Plasma
Females: 3.4 – 4.4 mmol/L
Urine (24 h) 25 -125 mmol/L

• Reagent Preparation
• Below 3.5 mmol/L is hypokalemia o Mix the content of bottle TPB with the content of
• Above 5.1 mmol/L is hyperkalemia bottle NAOH
▪ For smaller amounts of working reagent mix TPB
• The serum has higher upper value than the plasma due and NAOH in a ratio of 1 + 1.
to coagulation process that releases the potassium.
• The normal value is in SI unit o Allow to stand for 15 -30 minutes prior to use
o To convert it to conventional unit, multiply it by 1 ▪ PREC and STD are ready for use
mEq/L. ▪ STD is used undiluted directly in the
determination
Package Insert for Potassium
• Reagent Stability
o The reagents are stable up to the given expiry date
when stored at 2-25°C.
o The working reagent is stable for 30 days at 15-
25°C and 60 days at 2-8°C.
• Specimen
o Serum
o Lithium heparin plasma
o Wavelength: 578 nm, Hg 578 nm
o Temperature: 20-25°C
o Measurement: Against reagent blank
o Only one reagent blank per series is required.
Table 7. Procedure
Precipitation
Pipette into centrifuge tubes:

MACRO SEMI-MICRO
Specimen 100 μL 50 μL
PREC 1000 μL 500 μL
Mix carefully, centrifuge at high speed for 5-10 min
CATAPANG. CAGAS
Determination • Notes
Pipette into cuvettes: o Use non-hemolytic serum or heparin plasma as
specimen.
STD Sample STD Sample
o As red blood cells contain about 25 times the
Working amount of potassium, they have to be separated
2000 μL 2000 μL 1000 μL 1000 μL
reagent from the serum within one hour after blood
STD 200 μL - 100 μL -
collection. Otherwise, falsely elevate potassium
concentrations will be found.
Supernatant - 200 μL - 100 μL o Traces of detergents produce turbidity which leads
To produce a homogeneous turbidity, the STD or the clear to falsely elevated potassium concentrations. They
supernatant have to be added to the center of the surface of the therefore have to be avoided.
working reagent in the cuvette. Mix each cuvette carefully before o Contaminated glassware is the greatest source of
proceeding to the next sample. Allow to stand at least for 5 minutes. error. Glassware should therefore be thoroughly
rinsed with deionised water. Disposable plastic ware
Measure absorbance of the standard ( ∆𝜜 STD) and the sample
(∆𝜜sample) against working reagent blank between 5 and 30 minutes. may contain softeners which react with the reagent
and is therefore nor recommended.
Supernatant --- 200 μL --- 100 μL
MAGNESIUM
• Fourth most abundant cation in the body and second
• Procedure most abundant intracellular ion
o (1) Precipitation o 53% - bone
o (2) Mix carefully, centrifuge at high speed for 5 – 10 o 46% - muscle and other organs and soft tissue
minutes. o <1% - serum and erythrocytes
o (3) Determination
▪ Protein-bound (primarily albumin) – 33%
• To produce a homogeneous turbidity, the STD or the ▪ Free or ionized form – 61%
clear supernatant have to be added to the center of the ▪ In complexes with other ions – 5%
surface of the working reagent in the cuvette. Mix each
cuvette carefully before proceeding to the next sample. o Similar to other electrolytes, the free form of this ion
Allow to stand at least for 5 minutes. is physiologically active ion in the body
• Measure absorbance of the standard (∆𝛢STD) and the • Magnesium is a very essential cofactor of more than
sample (∆𝛢sample) against working reagent blank 300 enzymes used our body
between 5 and 30 minutes.
o Specifically, ATP production and anabolic processes
Formula
∆𝐴𝑠𝑎𝑚𝑝𝑙𝑒
𝐶 = 5𝑥 [𝑚𝑚𝑜𝑙 ⁄𝐿] 𝑜𝑟 [𝑚𝑒𝑞 ⁄𝐿]
∆𝐴𝑆𝑇𝐷
• Linearity
o The reaction is linear up to potassium levels of 10
mmol/l. Samples with higher concentrations have to
be diluted 1 + 1 with physiological saline (0.9%).
• This figure presents the distribution of Mg2+ and other
Multiply the result by 2
molecules in the plasma, Intracellular fluid (ICF),
▪ High potassium levels beyond 10 mmol/l cannot Interstitial fluid (ITF)
be read by the machine o Mg2+ is an intracellular ion
▪ The only way to read the concentration is to o The concentration is less in the blood plasma and
dilute using the concentration of potassium in the interstitial fluid (Extracellular fluid)
sample, then multiply the value of the diluted
sample by 2. Function of Magnesium
• Normal Values • Widespread
o Serum: 3.6 - 5.5 mmol/l o Essential cofactor of >300 enzymes
o Plasma: 4.0 – 4.8 mmol/l
▪ Glycolysis
• Quality Control ▪ Transcellular ion transport
o All sera with potassium values determined by this ▪ Neuromuscular transmission
method can be used. We recommend to use our ▪ Carbohydrate, protein, lipid, and nucleic acid
HumanTrol quality control sera based on animal synthesis
serum or our SERODOS based on human serum. ▪ Release and response to certain hormones
CATAPANG. CAGAS
• Richest source Table 8. Causes of Hypomagnesemia

o Raw nuts, dry cereal, and “hard” drinking water
o Vegetables, meat, fish, and fruit Causes of Hypomagnesemia
• Small intestine may absorb 20-65% of the dietary
Poor diet/starvation
magnesium Prolonged magnesium-deficient
Reduced Intake
intravenous therapy
Regulation of Magnesium Chronic alcoholism
Malabsorption syndrome
• Controlled largely by the kidney Surgical resection of small
intestine
o Nonprotein bound are filtered by the glomerulus Nasogastric suction
Pancreatitis
▪ Cannot be filtered if protein bound, because Decreased Absorption Vomiting
albumin is a large substance Diarrhea
▪ Proteins cannot be filtered in complex with an Laxative abuse
ion Neonatal
▪ 25-30% - reabsorbed by the PCT Primary
▪ 50-60% - reabsorbed in Ascending loop of Congenital
Henle Tubular disorder
Increased Excretion-Renal Glomerulonephritis
 Major renal regulatory site Pyelonephritis
Hyperparathyroidism
▪ 2-5% - reabsorbed in DCT
Hyperaldosteronism
Increased Excretion-
o Renal threshold: 0.60 – 0.85 mmol/L Endocrine
Hyperthyroidism
Hypercalcemia
▪ If Mg exceeds, it is filtered and excreted in the Diabetic ketoacidosis
urine Diuretics
Increased Excretion-Drug Antibiotics
o Only about 6% of filtered Mg is excreted in the urine Induced Cyclosporin
per day Digitalis
Excess lactation
• Parathyroid Hormone (PTH) Miscellaneous
Pregnancy
o Increases the renal reabsorption and intestinal
absorption of magnesium
o Both Calcium and Magnesium have an effect in the • In practice, the most common is gastrointestinal and
PTH drug induced, specifically in patients taking digitalis.
▪ Compared to Mg, Ca has a greater effect

Symptoms of Hypomagnesemia
• Aldosterone and Thyroxine
o Increases the renal excretion of magnesium Table 9. Symptoms of Hypomagnesemia
Clinical Significance Symptoms of Hypomagnesemia
Arrythmia
Hypomagnesemia Cardiovascular Hypertension
Digitalis toxicity
• Most frequent in hospitalized patient Weakness
o Intensive Care Unit (ICU) Cramps
Ataxia
o Diuretic Therapy or Digitalis Therapy for congestive
Tremor
heart failure or arrythmia Neuromuscular
Seizure
• Rare in non-hospitalized patient Tetany
Paralysis
o Their small intestine is efficient in absorbing daily Coma
intake of Mg Depression
Psychiatric Agitation
• Causes: Psychosis
Hypokalemia
o Reduce intake Hypocalcemia
o Decrease absorption Metabolic
Hypophosphatemia
o Increased excretion: Renal, Endocrine, and Drug Hyponatremia
induce
• Symptoms
o Asymptomatic patients are possible
o If the value falls below 0.5 mmol/L, the symptoms
are apparent to the patient
CATAPANG. CAGAS
Treatment of Hypomagnesemia Therapeutic Uses of Magnesium
• Oral supplementation • Preeclampsia

o Magnesium lactate • Cardiac arrhythmia
o Magnesium oxide • Myocardial infarction
o Magnesium chloride o Can be used due to the vasodilatory effect
o Aluminum-magnesium
• Common pathology
▪ Antitussive treatment of Gastrointestinal Reflux
(GERD) and Peptic Ulcer Disease (PUD) o Decreased blood flow
o Magnesium has a vasodilatory effect
• Parenteral supplementation
▪ Deliver the appropriate amount of oxygen
o Magnesium Sulfate needed by the tissues.
▪ Renal assessment should be done to avoid ▪ NOTE: Be careful because it can induce
induction of hypermagnesemia hypermagnesemia in the process of using
magnesium as a vasodilator.
Hypermagnesemia Treatment of Hypermagnesemia
• Causes: • Discontinue treatment for Drug induced
o Decreased excretion Hypermagnesemia
o Increased intake • Supportive therapy for symptomatic patients
o Miscellaneous • Hemodialysis: renal failure patients
o Patients with renal failure cannot secrete
Table 10. Causes of Hypermagnesemia magnesium through their kidneys, that’s why the
important thing to do is hemodialysis.
Causes of Hypermagnesemia • Diuretic and IV fluid: normal renal function
Acute or chronic renal failure

Specimen
Decreased Hypothyroidism
Excretion Hypoaldoosteronism
• Nonhemolyzed serum
Hypopituitarism (↓growth hormone)
Antacids o Preferred because although in a small amount,
Enemas erythrocytes contain 10 times magnesium compared
Increased Intake
Cathartics to extracellular fluid.
Therapeutic-eclempsia, cardiac arrythmia
Dehydration • Lithium Heparin Plasma
Miscellaneous Bone carcinoma
Bone metastases o Oxalate, EDTA, and citrate will bind with magnesium
• Most common cause is renal failure • 24-hour urine
o Defined by a Glomerular Filtration Rate (GFR) of o Preferred for analysis because of a diurnal variation
less than 30 ml per minute in excretion
o Must be acidified with HCl to avoid precipitation
Table 11. Symptoms of Hypomagnesemia
Colorimetric Methods
Symptoms of Hypermagnesemia
• Calmagite
Hypotension • Formazan dye
Cardiovascular Bradycardia • Methylthymol blue
Heart block
Flushing o Most methods use calcium shelter to prohibit
Dermatologic
Warm skin interference from the divalent cations, since
Nausea magnesium is also a divalent cation.
Gastrointestinal
Vomiting
Lethargy
Neurologic
Coma Table 12. Colorimetric Methods
Decreased reflexes
Dysarthria
Neuromuscular Calmagite Formazan Methylthymol
Respiratory depression
Paralysis
Metabolic Hypocalcemia Mg2+ binds with Mg2+ binds with the Mg2+ binds with
Decreased thrombin generation calmagite dye chromogen
Hemostatic
Decreased platelet adhesion Reddish-violet
Colored complex Colored complex
complex
• In life threatening symptoms, ECG changes such as
heart block, asystole, etc. can occur in magnesium 532 nm 660 nm -
levels of more than 5 mmol/l
CATAPANG. CAGAS
Atomic absorption Spectrophotometry (AAS)
• Reference method
• Limitations of testing: Total magnesium
o May not reflect the physiologically the active free
ionized magnesium
▪ 25% are protein-bound
▪ Magnesium, primarily an intracellular ion; Serum
concentrations will not necessarily reflect the
status of intracellular magnesium
 Maybe magnesium is high in the serum but
low in the cell (or vice versa)
Reference Range for Magnesium
• Serum, Colorimetric
o 0.63 - 1.0 mmol/L (1.26 - 2.10 mmol/L)
CATAPANG. CAGAS
[TRANS] LABORATORY UNIT 07: CHLORIDE, BICARBONATE, & LACTATE
CHLORIDE Urine
• Major extracellular anion • 24-hour urine collection

o Extracellular o The specimen of choice for urine chloride analyses
▪ Outside the cell or in
the plasma Sweat
o Anion • Analysis of chloride ion concentration in sweat is used
▪ Negatively charged to confirm the diagnosis of Cystic Fibrosis.
ion
• Cystic Fibrosis
o Different to sodium since
o the dysfunction of the CF transmembrane
sodium is the major
conductance regulator (CFTR).
extracellular cation
o CFTR – a chloride ion channel that moves chloride
• Functions: ions from inside the cell to outside the cell.
o Involved in maintaining osmolality, blood volume, ▪ If CFTR is dysfunctional, chloride is trapped
and electric neutrality inside the cell.
o Chloride shifts secondarily to a movement of sodium o Without the proper movement of chloride, water
or bicarbonate ion. cannot hydrate the cellular surface which leads to
Determination of Chloride mucus covering the cells to become thick and sticky
causing many of the symptoms associated with
• Specimen: cystic fibrosis.
o Blood • Chloride concentration in sweat is best determined by
o Urine colorimetric titration.
o Sweat
Methods
Specimen Consideration
Ion-Selective Electrodes (ISE)
Blood
• Most commonly used method in clinical chemistry to
• Lithium Heparin measure ion concentration like Chloride
• 3 major types:
o anticoagulant of choice
o Inert Metal Electrodes
• Serum or plasma may be used.
▪ In contact with a redox couple
o Serum is more commonly used in clinical
laboratories. o Metal Electrodes
▪ Participate in redox reaction
• Hemolysis does NOT cause a significant change in
serum or plasma values. o Selective Membrane Electrodes – ISE used in
chloride determination
o As a result of decreased levels of intracellular
chloride. ▪ Selective membrane
• Marked hemolysis  can be solid material like glass
o Chloride levels may be decreased as a result of  can be liquid like iron exchange electrode
dilutional effect.  can be special membranes such as gas
sensing and enzyme electrode.
• Whole blood samples may be used with some
analyzers. ▪ Type of ISE used with Ion exchange membrane
o Operations manual must be consulted first for • Principle:
acceptability
o An ion-exchange membrane (tri-n-
o Due to various factors that may be considered first
octylpropylammonium decanol) is used to selectively
depending upon the analyzer bind chloride ions.
ANTOYAN. NARAGA. OLIVA. VILLENA. CORTEZ. CAGAS BSMLS 3 1

LABORATORY UNIT 07: CHLORIDE, BICARBONATE, AND LACTATE
o Combination electrode is usually used to measure

ion and it has both indicator electrode and reference 𝐹𝑒𝑟𝑟𝑖𝑐 𝑖𝑜𝑛𝑠 𝑹𝒆𝒅𝒅𝒊𝒔𝒉 − 𝒃𝒓𝒐𝒘𝒏
𝐹𝑟𝑒𝑒 𝑡ℎ𝑖𝑜𝑐𝑦𝑎𝑛𝑎𝑡𝑒 + =
electrode. (𝑓𝑟𝑜𝑚 𝑓𝑒𝑟𝑟𝑖𝑐 𝑛𝑖𝑡𝑟𝑎𝑡𝑒) 𝒄𝒐𝒍𝒐𝒓
▪ Converts a specific ion with the activity of a • Free thiocyanate reacts with ferric ions, which is present
specific ion to electrical potential. in ferric nitrate to form a reddish-brown complex of
o Electrical potential depends on the activity of the ion, ferric thiocyanate.
so the greater the activity of the ion, the higher its
concentration. Reference Range for Chloride
Table 1. Reference Ranges for Chloride
Specimen Range
Plasma, serum 98-107 mmol/L

110-250 mmol/d, varies with
Amperometric-Coulometric Titration Urine (24 hr)
diet
• A method using coulometric generation of silver ions
which combine with chloride ion to quantitate Chloride
concentration. Package Insert for Chloride
o Silver chloride precipitate is formed
• Equivalence point
o When all the chloride has been precipitated or
combined with the silver ions
• Endpoint: the surge of free silver ions
o When free silver ions accumulate, the coulometric
generator and timer are turned off.
▪ Elapsed time – used to calculate the
concentration of chloride in the sample.
• Cotlove Chloridometer
o Instrument that uses this principle
Mercurimetric Titration (Schales and Schales)
• (1) PFF preparation (Protein Free Filtrate)

o Use tungstic acid as precipitating agent of protein
• (2) Principle
o PFF is titrated with standard solution of mercuric
ions (mercuric nitrate) to form a soluble compound
of mercuric chloride which does not dissociate to
mercuric ions.
• (3) Endpoint is detected colorimetrically.
𝐸𝑥𝑐𝑒𝑠𝑠 𝑚𝑒𝑟𝑐𝑢𝑟𝑖𝑐 𝑠-𝑑𝑖𝑝ℎ𝑒𝑛𝑦𝑙𝑐𝑎𝑟𝑏𝑎𝑧𝑜𝑛𝑒 𝑩𝒍𝒖𝒆-𝒗𝒊𝒐𝒍𝒆𝒕

+ =
𝑖𝑜𝑛𝑠 (𝑖𝑛𝑑𝑖𝑐𝑎𝑡𝑜𝑟 𝑑𝑦𝑒) 𝒄𝒐𝒍𝒐𝒓
• When excess mercuric ions combined with the indicator

dye (s-diphenylcarbazone), it will form a blue-violet
color
Mercuric Thiocyanate (Whitehorn Titration) Method

• Photometric Colorimetric Test for Chloride (TPTZ
• Specimen is mixed with a solution of mercuric Method)
thiocyanate to form Mercuric chloride o Determines the chloride ions concentration
o Mercuric chloride - final product

• Method o Use the prediluted [STD]/sample for the following

pipetting scheme
o Chloride ions react with a mercury (II)-2,4,6-tri-(2-
pyridyl)-s-triazine (TPTZ) complex to form mercury Table 3. Procedure
(II)-chloride.
o The liberated TPTZ reacts with iron (II) ions yielding Pipette into
a blue coloured complex. Macro Semi-micro
cuvettes:
o The resulting absorbance change at 590 nm is
directly proportional to the amount of chloride ions in STD Sample STD Sample
the sample. STD 50 μL - 20 μL -
Sample - 50 μL - 20 μL
Table 2. Contents
RGT 2000 μL 2000 μL 1000 μL 1000 μL
Content Measurement • Macro method
2 x100 mL Colour reagent o (1) Prepare two tubes, one for standard and one for
2.4.6-Tri-(2-pyridyl)-s-triazine (TPTZ) sample
[RGT] 0.986 mmol/L o (2) Dispense reagent first since it has larger volume
(partially as mercury (II) complex)
(2000 uL for both standard and sample)
Iron (II) sulphate 0.53 mmol/L o (3) Dispense 50 uL of standard and 50 uL of sample
3 mL Standard in the respective tubes
[STD] Chloride (Cl-) 100 mmol/L • Semi-micro method
or 355 mg/dL
o (1) Prepare two tubes for standard and sample
o (2) Dispense 1000 uL of reagent to both standard
• Reagent Preparation and sample
o [RGT] and [STD] are ready to use. o (3) Dispense 20 uL of standard and sample
• Reagent Stability • Mix, incubate for 5 minutes in the dark and measure the
absorbance of sample (∆Αsample) and STD (∆ΑSTD)
o [RGT] and [STD] are stable even after opening up to within 60 minutes against the reagent blank. Do not
the stated expiry date when stored at 2-25°C in the expose to light!.
dark.
o Contamination must be avoided. Calculation
• Specimen
• SI Unit:
o Serum, CSF, urine 𝑨𝒔𝒂𝒎𝒑𝒍𝒆
o Stability in serum at 2-25°C: 7 days 𝐜 = 𝟏𝟎𝟎 × [𝐦𝐦𝐨𝐥/𝐥]
o Stability in urine at 2-8°C: 7 days 𝑨𝑺𝑻𝑫
• Notes: • Conventional unit:

o Blood should be immediately processed (e.g. 𝑨𝒔𝒂𝒎𝒑𝒍𝒆
centrifuged within 1 hour), otherwise an electrolyte 𝐜 = 𝟑𝟓𝟓 × [𝐦𝐠/𝐝𝐥]
𝑨𝑺𝑻𝑫
exchange between serum and erythrocytes may
lead to a false low chloride determination. • The result may be converted according to the following
o Precipitates in urine may be redissolved by warming equations:
up.
𝑐 [𝑚𝑚𝑜𝑙/𝑙] = 𝑐 [𝑚𝑣𝑎𝑙/𝑙]
• Assay
o Wavelength: 590 nm (560-600 nm), Hg 578 nm 𝑐 [𝑚𝑚𝑜𝑙/𝑙] × 3.55 = 𝑐 [𝑚𝑔/𝑑𝑙]
o Temperature: 20-37°C 𝑐 [𝑚𝑔/𝑑𝑙] × 0.282 = 𝑐 [𝑚𝑚𝑜𝑙/𝑙]
o Measurement: Against reagent blank. Only one
blank per series is required. • 24 h urine:
𝑐 [𝑚𝑚𝑜𝑙/𝑙] × 𝑢𝑟𝑖𝑛𝑒 𝑣𝑜𝑙𝑢𝑚𝑒[𝑙] / 24[ℎ] = 𝑐[𝑚𝑚𝑜𝑙/24ℎ]
• Pipetting Scheme
o Before dispensing the [RGT] with the [STD] or the 𝑐[𝑚𝑚𝑜𝑙/24ℎ] × 0.0355 = 𝑐[𝑔/24ℎ]
sample, the [STD] and the sample should be
prediluted first with distilled water. Performance Characteristics
o Macro method: Predilute [STD] and the sample
1+40 with distilled water • Linearity
▪ E.g. 50 ul [STD]/sample + 2000 ul distilled water o The test is linear up to a chloride concentration of
o Semi-micro method: Predilute [STD] and the 500 mmol/l or 1775 mg/dl. Samples with higher
sample 1+50 with distilled water chloride concentration have to be further diluted 1+1
with distilled water.
▪ E.g. 20 ul [STD]/sample + 1000 ul distilled water. o Repeat the assay and multiply the results by 2.

Table 4. Reference Range for Chloride • Many current analyzers for CO2 determination do not
permit anaerobic sample handling due to some factors
SI unit Conventional unit that must be observed before handling anaerobic
Specimen [mmol/l] [mg/dl] sample:
Serum 95 –108 mmol/l 335–383 mg/dl o Must be capped/well-covered until the serum or
442–460 mg/dl plasma is separated and analyzed immediately
CSF 119 –130 mmol/l
3.9–8 g/24h • If the sample is left uncapped before analysis
Urine 110 –225 mmol/l
o CO2 escapes → levels can decrease by 6 mmol/L/h
• Quality Control o Thus, venous serum or heparinized plasma is
suitable for enhancements
o All control sera with chloride values determined by
this method can be employed. Methods
o We recommend to use our animal-based serum
HUMATROL. Quality control sera or our human Ion-Selective Electrodes (ISE)
serum based SERODOS.
• Uses an acid reagent to convert all the forms of CO2 to
• Automation CO2 gas
o Proposals to apply the reagents on analyzers are o Measured by a pCO2 electrode (Severinghaus
available on request. Each laboratory has to validate Electrode)
the application in its own responsibility.
• Notes Enzymatic Method
o The test detects chloride very sensitively. Usage of • Alkalinizes the sample to convert all forms of CO2 to
contaminated glassware and skin contact therefore HCO3- (bicarbonate)
can cause elevated chloride results. Use of • Uses a coupled enzymatic method
disposable plastic ware and gloves is highly
recommended.
• First enzymatic reaction
o Hemoglobin, bilirubin and ascorbic acid in
physiological concentrations do not interfere with the o Enzyme: Phosphoenolpyruvate Carboxylase
test.
▪ catalyzes the formation of oxaloacetate
o Lipemic samples interfere with the test (elevated
results) and should therefore be avoided. 𝑃𝐸𝐹 𝑐𝑎𝑟𝑏𝑜𝑥𝑦𝑙𝑎𝑡𝑒
𝑃ℎ𝑜𝑠𝑝ℎ𝑜𝑒𝑛𝑜𝑙𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝐻𝐶𝑂3 - → 𝑂𝑥𝑎𝑙𝑎𝑡𝑒 + 𝐻2𝑃𝑂4-
o RGT is sensitive to light, especially UV light. RGT
should be kept light-protected.
• Second enzymatic reaction
BICARBONATE
o Enzyme: Malate dehydrogenase
• 2nd most abundant anion in ▪ Oxaloacetate is formed with NADH and
the ECF, next to chloride ions hydrogen
• Function: ▪ NADH – consumed as a result of the action of
o Major component of the the enzyme (MDH)
buffering system in the 𝑀𝐷𝐻
blood 𝑂𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷𝑃𝐻 + 𝐻 + → 𝑀𝑎𝑙𝑎𝑡𝑒 + 𝑁𝐴𝐷+
• Uses same method for

• After the enzymatic reaction, monitor the change of
carbon dioxide determination
absorbance at 340 nm
o Since total carbon dioxide
o NADH produces a significant absorbance peak at
comprises of:
this wavelength
▪ Bicarbonate ion, Carbonic acid, Dissolved
• INTERPREATION: The decrease in the absorbance of
carbon dioxide
NADH at 340 nm is proportional to the total carbon
• Total CO2 measurement is indicative of Bicarbonate dioxide content
measurement
o Serum bicarbonate accounts for more than 90% of Reference Range for Bicarbonate
serum total CO2 at physiologic Ph
• CO2, venous 23 to 29 mmol/L (plasma, serum)
Determination of Total CO2
• Specimen LACTATE
o Venous serum or heparinized plasma • Is a by-product of an emergency mechanism that
o Anaerobic collection produces a small amount of ATP when oxygen delivery
is severely diminished.
▪ Specimen should be anaerobic to aim for the
highest accuracy

o When oxygen delivery is severely diminished → Reference Ranges for Lactate

leads to accumulation of excess NADH → activates
the conversion of pyruvate to lactate
Table 5. Reference Ranges for Lactate
▪ Pyruvate – normal end product of glucose
metabolism/ glycolysis.
Enzymatic Method, Colorimetric,
• Other function of Lactate: Plasma Whole Blood
o Main precursor for producing glucose 0.5 – 2.2 mmol/L 0.9 – 1.7 mmol/L
Venous
(4.5 – 19.8 mg/dL) (8.1 – 15.3 mg/dL)
Clinical Applications 0.5 – 1.6 mmol/L < 1.3 mmol/L
Arterial
(4.5 – 14.4 mg/dL) (<11.7 mg/dL)
• Measurements of blood lactate Cerebrospinal 1.0 – 2.9 mmol/L
o Useful for metabolic monitoring in critically ill fluid (9 – 26 mg/dL)
patients
o Indicating the severity of the illness
o For objectively determining patient prognosis.
Specimen Handling
• Tourniquet should NOT be used (ideally).

o Venostasis → increases the lactate level
o Can be used only on inevitable instances (e.g.,
difficult to locate veins/draw blood sample)
▪ Blood sample should be collected immediately
• After sample collection, anaerobic glycolysis will
occur.
o Anaerobic glycolysis – wherein glucose is converted
to lactose
o Should be prevented
• Heparinized blood
o must be delivered on ice and the plasma must be
separated quickly.
• Fluoride and iodoacetate
o inhibits glycolysis but the specific method directions
must be consulted.
Methods
Enzymatic Method
• Uses lactate oxidase to produce pyruvate and

hydrogen peroxide.
• Quantity of lactate is determined by the amount of
hydrogen peroxide produced
𝐿𝑎𝑐𝑡𝑎𝑡𝑒 𝑜𝑥𝑖𝑑𝑎𝑠𝑒
𝐿𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑂2 → 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝐻2𝑂2
• Addition of chromogen and catalyzed by peroxidase to

form a colored complex
𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒
𝐻2𝑂2 + 𝐻 𝑑𝑜𝑛𝑎𝑟 + 𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 → 𝑐𝑜𝑙𝑜𝑟𝑒𝑑 𝑑𝑦𝑒 + 2 𝐻20
• Chromogen is added to the hydrogen peroxide.

o Chromogen – precursor of biochemical pigment
o Hydrogen peroxide – detectable
• In the presence of peroxidase enzyme, the colored
complex formation is detected calorimetrically.

[TRANS] LABORATORY UNIT 8: CALCIUM AND PHOSPHATE
CALCIUM Sample Considerations

• The 5th most common element • Whole blood specimens should be analyzed within
• 2nd most prevalent cation in the human body next to 15-30 minutes of collection.
sodium
o If this is not possible, the specimen should be kept
Specimen in ice.
Total Calcium Determination ▪ Specimen will stable for at least 2 hours.

• If the specimen cannot be analyzed within 1 hour:
• Serum or lithium heparin if plasma
o the preferred specimen is serum.
• Citrate, EDTA or oxalate can interfere with the analysis,
thus, should NOT be used • Hemolysis and delayed plasma/serum separation
o The action of citrate and EDTA focus more on the lead to a decreased calcium concentration
chelation of calcium or binding of calcium → o Always process samples immediately.
unacceptable o Any samples upon prolonged standing will have
changes on the sample.
Ionized Calcium
• A decrease in plasma protein concentration will
• Must be collected anaerobically (without the presence result in decreased total calcium
of oxygen)
o Around 40-50% of calcium are bound to proteins
o Any change of the pH could affect the calcium level. (especially albumin). Thus, any changes in the
o With the presence of oxygen, as it increases → can protein concentration would also affect the calcium
change the pH strength → pH increases (alkaline) concentration.
→ promotes increase in protein binding (a lot of
calcium binds to protein) • Recumbent posture decreases calcium.
▪ Result to an increase calcium level = false o The sample should be drawn with the patient in a
decrease ionized calcium level sitting position.
o Acidosis or if the pH level is decreasing ▪ Standing increases the total calcium
concentration
▪ crystal protein binding will decrease = false
elevation in free calcium levels o Changes in plasma volume
• Best to use evacuated tube system to make sure that ▪ Every position change will have corresponding
the ionized calcium is collected anaerobically changes in the plasma volume.
▪ Plasma volume change of about 3.4% happened
o Less likely to be contaminated with oxygen as it is a
if from lying down to sitting. Around 14% if from
closed system
lying down to standing position. Around 9% from
• Heparinized whole blood (preferred) sitting to standing.
• Serum  Upon collection of calcium sample, it would be
• No liquid heparin best to let your patient rest first before
o Heparin concentration of 25 IU/mL, for example, drawing the blood.
decreases ionized Ca2+ by about 3% • Venous occlusions increase calcium
Timed urine collection o Fist-clenching or forearm exercise can lead to
falsely elevated ionized (free) calcium levels
• Much preferred (24-hour urine collection) (hemodynamic changes)
• Should be acidified with 6mol/L HCl
o Approximately 1 mL of the acid added for each 100
Laboratory Methods
mL of urine
o Purpose of adding HCl in urine: • Total Calcium Measurements
▪ Preservative o Includes measuring protein-bound calcium and
▪ Prevent calcium salt precipitation ionized calcium
o If the patient has hypoalbuminemia, it will lead to a
misleadingly low/ false decrease total calcium level,
thus, a correction must be made:
CATAPANG. CAGAS.
LABORATORY UNIT 08: CALCIUM AND PHOSPHATE (LABORATORY)
• Other materials:
o (1) Determine the albumin concentration of the

patient and subtract it to the normal reference range
of albumin.
o (2) Once you get the albumin, add it to the total
calcium measured.
o (3) Multiply by 0.8
o (4) Reporting of the results obtained.
Total Calcium Measurements Methods
• Ortho-cresolphthalein Complexone Method (O-CPC)

o O-CPC reacts with calcium to form a purple color in
alkaline solution
▪ Measured at near 580 nm
o Uses 8-hydroxyquinoline to prevent magnesium
interference
• Arsenazo III Dye Method
o Arsenazo III reacts with calcium to form a calcium- Preparation, Storage, & Stability
indicator complex
o Usually measured at near 650 nm • Mix equal volume of CPC reagent and diluent reagent
• Atomic Absorption Spectrophotometry (AAS) to get desired working reagent. Working reagent is
stable for 7 days at 2 – 8°C. The reagent kit should be
o Reference method stored at 2 – 8°C and is stable till the expiry date
indicated on the label.
• Ion Selective Electrode (ISE)
o The working solution can be prepared by mixing 1
volume of CPC reagent with 1 volume of diluent.
How To Perform Calcium Test in the Laboratory
(O-CPC Method) Assay Procedure
Materials Table 1. Contents of the Reagent Solution
• Use the kit manufactured by Blank Standard Test

Anamol Laboratories Pvt.
Ltd. Reagent 1000 μl 1000 μl 1000 μl
Standard NA 20 μl NA
• Kit Components: Sample NA NA 20 μl
o 1 bottle – Calcium CPC Mix the reagent and sample in the above-mentioned ratio and
reagent incubate for 5 mins at R.T.
o 1 bottle – Calcium CPC
diluent Aspirate reaction mixture into flow cell and record the absorbance.
o 1 bottle – Calcium
standard (10 mg/dL Final color is stable for 1 hour if not exposed to direct light.
concentration)
o Instructions for use • Preparation of 1000 uL Working Solution
▪ Users must read the o 500 uL of reagent + 500 uL diluent
instructions for use
thoroughly before
using the test kit. PREPARATION OF WORKING SOLUTION
• Take 500 uL of
• Clean & well-calibrated diluent without any
pipette of the following air bubble and add it
STEP 1.
volumes: to the first tube
labeled as the
o 1000 μl “blank”
o 100 𝛍l
CATAPANG. CAGAS.
• Take 500 uL of • Take 20 uL of

diluent without any patient sample and
STEP 2. air bubble and add it wipe the outside of
to the second tube STEP 11. the tip with a tissue
labeled as the paper and add it to
“standard” the third tube
labeled as the test.
• Take 500 uL of Mix it thoroughly
diluent without any
STEP 3. air bubble and add it • Start the incubation
to the third tube for 5 minutes at
labeled as the “test” room temperature
o Make sure that
the instrument is
STEP 12. switched on and
STEP 4. • Change the tip
open the
program for the
CPC reagent,
keeping it ready
• Take 500 uL of CPC
to read the blank
reagent without any
STEP 5. air bubble and add it
to the first tube • Once the incubation
labeled as the is complete, take the
“blank” STEP 13. tube of the blank
and aspirate to read
• Take 500 uL of it
reagent without any
STEP 6. air bubble and add it • Take the tube of the
to the second tube STEP 14. standard and
labeled as the aspirate to read it
“standard”
• Take the tube of the
• Take 500 uL of STEP 15. test and aspirate to
reagent without any read it
STEP 7. air bubble and add it
to the third tube
labeled as the “test • After a small lag
time the instrument
has already
• Mix the reagents calculated the
and working solution absorbance of the
is now ready STEP 16. standard and also
o The components the result
STEP 8.
of the tube o uL of diluent =
should not be 1000 μL working
mixed with the solution
tip
• Repeat the exact same procedure to run more samples

• Add the “standard” • Semi-automated machine is used
and the patient
sample into the
working solution by Package Insert of Calcium
taking 20 uL of
“standard” and wipe • Method
STEP 9. the outside of the tip
with a tissue paper o Calcium ions react with o-cresolphthalein-
and add the complexone in an alkaline medium to form a purple
standard to the colored complex.
second tube labeled o The absorbance of this complex is proportional to
as the standard.
the calcium concentration in the sample
• Mix it thoroughly
• Contents
o Sodium azide
STEP 10. • Change the tip
▪ Preservative
▪ Harmful
CATAPANG. CAGAS.
o Buffer Table 3. Pipetting Scheme

▪ Solutions that can resist any pH changes upon
the addition of either an acid or a basic Pipette into
Reagent blank Sample or STD
component cuvettes
▪ Able to neutralize small amounts of added acid
or base thus, maintaining the pH. Sample or STD --- 20 μl
o 8-Hydroxyquinoline Working reagent 1000 μl 1000 μl

▪ Prevent any magnesium interference. Mix and measure the absorbance of sample (∆Asample) and
▪ Magnesium can affect the overall assay or standard (∆ASTD) against the reagent blank within 5 to 30
process. minutes.
Table 2. Contents of the Reagent Solution Calculation of the Calcium Concentration
Contents Concentrations ∆𝑨𝒔𝒂𝒎𝒑𝒍𝒆

𝒄=𝟖𝒙 (𝒎𝒈/𝒅𝒍)
∆𝑨𝑺𝑻𝑫
100 mL Buffer Solution
or
Lysine buffer (pH 11.1) 0.2mol/l
∆𝑨𝒔𝒂𝒎𝒑𝒍𝒆
𝒄=𝟐𝒙 (𝒎𝒎𝒐𝒍/𝒍)
Sodium azide 0.095% ∆𝑨𝑺𝑻𝑫
100mL Colour Reagent Performance Characteristics
8-Hydroxyquinoline 14mmol/l • Linearity

o The test is linear up to a calcium concentration of 15
o-Cresolphthalein-complexone 0.1mmol/l mg/dl or 3.75 mmol/l.
o Samples with a higher concentration have to be
Hydrochloric acid 40mmol/l diluted 1+1 with distilled water.
o Repeat the assay and multiply the results by 2.
3mL Standard
• Normal values
Calcium (II) 8mg/dl or 2mmol/l o Serum/plasma: 8.1-10.4 mg/dl or 2.02-2.60 mmol/l
Sodium azide 0.095%

• Quality Control
o All control sera with calcium values determined by
this method can be employed.
• Reagent Preparation o We recommend to use our quality control sera
HumaTrol based on animal serum or our SERODOS
o Add reagent to buffer in equal volumes as required, based on human serum
mix and allow to stand for 30 minutes at room
temperature before use. • Notes
• Reagent Stability o Contaminated glassware is the greatest source of
error.
o The reagents and the standard are stable even after
opening up to the stated expiry date when stored at ▪ Disposable plastic ware is recommended for the
2-25 °C. test.
o Contamination must be avoided.
o The test is not influenced by hemoglobin up to 200
o The working reagent is stable for 7 days at 2-8 °C mg/dl and bilirubin up to 20 mg/dl.
(refrigerator) and for 3 days at 15-25 °C (room
temperature) ▪ There is no problem as long as it does not reach
that amount
• Specimen ▪ It’s better not to use hemolyzed sample to get
o Serum, heparinised plasma accurate result
o Stability in serum: 10 days at 2-25 °C ▪ Most error occurs at pre-analytical phase
• Assay o Lipemic and haemolytic samples require a sample

blank.
o Wavelength: 570 nm, Hg 578 nm
o Optical path: 1 cm ▪ By using the same pipetting scheme mix 20 µl of
o Temperature: 20-25 °C sample with 100 µl of distilled water and
measure the absorbance (∆Asample blank) against
• Measurement distilled water.
o Against reagent blank. ▪ ∆Asample blank has to be subtracted from ∆Asample
o Only one reagent blank per series is required. blank.
CATAPANG. CAGAS.
o BUF and STD contain sodium azide (0.095%) as Specimen

preservative.
• Serum
▪ Do not swallow.
▪ Avoid contact with skin and mucous membranes o Preferred specimen for the phosphate determination
▪ It is harmful o If not available, use of plasma
• Lithium heparin plasma
Table 4. Reference Ranges for Calcium (Bishop)
o The anticoagulant to be used is Lithium heparin
o Oxalate, citrate, or EDTA can interfere with the
Reference Range analysis
o Hemolysis should be avoided
o Circulating phosphate levels are subject to circadian
Total Calcium-Serum, Plasma
rhythm
Child, <12 y 2.20-2.70 mmol/L (8.8-10.8 mg/dL) ▪ Circadian rhythm
Adult 2.15-2.50 mmol/L (8.6-10.0 mg/dL)  the variation of concentration that depends on
the time of the day
Ionized Calcium-Serum ▪ Highest concentration in the late morning and
lowest concentration in the evening
Child 1.20-1.38 mmol/L (4.8-5.5 mg/dL)
• 24-hour urine
Adult 1.16-1.32 mmol/L (4.6-5.3 mg/dL)
o Recommended urine sample
Ionized Calcium-Plasma Sample Considerations
Adult 1.03-1.23 mmol/L (4.1-4.9 mg/dL) • Phosphate concentrations in plasma or serum are
increased by prolonged storage with cells at room
Ionized Calcium-Whole Blood temperature or 37 °C
Adult 1.15-1.27 mmol/L (4.6-5.1 mg/dL) o Process samples immediately rather than letting it
stay for a longer period of time.
Total Calcium-Urine 2.50-7.50 mmol/d (100-300 mg/d),
(24hr) varies with diet • Inorganic phosphate increases by 4-5 mg/dL or 1.61
mmol/L per day in hemolyzed specimens stored at 4 °C,
more rapidly at room temperature or 37 °C
• Phosphate is stable in separated serum for days at 4 °C
and for months when frozen, provided evaporation is
prevented.
Laboratory Methods
• Involve the formation of an ammonium

phosphomolybdate complex
o Fiske-Subbarow Method
▪ Specific name of ammonium phosphomolybdate
• Can be measured by:
o UV at 340 nm
o Reduced to molybdenum blue, which is read
between 600 and 700 nm
▪ Reducing agents used in order to reduce
ammonium phosphomolybdate to become
molybdenum blue:
 Pictol – amino naphthol sulfonic acid
 Elon – methyl amino phenol
PHOSPHATE  Senidine
• Calcium and phosphate is similar to the relationship of  Ascorbic Acid

sodium and potassium. ▪ Molybdenum blue will be measured in order to
get the concentration of the phosphate.
o There is an interchange or exchange between the
two • Formation of phosphomolybdate complex is ph-
o If one of them increase/decrease, then there is dependent
interchange with their levels and position • Rate of its formation is influenced by protein
(extracellular and intracellular) concentration
CATAPANG. CAGAS.
Enzymatic method o Sulfuric acid
• Catalyzed by glycogen phosphorylase, ▪ Act as preservative and precipitating agent

phosphoglucomutase, and G6PD o Detergent
o Enzymes present in the reaction principle ▪ Act as a buffer
• NADPH produced can be quantitated fluorometrically or • Reagent Preparation
spectrophotometrically
o Reagent and standard are ready for use
o Measures the presence of NADPH
• Reagent Stability
Package Insert for Phosphate
o The reagents are stable, even after opening, up to
the stated expiry date when stored at 2-25°C
• Specimen
o Serum
o Plasma must not be used. Anticoagulants may
cause false low results.
o Stability in serum:
▪ 7 days at +4°C
▪ 2 days at 20-25°C
o NOTE:
▪ It depends on the test whether we can use
plasma as a sample.
▪ E.g. Machines
 The requirement of the machines varies, e.g.
blood gas analysis. There are some machines
that accept the lavender top tubes. It is
common and advantage for the patient with
required CBC results because you do not
need to collect a lot of blood samples and
place it in different tubes.
o Wavelength: 340 nm Hg or 334 nm
o Optical path: 1cm
o Temperature: 20-25°C
o Measurement: Against reagent blank, one reagent
blank per series is required
Table 6. Procedure
• Method
Pipetting Scheme
o Phosphate reacts with molybdate in strong acidic
medium to form a complex. The absorbance of this Pipette into cuvettes:
complex in the near UV is directly proportional to the
phosphate concentration. Sample or
Reagent blank
Standard
Table 5. Contents of the Reagent Sample / STD 10 μL

Reagent 1000 μL 1000 μL
Reagent Solution Measurement Mix, incubate at least 1 minute at room temperature. Measure the
absorbance of the sample and the standard against the reagent
2 x 100 ml Reagent blank with 60 minutes (∆𝐀).
Ammoniumheptamolybdate 0.3 mmol/l
• Procedure
Sulphuric acid (pH < 1.0) 160 mmol/l o (1) Pipetting
Detergent 1%
o (2) Incubation – at least 1 minute at room
temperature
Activators and stabilizers o (3) Measure the absorbance against the reagent
blank
1 x 5 ml Standard
▪ The mixture of sample and reagent is stable up
10 mg/dl or 3.2 to 60 minutes
Phosphorus
mmol/l
CATAPANG. CAGAS.
Calculation
𝑪 = 𝟏𝟎 𝒙 [𝐦𝐠/𝐝𝐥]
∆𝑨𝑺𝑻𝑫
or
𝑪 = 𝟑. 𝟐 𝒙 [𝒎𝒎𝒐𝒍⁄𝑳]
∆𝑨𝑺𝑻𝑫
• Linearity
o The test is linear up to a phosphorus concentration
of 20 mg/dl or 6.4 mmol/l. Dilute samples with a
higher concentration 1+1 with distilled water.
o Multiply the results by 2
• Normal Values
o Adults: 2.5 – 5.0 mg/dl or 0.81 – 1.62 mmol/L
o Children: 4.0 – 7.0 mg/dl or 1.30 – 2.26 mmol/L
• Quality Control
o All control sera with values determined by the
method can be used. We recommend the use of
HUMATROL quality control serum based on animal
serum or our SERODOS based on human serum.
• Automation
o Special applications for automatic analyzers are
• Notes:
o Icteric and slightly lipemic samples require a sample
blank. By using the sample pipetting scheme, mix 10
μL sample with 1000 μL distilled water and measure
the absorbance against distilled water. The
absorbance ∆𝐴𝑠𝑎𝑚𝑝𝑙𝑒 𝑏𝑙𝑎𝑛𝑘 has to be subtracted from
∆𝐴𝑠𝑎𝑚𝑝𝑙𝑒 .
o Strong lipemic and hemolytic sera should not be
used.
o Contaminated glassware is the greatest source of
error. Disposable plastic ware is recommended for
the test.
o Reagent contains sulphuric acid. If skin or mucous MABAGSAK ANG MAG LEAK ANI
membranes come into contact with the reagent
wash thoroughly with water and consult a doctor.
Table 7. Reference Ranges for Inorganic Phosphorus
Serum Measurement
Neonate 1.45 – 2.91 mmol/L (4.5 – 9.0 mg/dl)
Child ≤ 15 y 1.07 – 1.74 mmol/L (3.3 – 5.4 mg/dl)
Adult 0.78 – 1.42 mmol/L (2.4 – 4.4 mg/dl)
Urine (24 h) 13 – 42 mmol/d (0.4 – 1.3 g/d)
CATAPANG. CAGAS.
[TRANS] LABORATORY UNIT 09: HYPOTHALAMUS AND PITUITARY GLAND
HYPOTHALAMUS
• Part of the brain that is located under the 3rd ventricle • Hypothalamic-Hypophyseal
and directly above the pituitary gland Portal System
o The hypothalamus and pituitary gland are located o 2 capillary beds directly
near each other joined by blood vessels
PITUITARY GLAND
• Located within the confines of the sella turcica
o Also known as “Turkish saddle”
• Connected to the median eminence of the
Neurosecretory cells hypothalamus by the infundibular stalk
Structure of the Pituitary Gland
• Specialized neurons
• Releasing and inhibiting
hormones
• Modify the action of the
pituitary gland
• Two distinct lobes:

o Anterior Pituitary
▪ Also called Adenohypophysis / Pars distalis
▪ True endocrine tissue
▪ Secretes classic hormones
o Posterior Pituitary
▪ Also called Neurohypophysis / Pars nervosa
▪ Neural tissue
▪ Secretes neurohormones but does NOT
synthesize it
Hormones of the Pituitary Gland
• Anterior pituitary (Adenohypophysis)

o 5 Hormone-synthesizing and secreting cell
Releasing Hormones o Growth hormone (somatotropin) (GH)
o Thyroid – stimulating hormone (thyrotropin) (TSH)
• Secreted by hypothalamic neurons and transported to o Gonadotropins
the anterior pituitary by the hypothalamic-hypophyseal o Follicle-stimulating hormone (FSH)
portal system o Luteinizing hormone (LH)
• Function as trophic hormones to either stimulate or o Proopiomelanocortin (POMC)
inhibit release of anterior pituitary hormones o Adrenocorticotropin (ACTH)
• Seven (7) releasing hormones: o β-lipotropin
o Thyrotropin-releasing hormone (TRH) o β-endorphin
o Corticotropin-releasing hormone (CRH) o Prolactin (PRL)
o Gonadotropin-releasing hormone (GnRH)
• Posterior pituitary (Neurohypophysis)
o Growth Hormone-releasing hormone (GHRH)
o Growth Hormone-inhibiting hormone (GHIH) o Arginine vasopressin (AVP) = Antidiuretic hormone
o Prolactin-releasing hormone (PRH) (ADH)
o Prolactin-inhibiting hormone (PIH; dopamine) o Oxytocin
ANTOYAN. GABAESIN. IBONES. LATONIO. MACADANGDANG. MERCADO. NARAGA. OLIVA. RELATOR. VILLENA. AQUINO. BSMLS 3 1
CATAPANG. CAGAS
LABORATORY UNIT 09: HYPOTHALAMUS AND PITUITARY GLAND
Anterior Pituitary Hormones (3) Thyrotrophs – Thyroid-stimulating Hormone
• 5 Hormone-synthesizing and • Secretes thyroid-stimulating

Secreting cells hormone (TSH)
• TSH (thyrotropin or
o Somatotrophs – GH thyrotrophin) stimulates the
o Lactotrophs – PRL production of T4 and T3
o Thyrotrophs – TSH
o Gonadotrophs – FSH, LH o T4 – Thyroxine
o Corticotrophs – POMC o T3 – Triiodothyronine
(4) Gonadotrophs – FSH and LH
(1) Somatotrophs – Growth Hormone
• Secretes follicle-stimulating
• Somatotropin hormone (FSH) and the
luteinizing hormone (LH)
o Secretes growth
hormone o Both acts on the gonads
o Stimulates tissues to
secrete hormones that • Luteinizing hormone (LH)
stimulate body growth and regulate metabolism o Stimulates the secretion of estrogen and
progesterone
• Growth hormone o Responsible for the maturation of egg cells in the
o Naturally occurring ovaries
o Important for growth, cell regeneration and cell o Stimulates sperm production and secretion of
reproduction testosterone in the testes
o Helps speeding up the healing process and in
repairing muscle tissue • Follicle stimulating hormone (FSH)
o Helps build muscle mass and boost up metabolism o For the sexual development
o Helps burn fats o Women: Control the menstrual cycle and stimulate
o Slows aging process the growth of eggs in the ovaries
o According to some studies, it can treat age-related
diseases ▪ Ovulation – high FSH level before the egg is
release in the ovary
• Synthetic Human Growth Hormone
o Men: Control the production of sperm cells and FSH
o Used to treat poor growth in children and adults level do not change much
o Can treat adults with short bowel syndrome or o Children: Low FSH level until puberty
muscle loss due to HIV/AIDS
▪ When the levels begin to rise in girls, it helps
(2) Lactotrophs – Prolactin signal the ovaries to make estrogen and for the
boys, it signals the testes to make testosterone
• Lactotropin
(5) Corticotrophs - Proopiomelanocortin
o Secretes prolactin which
initiates milk production in • Secretes adrenocorticotropic
the mammary glands hormone (ACTH)
• Prolactin test o Stimulates the adrenal cortex to
secrete glucocorticoids (e.g.,
o Measures the level of prolactin in the body cortisol)
o Increased prolactin may indicate:
• POMC (Proopiomelanocortin)
▪ Pregnancy or new mother
o From the precursor preproomiomelanocortin
o Increased prolactin in individuals that are NOT o Precursor of melanotropin or tortora, corticotropin,
pregnant may indicate: lipotropin, and endorphin
▪ Prolactinoma – Tumor in the pituitary gland
Posterior Pituitary Hormones
 Makes the gland produce too much prolactin
▪ Causes production of breast milk in men and in • 2 hormones released by the
women magnocellular neurons of the
hypothalamus:
o Increased prolactin levels in women that are NOT
pregnant may cause: o Paraventricular nuclei
o Supraoptic nuclei
▪ Menstrual problems
▪ Infertility • Neurohypothesis does not synthesize hormones
o Increased prolactin levels in men may cause: o Only store and release two hormones
o Transported along the axons
▪ Low sex drive o Stored in the nerve terminals that end in the neuro
▪ Erectile dysfunction hypothesis
CATAPANG. CAGAS
(1) Arginine vasopressin or Antidiuretic hormone Growth Hormone

• Decreases urine volume to conserve water • Synthesized, stored, and secreted by somatotrophs
• Decreases water loss through sweating • On long bone growth in children
• Raises blood pressure by constructing the arterioles • Secreted in pulsatile fashion
o Greatest in puberty and steady decline with
(2) Oxytocin increasing age
• “Love drug” • 70% of growth hormone secretion occurs during stage 4
• Stimulates smooth muscle or slow-wave sleep
• Contractions of uterus during childbirth
• Growth hormone increases the:
• Ejection in the mammary gland
o Rate of protein synthesis in all body cells
Abnormalities of the Pituitary Function o Mobilization and use of fatty acids from adipose
tissue for energy
• Hormonal Excess
• Growth hormone decreases the:
o Clonal expansion of a distinct population of cells
o Can result from an increase in trophic hormones o Rate of glucose utilization throughout the body
from the hypothalamus or ectopic sites ▪ i.e., enhances body protein, uses up fat stores,
and conserves CHO
• Hormonal Deficiency
o Rate of glucose utilization throughout the body
o More varied cause
o Deficiency of one or more hormones, often with ▪ i.e., enhances body protein, uses up fat stores,
continued and progressive loss of other hormones and conserves CHO (carbohydrate)
over time
Stimulant
• Somatocrinin
o Growth hormone-releasing hormone (GHRH)
• Ghrelin
o Stimulates the secretion of GHRH and growth
hormones
o It is the hunger hormone by gastric neuroendocrine
cells and hypothalamus
Inhibitor
• Somatostatin
o aka Growth Hormone-inhibiting Hormone (GHIH)
• (Photo) Hypothalamus and the different pituitary cells o Paraventricular and arcuate nuclei of the
that secrete hormones and their target organs and the hypothalamus
action being done o Inhibits:
HORMONES OF THE ANTERIOR PITUITARY ▪ GH and TSH, insulin, gut hormones (motilin,
secretin, and gastrin)
Melanocyte-stimulating Hormone (MSH)
Metabolic Effects of Growth Hormone
• Also called α-MSH, α-intermedin, α-melanophore
• Protects the skin from UV rays in the development of • Has direct action on long bone growth in children;
pigmentation and control of appetite
• But most of its anabolic and metabolic actions are
o Too much MSH = hyperpigmentation or abnormal mediated indirectly through an intermediary, IGF-1
darkening of skin (also called somatomedin C)
o Too little MSH = lack of skin pigmentation and loss
o IGF is synthesized in the liver and it negatively feeds
of natural protection from the UV rays
back
• MSH and ADH are
• Growth hormone is anabolic
connected
o Anabolic – refers to muscle building
o If ADH is low = MSH is
also low • Increases fat breakdown
• Increases hepatic glucose output
▪ Leads to thirst and
frequent urination
CATAPANG. CAGAS
Stimuli • (5) If blood glucose continues to increase,

hyperglycemia inhibits the release of GHRH
• Major stress (surgery, sepsis) • (6) High blood glucose (hyperglycemia)
• Fasting o stimulates the release of GHIH
• Sex steroids
• Chronic malnutrition • (7) GHIH
• Apomorphine o inhibits secretion of hGH by somatotrophs
• Levodopa
• High-protein meals • (8) Low level of hGH and IGFs
o decreases the rate of glycogen breakdown in the
liver and glucose enters the blood more slowly
• (9) Blood glucose level falls to normal (about
90mg/100mL)
• (10) If blood glucose continues to decrease,
hypoglycemia inhibits the release of GHIH
Detection of Growth Hormone
• Undetectable for most of the day in healthy, non-

stressed individuals
• Single sampling is difficult to interpret
o Due to the episodic nature of hormone secretion
Diagnosis
• Growth Hormone Deficiency

o With the use of growth hormone measurements
Table 1. GH – UGF axis following pharmacologic stimulation
• Growth Hormone Excess
Growth Hormone Effect
o Confirmed by failure of GH suppression following an
Deep sleep, α-Adrenergic, Fasting,
oral glucose load
Stimulation Acetylcholine, Sex steroids, Stress, Amino
acids, Hypoglycemia • Laboratory Technique
Obesity, β-Andregenic, Glucocorticoids, High
Suppression
FFA, Hyperglycemia, Hypothyroidism, IGF-I o Chemiluminescent immunoassay
Undernutrition, Acute illness, Chronic illness,
Inhibition GH receptor deficiency, GHR antibodies,
Clinical Significance of Growth Hormone Deficiency
IGF-I receptor deficiency
• Idiopathic in children
Regulation of Growth Hormone Secretion o Dwarfism
• (1) Low blood glucose • Pituitary adenoma in adults

(hypoglycemia) o Individuals with childhood onset of idiopathic GH
o Stimulates the deficiency are less likely to have permanent GH
release of GHRH deficiencies
• (2) GHRH • Dx: GH measurements following pharmacologic

stimulation
o Stimulates
secretion of hGH • Insulin Tolerance Test
by somatotrophs
o Gold standard test for GH deficiency
• (3) hGH and IGFs o Failure of GH to rise above 5 ng/ml (adults) and
above 10 ng/ml (children) is abnormal
o Speed up
breakdown of liver ▪ However, it is most unpleasant for the patient
glycogen into and requires the attendance of the physician
glucose, which throughout the testing period
enters the blood ▪ It is contraindicated with those with history of
more rapidly seizures or cardiac or cerebrovascular disease
• (4) Blood glucose
level rises to normal (about 90 mg/100mL)
CATAPANG. CAGAS
Clinical Significance of Growth Hormone Excess Laboratory Methods for Prolactin

• Gigantism in children • Screening: 3 specimens should be obtained at 20-30-
• Acromegaly in adults minute intervals
o Can be due to growth hormone • Immunometric assay
overproduction o Each sample could either be analyzed separately
o Screening test for clinically and the results are averaged
suspected acromegaly: o Or, alternatively, an equal aliquot from each sample
▪ Randomly collected IGF-1 could also be pulled into a single specimen for
(Insulin-like growth factor 1) analysis
• Dx: confirmed by failure of GH Functions of Prolactin

suppression following an oral glucose
load • Acts on breast tissue, in setting of estrogen priming
• Stimulates lactation
• Tests Performed for GH Excess: • Inhibits GnRH
o IGF-1 Level o Decrease LH and FSH
▪ Screening
▪ A normal IgF-1 with respect to the appropriate Clinical Significance of Prolactin
age and gender matched reference ranges rules
out the diagnosis of acromegaly • Deficiency
o Oral Glucose Tolerance Test o Pituitary necrosis or infraction
▪ Confirmatory • Excess
▪ (1) 75 g glucose o Inhibition of GnRH
▪ (2) Obtain blood samples at baseline
▪ (3) Every 30 minutes over the next 2 hours for ▪ A decrease in estrogen and progesterone failure
glucose and GH of ovarian follicular maturation (ovulation)
▪ Normal response: suppression of GH (˂1 ng/ml ▪ Sexual dysfunction and infertility in both men
or 1 ug/L) and women
▪ If the GH fails to drop below 1, the patient is ▪ Women (♀) – Luteal phase abnormalities,
diagnosed as having acromegaly Oligomenorrhea or amenorrhea
Prolactin  Luteal phase - when egg exits the ovary and
uterus thickens
• Produced by lactotrophs in the anterior pituitary gland
▪ Men (♂) – Hypoandrogenemia, Decreased
• Initiation and maintenance of lactation
libido, Impotence, Galactorrhea
• Inhibited by dopamine (hypothalamus)
• Circadian secretion: o Galactorrhea
o highest (zenith) levels attained during sleep and a
nadir occurring between 10 am and noon
• Pulsatile secretion:
o The amplitude and frequency of which not only vary
throughout the day but are influenced by a variety of
physiologic stimuli
o Stimuli:
▪ Stress, sleep postprandially, pain
▪ Coitus, pregnancy, nipple stimulation or nursing
• Serum half-life: 26-47 minutes
Table 2. Hormones of the Anterior Pituitary
Hypothalamic Releasing Hormone Hypothalamic Inhibiting Hormone

Hormone Secreted by
(Stimulates Secretion) (Suppresses Secretion)
Human growth hormone (hGH) also known Growth hormone-releasing hormone Growth hormone-inhibiting hormone
Somatotrophs
as somatotropin (GHRH), also known as somatocrinin (GHIH), also known as somatostatin
Thyroid-stimulating hormone (TSH), also Growth hormone-inhibiting hormone
Thyrotrophs Thyrotroponin-releasing hormone (TRH)
known as thyrotropin (GHIH)
Follicle-stimulating hormone (FSH) Gonadotrophs Gonadotropin-releasing hormone (GnRH) ----
Luteinizing hormone (LH) Gonadotrophs Gonadotropin-releasing hormone (GnRH) ----
CATAPANG. CAGAS
Prolactin-inhibiting hormone (PIH),

Prolactin (PRL) Lactotrophs Prolactin-releasing hormone (PRH)*
which is dopamine
Adrenocorticotropic hormone (ACTH), also
Corticotrophs Corticotropin-releasing hormone (CRH) ----
known as corticotropin
Melanoctyte-stimulating hormone (MSH) Corticotrophs Corticotropin-releasing hormone (CRH) Dopamine
*Thought to exist, but exact nature is uncertain
HORMONES OF POSTERIOR PITUITARY GLAND Oxytocin
• Stimulated by stretching of the cervix and vagina during

Table 3. Summary of Posterior Pituitary Hormones parturition (Fergusson reflex)
o Fergusson reflex (positive feedback)
Hormone and Principal
Control of Secretions ▪ A neuroendocrine reflex comprising the self-
Target Tissues Actions
sustaining cycle of the uterine contractions
Stimulates ▪ Initiated by pressure at the cervix or vaginal wall
contraction of
Oxytocin (OT)
smooth muscle • Uterine contractions in labor both by direct action on:
Neurosecretory cells of cells of uterus
hypothalamus secrete during childbirth o Myometrium
OT in response to o Stimulation of prostaglandin secretion by the
uterine and stimulation Stimulates
contraction of
decidua
of nipples
myoepithelial • Stimulates the myoepithelial cells surrounding the
cells in mammary
mammary glands and lactiferous ducts to contract,
glands to cause
milk ejections resulting in milk ejection (“letdown”)
Antidiuretic Neurosecretory cells of Conserves body o Suckling of the nipple
hormone (ADH) hypothalamus secrete water by o Anticipation of nursing
or vasopressin ADH in response to decreasing urine
elevated blood osmotic volume
o Hearing a baby cry
pressure, dehydration,
Decrease water NOTE: Stressful situations, can inhibit secretion, resulting in
loss of blood volume,
loss through decreased milk ejections.
pain or stress; inhibition
perspiration;
of ADH secretion include
raises blood
low blood osmotic
pressure by Control of Oxytocin Secretion
pressure, high blood
constricting
volume and alcohol
arterioles
• Small oligopeptides (9
amino acid residues)
• Synthesized in the nerve
cell bodies within the
hypothalamus
• Transported along axons to
the nerve terminals within
the posterior pituitary gland
• Stored in secretory
vesicles
• (1) Stretch of the uterus and the uterine cervix of the

breasts’ nipples increases action potentials in axons of
oxytocin-secreting neurons.
• (2) Action potentials are conducted by sensory neurons
from the uterus and breast to the spinal cord and up
ascending tracts to the hypothalamus.
• (3) Action potentials are conducted by axons of
oxytocin-secreting neurons in the
hypothalamohypophysial tract to the posterior pituitary,
where they increase oxytocin secretion.
• (4) Oxytocin enters the circulation, increasing
contractions of the uterus and milk ejection from the
lactating breast.
CATAPANG. CAGAS
Clinical Significance of Oxytoxin • Secretion is much more sensitive to changes in

osmolality than to changes in intravascular volume.
• Excess or deficiency pathologic conditions are rare and o 1% or 2% increase in osmolality will cause an
are limited to case reports. automatic rise in ADH secretion.
• Clinical demand for measurement is extremely rare.
• Half-life of 3-5 minutes • Stimuli
o Rapid degradation by oxytocinase o Nausea, Cytokine, IL-6, Hypoglycemia, Hypercarbia,
Nicotine
AVP or ADH • Basal plasma vasopressin
• Synthesized within the paraventricular and supraoptic o 0.5 – 2 pg/μL
nuclei of the hypothalamus. • In concert with ANP, thirst, and RAA axis, maintenance
• Maintains osmotic homeostasis by regulating the water of:
balance.
o Blood pressure
o Stimulating the V2 receptors on principal epithelial o Volume
cells that line the cortical collecting ducts of the o Tonicity
kidney.
▪ The ability of extracellular solution to move water
▪ V – Vasopressin by osmosis.
o Induces the increase in the production of cAMP, • Plasma Osmolality
which, in turn, causes the water channels
(aquaporins) to fuse with the apical membrane of o 280-295 mOsm/kg
the principal cells → increasing water permeability
• Plasma Sodium
and reabsorption.
o 135-145 mmol/L
• Act as a potent pressor by causing vasoconstriction
(V1 receptor) and stimulates the production of clotting • Freezing-point depression osmometry or the plasma
factor VIII. vapor pressure
o Clotting Factor VIII is the antihemophilic factor. • Indirect: plasma sodium, BUN, and glucose
• Water channels:
• Modulated by changes in serum osmolality and by
alterations in intravascular volume.
Serum osmolality
• > 295 mOsm/kg – maximal stimulation

• < 284 mOsm/kg – supressed
• Thirst receptors
o Within the hypothalamus
Clinical Significance of AVP (ADH)
• Osmoreceptors
(1) Diabetes insipidus
o Stimulate ADH
o Anterior hypothalamus • Characterized by the passage of large volumes of
diluted urine
Intravascular Volume
• More than 2.5L per day in the phase of an inappropriate
• Detected by baroreceptors plasma osmolality
o Low-pressure volume receptors • Central/Neurogenic
▪ Right atrium and pulmonary venous system o Deficiency of ADH caused by failure of the
hypothalamus to produce the hormone
o High-pressure arterial receptors
▪ Pituitary tumor, traumatic injury, autoimmune,
▪ Carotid sinus and aortic arch idiopathic
o These receptors are normally under chronic or o ADH is low, and the kidney rapidly acts to conserve
sustained inhibition water in response to exogenous ADH administration
▪ A drop in • Nephrogenic
intravascular
volume removes o Failure of the kidney to respond to ADH
the inhibition and ▪ Renal failure, drugs, congenital defects in the
results in a rise receptors in the DCT
inner ADH →
increased water o Associated with normal or increase levels of ADH
reabsorption and administration of additional ADH; it has little or
no effects on the renal absorption.
CATAPANG. CAGAS
• Diabetes Mellitus Vs. Diabetes Insipidus Syndrome of Inappropriate ADH (SIADH)

o DM = Insulin Deficiency
o DI = ADH Deficiency • Characterized by hypersecretion of ADH resulting to
excess water reabsorption, dilutional effect on plasma
Laboratory Tests components and hypoosmolality
• Characterized by euvolemic hyponatremia and
• Water deprivation test associated with hyperosmolar urine
• Expected lab results:
o Preferred diagnostic test
o Response to either endogenous or exogenous AVP o Sodium level
or ADH o Urine Osmolality
o Patients with mild polyuria may be instructed with o Plasma Osmolality
fluid intake for 10 PM onwards
o For severe polyuria, of which produces 8 to 10L a
day, water deprivation must start early in the
morning with close observation.
Table 4. Tests in Differential Diagnosis of Disorder of Water Homeostasis
BASELINE AFTER 12-HOUR FLUID RESTRICTION Urine

Osmolality
Disorder Serum Na+ Serum Na+
Urine Na+ and Urine Na+ and Post-AVP
and Serum ADH and Serum ADH Challenge
Osmolality Osmolality
Osmolality Osmolality
Normal control N N N N High High Same
SIADH Low N-High High Low-N High High -
Neurogenic DI N-High Low Low High Low-N Low Increased
Nephrogenic DI N-High Low N-High High Low-N High Same
Psychogenic DI Low-N Low Low N N-High N-High Same
CATAPANG. CAGAS
[TRANS] LABORATORY UNIT 10: THYROID GLAND
THYROID GLAND
Anatomy and Development • Shaped like a butterfly
o Resembles like “H”
Thyroid Anatomy
letter
• Two lobes
o Resting on each side of
the trachea
• Isthmus
o Bridges lobes by a band
of thyroid tissue
• Positioned in the lower anterior neck o located anterior to the
o level of C5 and T1 vertebrae trachea
o Neck cross section
▪ thyroid gland is located at the lower anterior • Posterior to the thyroid
neck of the human gland are the following:
o PTH gland (parathyroid
gland)
▪ To regulate serum
calcium levels
o Recurrent laryngeal
nerves
▪ Innervates the vocal
cord
• Parathyroid Gland and Laryngeal nerves are surgically
significant
• Fully developed thyroid gland
o Care is needed to avoid injury that could lead
o Normal: 15-20 g hypocalcemia or permanent hoarse voice
o Disease states: several hundred grams
• Histology of the Thyroid Gland (cross-section)
• Fibrous capsule
o Covers Thyroid gland
for protection
• Pyramidal lobe
o A remnant of the
thyroglossal duct
o Normal component of
thyroid gland
o 10-30% of the population Structures of the Thyroid Gland
have which is considered
• Thyroid follicle (acinus)
as a rare third lobe
o Basic or secretory unit of thyroid gland
o Follicle – functional unit of thyroid gland
BAGAMANO. DUMAGONOT. LAMOSTE. PAÑA. SARGADO. VILLAREAL. CORTEZ. CAGAS BSMLS 3 1

LABORATORY UNIT 10: THYROID GLAND
• Follicular cells • Summary of the Anatomy of Thyroid Gland

o Surrounds each follicle
o Encloses colloid
Thyroid Development
• Fetal Thyroid
• Colloid – amorphous material o develops from an outpouching of the foregut at the
base of the tongue
o Mainly made up of thyroglobulin
o Outside the follicular cells: basal lamina • First 4-8 weeks of gestation
• Parafollicular cells (C cells) o Fetal thyroid originates as an
endodermoproliferation at the tip of the foramen
o Secretes calcitonin
cecum of the developing tongue
o involved in calcium homeostasis
▪ Migrates to its final location over the thyroid
cartilage
o Migrates inferiorly to
its final site: anterior
and inferior to the
larynx
▪ The arrow pointing
downwards:
foramen cecum
▪ Thyroid
diverticulum is
going down
• Early 5th week of

gestation
o The thyroid remains
connected to the
foramen cecum by the
thyroglossal duct
• Late 5th week of

gestation
o Thyroglossal duct
starts breaking down
compared to the early
5th week
• 7th to 8th week of

gestation
o The gland reaches its
final site on the 7th
• Between the follicles are the capillaries and fibroblast week
which are not visible in the picture o Bishops (ref book) –
around 8th week

• 11th week of gestation

o Thyroid gland begins to produce measurable
amounts of thyroid hormone
▪ Iodine - essential component of thyroid hormone
 Main source: dietary intake
• Thyroid hormone – critical to fetal neurological
development
o Severe iodine deficiency
▪ Caused by neither the fetus nor the mother
produces insufficient thyroid hormone
 Leads to hypothyroidism
 Hypothyroidism leads cretinism and mental
retardation
• Congenital Hypothyroidism
o Inadequate thyroid hormone production of newborn
infants
o Can also occur because of anatomic defect in the
gland or inborn error of thyroid metabolism
▪ Screening test is performed in newborns to
detect these neonatal and endocrine disorder
CHECKPOINT
o (1) Hypothalamus will sense the low blood levels of
• The thyroid gland is located at the _______ T3 (triiodothyronine) and T4 (thyroxine), and these
of the neck. will stimulate the synthesis of TRH
o Lower anterior of the neck
▪ Thyrotropin-releasing hormone (TRH)
• What is the functional unit of the thyroid  Synthesized by the supraoptic and
gland? supraventricular nuclei of the hypothalamus
o Thyroid follicle or the follicle  Stored in the median eminence of
hypothalamus
• What mainly composes the colloid?
o thyroglobulin  Positive feedback
o (2) TRH will stimulate the anterior pituitary gland
• ____ is an essential component of the thyroid to synthesize and release TSH by the thyrotrophs.
hormone
o Iodine  Thyrotrophs – cells of pituitary gland that
produce TSH
• At what week does the thyroid gland begin to  Positive feedback
produce measurable amounts of thyroid ▪ TSH is released in the blood stream, and it will
hormone? go in the thyroid gland
o 11th week of gestation
 It stimulate the thyroid follicular cells in the
thyroid gland and produce the T3 and T4,
which can also be release in the blood stream
Physiology
o (3a) T3 and T4 are released to the bloodstream
• Control of Thyroid Hormone Secretion o (3b) T3 and T4 are circulated first in the liver and
• Actions of Thyroid Hormones kidney
▪ T3 and T4 will be increased in the blood stream.
Control of Thyroid Hormone Secretion o (4) Increased T3 and T4 inhibits the TSH release
directly to the pituitary gland and indirectly decrease
• Hypothalamic–Pituitary–Thyroid Axis
TRH release through the hypothalamus.
o Central to the regulation of thyroid hormone
production and is governed by the feedback  Negative feedback mechanism
mechanism.

Actions of Thyroid Hormones o Major Binding Proteins:

▪ Thyroxine-binding protein (70%)
Overview of the Secreted Thyroid Hormones
 T3 bound on 10-fold reduced affinity than T4
▪ Thyroxine-binding prealbumin (20%)
▪ Albumin (10%)
o Protein Concentration
▪ Affects circulating quantities of circulating thyroid
hormone
 Thus, protein and thyroid hormone complex is
significant
▪ E.g. Pregnant patient
 An estrogen level is high during pregnancy, it
increases the production of thyroxine binding
in the liver having more protein bound
hormone → it increases the thyroid hormone
level.
• Abnormal Binding Protein Levels
o Requires measurement of free thyroid hormone
• (1) Release of Thyroid hormone into the bloodstream
▪ Although in an individual usually have a normal
o Thyroid hormone secreted from the thyroid gland thyroid hormone level, there are instances that
goes into the blood stream, it circulates in the body free thyroid hormone is measured.
until it reaches the target cell. ▪ This is due to abnormal binding protein level.
▪ Thyroxine (T4) and Triiodothyronine (T3)  May alter the amount of thyroid hormone in
the circulation
• (2) Travels into the target cell entering its cytoplasm
o Determine the true amount of thyroid hormone in the
o When it reaches the target cell, it diffuses into the
circulation
interstitial fluid and through the lipid bilayer of the
cell membrane Thyroid Hormones in the Cytoplasm
▪ lipid-soluble hormone
• (3) Production of mRNA
o Inside the cell, it produces mRNA and proteins
• (4) Production of Proteins
Thyroid Hormones in the Blood • (1) Right after it enters the cytoplasm, thyroxine
(inactive form) is deiodinated into triiodothyronine
• Free Thyroid Hormone (active form) that will be able to cross the cell
membrane
o Can travel across the membrane
o Made up of: o 5’-monodeiodonation enzyme play its crucial role
▪ Thyroxine (0.03%)
▪ Triiodothyronine (0.3%)
o The body produce 110 nmol of T4 and 10 nmol of
T3 produced daily.
▪ Thyroxine
 100% produced by the thyroid (thyroidal in
origin)
▪ Triiodothyronine
• (2) Inside the cell, the thyroid hormone binds with the
 20% produced by the thyroid (thyroidal in
thyroid receptor in the cytosol, where it will synthesize
origin)
protein.
 80% produced by enzymatic reaction and • (3) The DNA is transcribed, producing messenger RNA.
nonthyroidal tissues, acted upon by enzyme
5’-monodeiodination o It releases in the nucleus and enters the cytosol,
where it will synthesize protein.
• Protein Bound Hormone
• (4) The newly produced proteins will then carry out the
o Thyroid hormone bound to protein cell activity causing the hormone effects.

• Excess Thyroxine (T4)

CHECKPOINT
o Release of too much thyroxine can cause
Thyrotoxicosis
• What is synthesized by the supraoptic and
supraventricular nuclei of the hypothalamus? ▪ Can be correlated to Hyperthyroidism
o TRH
 Over activity of the thyroid gland produces too
• TRH stimulating the pituitary gland to much thyroxine
produce TSH is what kind of feedback ▪ Thyrotoxicosis is recognized by a goiter
mechanism?
 Goiter – a swelling of the neck due to
o Positive feedback
enlargement of the thyroid gland
• Some T3 and T4 are circulated in the kidney o Symptoms
and liver, what hormone is mostly converted
in these organs? ▪ Intense heat sensitivity
o T4 ▪ Weight loss
▪ Increase appetite
• What are the three (3) major binding protein? ▪ Increase bowel movement
o Thyroxine-binding protein, Thyroxine- ▪ Irregular menstrual cycle
binding pre-albumin, and Albumin ▪ Rapid or irregular heartbeat
▪ Palpitations
• Thyroxine is deiodinated by enzyme. ▪ Tiredness
o 5’ monodeiodonation enzyme ▪ Irritability
▪ Tremor
▪ Retraction of the eyelid
Hormones Produced
• Deficient Thyroxine (T4)
Hormones Produced and its Properties o Little production of thyroxine can cause
Hypothyroidism.
(1) Thyroxine / T4 / Tetraiodothyronine
▪ Usually caused by autoimmune diseases, poor
• Major hormone discharged into the bloodstream iodine intake, or caused by the use of certain
drugs, and sometimes the cause is unknown.
o Necessary for digestion, heart and muscle function,
brain development, and bone maintenance o Causes mental disability and stunted growth if not
treated before birth or during childhood
• Considered to be inactive form o Signs and Symptoms of Hypothyroidism:
o Organs such as liver and kidneys convert it to its ▪ Fatigue
active form, triiodothyronine ▪ Intolerance to cold temperatures
• Thyroid hormones serve a critical function in body’s ▪ Low heart rate
metabolic rate regulation or the rate of energy that our ▪ Weight gain
body is using. ▪ Reduced appetite
▪ Impaired memory
• Control of Thyroxine ▪ Sadness
▪ Muscle stiffness
o Controlled by the feedback loop system which
▪ Reduced fertility
involves:
(2) Triiodothyronine / T3
▪ Hypothalamus
 Secreted Thyrotrophin Releasing Hormone • Thyroid gland secretes 3 hormones: T3, T4, calcitonin
(TRH) which stimulates the pituitary gland o Thyroid releases large amounts of T4 hormone
▪ Pituitary gland (inactive)
 Stimulated by TRH to secrete Thyroid ▪ Needs to be converted into active form, T3, to be
Stimulating Hormone (TSH) which stimulates used
the thyroid gland.
• Deiodination pathway: converts T4 → T3
▪ Thyroid gland
o (1) Thyroid will secrete T4
 produces the thyroid hormones Thyroxine and o (2) T4 will be acted by the deiodinase enzyme which
Triiodothyronine in response to TSH controls organs, such as the liver, to convert it into
o Governed by a feedback loop T3
o (3) T3 and reverse T3 will now be produced
▪ Prevents the release of both thyrotrophin
releasing hormone and thyrotropin stimulating • Reverse T3 (RT3)
hormone when thyroid hormone levels rise
o Inactive form of thyroid hormone, just like T4
o This system enables the body to maintain a steady o Functions to block T3 receptor sites to maintain
quantity of thyroid hormone. regulation of thyroid activity

• T3 or Triiodothyronine • Function: Regulates blood calcium levels along with

Parathyroid Hormone
o Found in most cells
o Function: o Decrease blood calcium levels
o Increase bone calcium levels
▪ Regulates body metabolism, heat and energy
production, and heart rate. ▪ Osteoclast is inhibited
▪ Osteoblast is activated
o Two Forms:
 Osteoblasts takes the calcium from the blood
▪ Bound T3 and deposits it to the bone
 Attaches to protein to help circulate hormone  Makes the calcium levels in our blood
in the body decrease and the calcium levels in our bone
▪ Free T3 increase
 Moves freely in the body and does not attach Functions of the Thyroid Hormones
to any proteins
• T3 is controlled by feedback loop of the hypothalamus, T3 and T4
pituitary gland, and thyroid gland • Control basal metabolic rate and calorigenesis
o E.g., Thyroid secretes a large amount of T3 o Control basal metabolic rate
▪ (1) T3 signals the hypothalamus to prevent the ▪ Increases cellular metabolism
release of Thyrotropin-releasing hormone (TRH)
and Thyroid-stimulating hormone (TSH) in the o Thyroid calorigenesis
pituitary gland.
▪ Mediated by the simulation of active sodium and
 Maintains the regular amount of T3 in the potassium activated adenosine triphosphatase
body circulation activity
▪ (2) Both TRH and TSH stimulates the thyroid • Stimulate synthesis of proteins and carbohydrate
glands to produce T3 metabolism
 Needs to be regulated o Via cardiomyocyte
 Little or too much T3 results to health
▪ Thyroid hormones activate the ATP and mTOR-
problems and conditions
p17(S6K) pathways
• T4 and T3 have the same functions
• Enhancement of mitochondrial metabolism
o Both are controlled by a feedback loop
o Via stimulating the mitochondrial replication and
o Both also have iodine as its major component
energy production within the mitochondria
o They differ in coupling of diiodotyrosine (DIT) or
monoiodotyrosine (MIT) ▪ By switching metabolism from glycolytic pathway
to more efficient oxidative phosphorylation
▪ Coupling happens when iodinated tyrosines are
cleaved • Stimulation of adrenergic energy & enhancing the
 Forms DIT and MIT sensitivity of adrenergic receptors to
catecholamines
▪ T4 formed by 2 DIT
▪ T3 formed by 1 DIT and 1 MIT o Increasing the heartrate and myocardial contractility
and regulation of the amount of cardiac beta-
(3) Calcitonin adrenergic receptors
• Synthesized and secreted by specialized C cells • Increase use of glucose and fatty acids for ATP
(parafollicular cells) production and enhance degradation of cholesterol
o Parafollicular cells are seen in spaces between through excretion
thyroid follicles o ATP production
• Controlled by negative feedback system ▪ increased by a thyroid hormone-induced
o Acts when there is a humoral stimulus increase in the adenine nucleotide translocation
across the mitochondrial membrane
▪ Humoral stimuli are due to ions, nutrients and
o Helps in liver process by removing excess
different chemicals
cholesterol from the body
 Stimulus: calcium levels
• Increase lipolysis
▪ E.g., High blood calcium levels (Hypercalcemia)
o T3 regulates several genes involved in lipid
 Large amount of calcium would enter through
mobilization, storage and in thermogenesis
either receptor or channels of parafollicular
cells → eventually stimulates the parafollicular ▪ Targets the white and brown adipose tissues
cells to secrete calcitonin → inhibit the activity which regulates the genes that are crucial for
of osteoclasts → ↓ blood calcium levels their functions (E.g., lipolysis)

• Accelerate body growth and sexual maturation ▪ A large glycoprotein that is produced in the
rough endoplasmic reticulum, modified in the
o Thyroid hormones
Golgi complex, and packaged into secretory
▪ Direct effect on osteoblasts by interaction with vesicles.
thyroid hormone receptors o The vesicles then undergo exocytosis, which
▪ Indirect effect by interacting with growth releases TGB into the lumen of the follicle.
hormone synthesis and the action of insulin-like
growth factors • (3) Oxidation of iodide
• Contribute to the development of the nervous o Some of the amino acids in TGB are tyrosines that
system will become iodinated.
o However, negatively charged iodide ions cannot
o Glial cells, astrocytes, and the main target cells:
bind to tyrosine until they undergo oxidation
neurons and maturating oligodendrocytes
(removal of electrons) to iodine: 2 I → I2.
▪ T4 is converted into its active hormone T3
▪ As the iodide ions are being oxidized, they pass
▪ T3 will act to the nuclear receptors to control the
through the membrane into the lumen of the
expression of genes involved in:
follicle.
 Myelination, Cell differentiation, Migration,
Signaling • (4) Iodination of tyrosine
Calcitonin o As iodine molecules (I2) form, they react with

tyrosines that are part of thyroglobulin molecules.
• Lowers blood levels of Ca2+ and HPO42- through two o Binding of:
mechanisms: ▪ one iodine atom → yields monoiodotyrosine
o (1) Inhibiting activity of osteoclasts (T1)
▪ second iodination → produces diiodotyrosine
▪ Osteoclasts (T2).
 cells responsible for breaking down of bones
o Colloid
▪ When the bone is broken down, the calcium
contained in the bone is released to the blood ▪ The TGB with attached iodine atoms
stream. ▪ A sticky material that accumulates and is stored
▪ If osteoclasts are inhibited, the amount of in the lumen of the thyroid follicle
calcium released also reduces • (5) Coupling of T1 and T2
o (2) Decreasing resorption of calcium and o During the last step in the synthesis of thyroid
phosphate in kidneys hormone,
▪ Reduces calcium blood levels ▪ Two T2 molecules join to form T4
▪ One T1 and one T2 join to form T3
Synthesis of Thyroid Hormones • (6) Pinocytosis and digestion of colloid
• Thyroid hormones o Droplets of colloid re-enter follicular cells by
o Synthesized by attaching iodine atoms to the amino pinocytosis and merge with lysosomes.
acid tyrosine. ▪ Digestive enzymes in the lysosomes break down
o 2 primary hormones: Thyroxine (T4) and TGB, cleaving off molecules of T3 and T4.
Triiodothyronine (T3)
• (7) Secretion of thyroid hormones
▪ Produced by follicular cells
o Because T3 and T4 are lipid-soluble, they diffuse
• Thyroid gland through the plasma membrane into interstitial fluid
o Only endocrine gland that stores its secretory and then into the blood.
product in large quantities, normally about a 100-day o T4 normally is secreted in greater quantity than T3,
supply. but T3 is several times more potent.
▪ Moreover, after T4 enters a body cell, most of it
Formation, Storage & Release is converted to T3 by removal of one iodine.
• (1) Iodide trapping • (8) Transport in the blood
o Thyroid follicular cells trap iodide ions (I) by actively o More than 99% of both the T3 and the T4 combine
transporting them from the blood into the cytosol. with transport proteins in the blood, mainly
thyroxine-binding globulin (TBG).
▪ As a result, the thyroid gland normally contains
most of the iodide in the body.
• (2) Synthesis of thyroglobulin
o While the follicular cells are trapping Iodide ions,
they are also synthesizing thyroglobulin (TGB)

(1) Hyperthyroidism
• Signs and Symptoms

o Heat intolerance
▪ Feeling of being overheated
▪ One of the functions of thyroid hormone is to
increase heat production, since the hormones
produced are excessive, the person feels
overheated
o Tachycardia
▪ The medical term for heart rate over 100 beats a
minute
o Weight loss despite normal or increased appetite
o Hyperdefecation
o Weakness
o Emotional lability
o Tremor
• Diseases associated with Hyperthyroidism
o Graves’ Disease – an autoimmune disease in
which antibodies are produced that activate the TSH
receptor.
▪ Most common disease associated with
hyperthyroidism
▪ Features include thyrotoxicosis
 The latter form of thyrotoxicosis is called
hyperthyroidism
▪ Can be a result of:
 Excessive thyroid hormone ingestion
 Leakage of stored thyroid hormone in the
thyroid follicles
 excessive thyroid gland production
Clinical Significance of the Thyroid Gland ▪ Other features also include:
 Goiter
Effects of Thyroid Hormones
 Ophthalmopathy – changes associated with
• Tissue Growth and Brain Maturation inflammation and infiltration of periorbital
• Increased Heat Production tissue
• Increased Oxygen Consumption  Dermopathy – skin changes in the lower
• Increased B-adrenergic Receptors extremities that have an orange, pale texture
o Are receptors that regulate many aspects of airway o Other diseases that lead to hyperthyroidism include
function, including mast cell mediator release and toxic adenoma and rarely, thyroid carcinoma.
plasma exudation
o If the thyroid gland is producing excessive or Table 1. Results of Thyroid Function Tests
deficient hormones, thyroid diseases will occur
Thyroid Diseases: Classified based on Functionality Disorders TSH T3 T4 FT4
• Hyperthyroidism
Graves’ disease ↓ ↑ ↑ ↑
o When the thyroid gland makes too many hormones
Neonatal Graves’
↓ ↑ ↑ ↑
• Hypothyroidism Disease
o When the thyroid gland does not make enough Subclinical

↓ N N N
hormones. hyperthyroidism
• Euthyroidism
• Patients with hyperthyroidism
o When the thyroid gland is functioning normally.
o Patients have various disease that do not directly o have suppressed TSH values
involve the thyroid gland. o with the exception of those few individuals who
have secondary hyperthyroidism caused by TSH-

producing pituitary tumors, or other rare disorders Table 3. Results of Thyroid Function Tests
such as pituitary resistance to thyroid hormones.
• Subclinical hyperthyroidism TSH FT4 T3
o refers to a condition where TSH is low (<0.1 μIU/mL)
Sick Euthyroid Syndrome N N or ↑ ↓
with levels of T4 and T3 within the reference values,
and with no signs or symptoms of hyperthyroidism
(2) Hypothyroidism • Other diseases that lead to euthyroidism include goiter,
thyroid adenoma and thyroid carcinoma.
• Signs and Symptoms
o Cool and/or dry skin Laboratory Test
o Bradycardia
o Weight gain • In clinical practice, thyroid function tests are routinely
o Increased sleeping measured to diagnose disorders of the thyroid.
o Congestive heart failure • Almost all laboratory tests or thyroid functions are
o Myopathy commercially available in either kit form or on
o Severe hypothyroidism - Coma may rarely occur automated immunoassay instruments.
• Three types: primary, secondary, tertiary Thyroid Stimulating Hormone (TSH)
o Primary – occurs where the production of T4 and • Thyrotropin
T3 is impaired. • Best thyroid function screening test
o Secondary – result of pituitary or hypothalamic
disease o Initial test for suspected thyroid disease
o Tertiary – occurs as a result of hypothalamic
• Immunoassay
dysfunction
o Method of choice for the measurement of serum
TSH in the clinical laboratory.
Table 2. Results of Thyroid Function Tests
• Used to follow patients on thyroid hormone therapy
• Used in conjunction with thyroxine to manage patients
Disorders TSH FT4 T3 with graves’ disease
Primary hypothyroidism ↑ ↓ ↓ Serum T4 and T3

Cretinism (Congenital
hypothyroidism)
↑ ↓ ↓ • Serum Total Thyroxine (T4)
o Immunoassays and instrument-based methods
Hashimoto’s thyroiditis /
↑ N or ↓ No↓
hypothyroidism ▪ used to measure thyroxine in biological fluids,
predominantly serum or plasma.
• TSH is low in patients with secondary hypothyroidism o Used to make a diagnosis of underactive or
• Cretinism (congenital hypothyroidism) overactive thyroid when Thyroid Stimulating
Hormone is abnormal
o Most common disorder in hypothyroidism o Used for monitoring patients with graves’ disease
o Caused by the in deficiency of the thyroid hormone and for newborn screening test for hypothyroidism
resulting to a mental retardation. o Fairly accurate in patients with no protein
abnormalities and not pregnant
▪ An elevated TSH is the most common and
sensitive test in diagnosing cretinism. • Serum Total Triiodothyronine (T3)
• Hashimoto’s thyroiditis/hypothyroidism o Immunoassays are the techniques of choice to
o Primary hypothyroidism measure triiodothyronine
o Thyroid gland is attack by a cell-mediated o It is used to diagnose hyperthyroidism when the
autoimmune response thyroid-stimulating hormone is low and thyroxine is
still normal
• Other conditions that lead to hypothyroidism include
myxedoma coma. • Measured by immunoassay
(3) Euthyroidism
Free Thyroxine (FT4)
• Signs and Symptoms (similar to hypothyroidism)
• Free thyroxine is the metabolically active thyroid
o Fatigue and weakness hormone that is NOT bound to protein
o Bradycardia • Reliable methods for measuring free thyroxine in serum
o Weight gain include those that separate free and bound hormone
o Hypotension fractions by Direct equilibrium dialysis or
o Cold intolerance Ultrafiltration
o Joint and muscle pain

o After separation, the free concentration is measured • Serum TBG level test is a blood test sometimes referred
by a sensitive analytical method, such as to as Thyroxine-Binding Globulin Test
immunoassay or mass spectrometry.
o It measures the amount of the TBG protein in the
• Should be ordered when TSH is abnormal to determine blood
thyroid hyperfunction or hypofunction o Free T3 and T4 are the thyroid hormones that is not
bound to the TBG
Reverse Triiodothyronine (rT3) • TBG is measured via:
• A major metabolite of thyroxine, produced by 5- o Heterogenous method
deiodination of thyroxine.
▪ Measures the competition between endogenous
o High in patients with non-thyroidal illness TBG and labeled TBG for binding with a
mobilized anti-TBG antibody
• Serum rt3 is increased in:
o Solid Phase Second Antibody
o healthy newborns
o patients with hyperthyroidism ▪ Conducted via chemiluminescence assay or the
o patients taking amiodarone and propranolol addition of luminol and hydrogen peroxide to the
bound conjugate
• Radioimmunoassays are the techniques of choice to
measure reverse triiodothyronine o Enhanced microparticle turbidimetry
▪ Inhibition of the cross-linking antigen
Thyroglobulin (Tg) microparticle complex by the endogenous
substance anti-TBG antibody
• Protein made by follicular cells of the thyroid gland
 decreasing the turbidity of the reaction
o Used by the thyroid gland to produce T3 and T4
Thyroid autoimmunity
• If increased, it is caused by variety of disorders such as
Graves’ disease, Hashimoto’s thyroiditis, or nodular • Most common auto-antibody:
goiter. o TPO antibodies
• Purpose of Tg test o TgAb
o To measure the amount of thyroglobulin in the blood o TSH receptor antibodies
o Routinely ordered to manage patients with the
• Thyroid Peroxidase antibodies (TPOAb)
history of papillary or follicular thyroid cancer – two
of the most common types of thyroid cancer. o Thyroid peroxidase is an enzyme that is crucial to
the production of thyroid hormones
• Testing may be performed for several reasons: o TPO antibodies may interfere with the action of this
o Extensive evaluation enzymes
o Monitoring recurrence
▪ Almost all of the patients with Hashimoto
o Prognosis
thyroiditis have high levels of TPOAb
• Competitive Immunoassay ▪ Needs further autoantibody test
o ELISA • Thyroglobulin antibodies (TgAb)
▪ Accuracy of the test depends on how strong the o Thyroglobulin is a protein by the thyroid gland
binding is between the thyroglobulin and ▪ TgAb may be present when thyroid is damage
antibody used.
▪ If the thyroglobulin body seems abnormal, the o TgAb are often measured in addition oof
ELISA test should be done again to ensure the thyroglobulin test after the patient completes
results are accurate. treatment for thyroid cancer
o TPOAb and TgAb are detected by:
• Other tests
▪ Indirect immunofluorescence
o Results of the test depend on which type of test was ▪ Agar gel diffusion precipitin technique
done when comparing test results ▪ Agglutinationn
o It is important that the test were the same type and ▪ RIA
done in the same laboratory ▪ Chemiluminescence-based immunometric
▪ Reference ranges of every laboratory varies assays
• TSH receptor antibodies (TRAb)
Thyroxine-Binding Globulin (TBG)
o Antibodies that bind the receptor on thyroid cells
• A protein produced by the liver normally activated by the Thyroid-stimulating
• Purpose: Bind to the thyroid hormones (T3 and T4) hormone
produced by the thyroid gland o Graves’ disease – Thyroid-stimulating
immunoglobulin (TSI) binds to the TSH receptor
o It can regulate metabolism and perform other and mimics the action of TSH
important functions

▪ This will cause a constant stimulation on the After review of her previous laboratory tests, it was
thyroid gland, releasing to much thyroid hormone found that her thyroid function tests, including TSH and free
→ abnormal metabolism in the bloodstream T4 levels, were previously normal on several occasions.
▪ Stimulation by the TSI can also cause abnormal
In a follow-up visit, the patient denied recent
growth of the thyroid gland pregnancy, iodine exposure, neck pain or fever, recent acute
o To measure: illness, and symptoms of thyrotoxicosis. In addition, she
denied receiving any new medication, specifically
▪ Autoantibodies are directed against several amiodarone or lithium.
thyroid and thyroid hormone antigens
What is the most appropriate next step?
Table 4. Reference Range of the Different Tests a) Thyroid scan and uptake and a neck ultrasound
b) TSH receptor antibodies
c) Initiate treatment for Graves’ disease
TESTS REFERENCE VALUES d) Stop all supplements and repeat thyroid function
tests after a week
- The patient has been drinking oral over-the-
Thyroid-stimulating Hormone 0.5 - 5.0 mIU/L
counter drugs for her hair loss, which has major
component of biotin. Biotin is widely used for
Serum T4 and T3 component of immunoassays and biotin-
• Serum Total Thyroxine 5-12.6 ug/dL streptavidin immunoassays. If consumed, it may
(T4) interfere with the common assays used to
• Serum Total 60-160 ug/dL measure the thyroid function tests, leading to
Triiodothyronine (T3) (0.9-2.46 nmol/L) falsely increase or decrease level of TSH.
- The supplement sometimes also causes
Free Thyroxine 0.9-2.3 ng/dL (12-30 pmol/L) biochemical profile that is indistinguishable with
Graves’ disease. In addition to suppress TSH and
elevated free T4, biotin can interfere with assays
Reverse Triiodothyronine 10-24 ng/dL (230-540 pmol/L)
used to identify TSH receptor antibodies, which is
a characteristic of Graves’ disease.
Thyroglobulin (Tg) 30 ng/mL (45 pmol/L) - Thyroid function tests return to normal within a
week if the patient will not continue taking the
TBG 13-39 ug/dL (150-360 nmol/L) supplements. Though some studies suggests that
this might normalize after 24-48 hrs.
- As biotin can mimic the biochemical pattern of
Thyroid Autoimmunity Graves’ disease with low TSH, increased free T4,
• TPO and positive TSH receptor antibodies, this may
• TgAb lead to unnecessary treatment, which could
• TSH receptor antibodies induce hypothyroidism. However, the biochemical
(TRAbs) abnormality secondary to biotin has no clinical
significance and is not associated with any
symptoms.
ADAPTIVE CASE STUDY
A 36-year-old woman with a past medical history of

fibromyalgia presented to her general physician for a regular
check-up. The patient reported that her mother was recently
diagnosed with hypothyroidism and requested that her
scheduled blood work include thyroid function tests. The
patient said she has been well; even her symptoms related
to fibromyalgia had been under control. Her only complaint
was some hair loss, but it had slightly improved since she
began using over-the-counter supplements 3 months before
her appointment.
In physical examination, the patient appeared to be

healthy: her vital signs were normal, there was no evidence
of thyroid eye disease, tremor, or tachycardia; and her
thyroid gland was not enlarged.
Blood work completed the same day revealed

normal complete blood count and liver and kidney function
tests, but thyroid-stimulating hormone (TSH) was low,
measuring 0.02 µIU/mL (reference range, 0.4-4.6 mIU/L).
Free thyroxine (T4) was measured at 24 pmol/L (reference
range, 10-19 pmol/L), and free triiodothyronine was
measured at 7.1 pmol/L (reference range, 3.5-6.5 pmol/L).

[TRANS] LABORATORY UNIT 11: ADRENAL CORTEX
ADRENAL CORTEX Adrenal (Suprarenal) Glands

• Nervous and endocrine system
o constantly trafficking
information, making • Paired, pyramid-shaped
demands, controlling organs atop the kidneys.
ever move but they • Located superiorly or above
have different ways the kidneys
of doing it. • Weighs 4 to 6 grams
Table 1. Nervous and Endocrine System

Cross Sections
Feature Nervous System Endocrine System
Electrical impulses Chemical impulses

Signals
(action potentials) (hormones)
Transmission by
Pathways Transported by blood
neurons
Speed of
Fast Slow
Information
Duration of
Short lived Shor or long lived
effect
Type of action Voluntary or
and response involuntary
Always involuntary • Structurally and functionally, two glands in one:
Localized (cells Often distant (many o Adrenal Cortex
Target
connected to neuron) cells can be affected) o Adrenal Medulla
▪ Formed from neural ectoderm, can be
• Nervous System considered a modified sympathetic ganglion.
▪ Functions as an atypical sympathetic ganglion
o Acts quickly in delivering messages ▪ Contributes to 10-20% of the total adrenal weight
o Spray nerve impulses to specific cells and organs
Adrenal Cortex
• Endocrine System
o Prefers slower and wider stream of data that lasts a • Synthesizes and releases
whole lot longer steroid hormones
o Secretes hormones or chemical impulses that (corticosteroids)
travels through circulation • Corticosteroids – are
steroid hormones that are
▪ That is why it has slower movement compare to produced in the adrenal
nervous system, the kind of lightning fast. cortex
o Endocrine system is scattered all over the body • Three functionally distinct
zonal layers:
▪ Unlike other organ systems that are nestled
together meaning combined in one location. o Zona Glomerulosa, Zona Fasciculata, Zona
▪ From brain, throat, kidneys and genitals Reticularis
o Not directly linked by nervous system, the two ▪ Different corticosteroids for each layer derived
interact by the effects of the hypothalamus on the from the common precursor cholesterol
pituitary gland
▪ Pituitary gland STERIOD HORMONE SYNTHESIS
 Known as master gland Types of Hormones
 Produces and secretes many hormones that
• Hormones can be categorized into three distinct groups
signals other glands like thyroid, parathyroid
according to their chemical composition of peptide,
and adrenal glands to make their own
steroid, and amino acid derivative
hormones
ANTOYAN. GABAESIN. LATONIO. MACADANGDANG. NARAGA. SARGADO. VILLAREAL. AQUINO. CATAPANG. CAGAS BSMLS 3 1
LABORATORY UNIT 11: ADRENAL CORTEX
Table 2. Types of Hormones
PEPTIDE STEROID AMINO ACID DERIVATIVE
Synthesised as prohormones
Synthesised in a series of reaction
Synthesis requiring further processing (e.g., Synthesised from the amino acid tyrosine
from cholesterol
cleavage) to activate
Stored in vesicles (regulatory Released immediately (constitutive Storage before release (storage mechanism
Storage
secretion) secretion) varies)
Most are polar and water soluble, Generally non-polar and require Some are polar (adrenaline), others must be
Solubility
can travel freely in the blood carrier proteins to travel in blood protein-bound
Bind receptors on cell membrane Adrenaline acts on membrane receptors, while
Bind to intracellular receptors to
Receptors transduce signal via the use of thyroid hormones act directly on nuclear
change gene expression directly
second messenger systems receptors
Often fast onset transient changes in Alterations in gene expression; Adrenaline functions like peptides, thyroid
Effects protein activity, through gene slower onset but longer duration hormones function in a similar manner to
expression changes can occur than peptide hormones steroids
Insulin, glucagon, prolactin, ACTH, Cortisol, aldosterone, estrogen,
Examples Adrenaline, thyroxin, triiodothyronine
gastrin parathyroid hormone progesterone, testosterone
• Steroid hormones are hydrophobic and lipophilic ▪ This free cholesterol released from the lipid
droplets can now be utilized for steroid hormone
o Requires a carrying proteins to travel in blood production
o Binds to intracellular receptors
o (3) Plasma lipoprotein-derived cholesteryl esters
• Why is Steroid Hormone Receptor Located Inside
the Cell? ▪ Like HDL or LDL
o Cell membrane are also lipophilic
o Steroid hormone can freely diffuse across the
plasma membrane of the cell
o They bind to receptors in either in the cytoplasm or
nucleus of the target cell to form an active receptor
hormone complex
• Synthesis of Steroid Hormones or Corticosteroids
Steroid Hormone o There are lots of regulators or stimulants to

influence adrenal cortex to secrete corticosteroids
▪ e.g., RAAS, plasma concentration NA/K, ACTH
regulator
o In response to long term stress, corticotropin-
releasing hormone (CRH) is secreted from the
hypothalamus → causing the release of ACTH in the
anterior pituitary gland
o ACTH stimulates the transport of free cholesterol
• Steroids are derivatives of cholesterol into the adrenal mitochondria and then initiating sear
• The cholesterol utilized for steroidogenesis is derived production
from a combination of sources: o Inside the adrenal mitochondria, cholesterol will be
o (1) Cholesterol can be made within the cell from converted into pregnenolone in the presence of
acetyl CoA (de novo synthesis) cholesterol side chain cleavage enzyme
o (2) The mobilization of cholesterol esters stored in • Synthesized and secreted on demand (not stored).
lipid droplets through cholesterol ester hydrolase
o The first and rate-limiting step in the synthesis of all
▪ The adrenal cortex contains lipid droplets that steroid hormones is conversion of cholesterol to
are reached in esterified cholesterol pregnenolone by the enzyme cholesterol
▪ The presence of cholesterol ester hydrolase desmolase
releases free cholesterol from the lipid droplets
Cholesterol Desmolase • Major function: Blood pressure and K homeostasis

o Regulates blood pressure and concentrations of
• Also known as cholesterol side minerals particularly sodium and potassium in the
chain cleavage extracellular fluid
• A cytochrome P450 (CYP450)
enzyme that removes 6 carbon Aldosterone
side chain from cholesterol.
• There are multiple regulators that • Most important mineralocorticoid
influence corticosteroid secretions • Maintains Na+ balance by
such as the stAR and ACTH reducing excretion of sodium
from the body.
o Both facilitates the transport of cholesterol towards
• Stimulates reabsorption of Na+
adrenal mitochondria
by the kidneys and K+ excretion
o Once inside the adrenal mitochondria, cholesterol
• Aldosterone secretion is
will be converted to pregnenolone by the enzyme
stimulated by:
cholesterol side chain cleavage
o Decreasing blood volume or pressure (renin-
• 6 carbon side chain angiotensin system) is the major stimulant.
o Once the cholesterol is o Low blood Na+
converted to pregnenolone, o Rising blood levels of K+
the 6 carbon side chain will o ACTH
be removed
o This is due to the action of The Four Mechanisms of Aldosterone Secretion
the cholesterol desmolase
• (1) Renin-angiotensin Mechanism
enzyme
o Main regulator
o Occurs when kidneys release renin (a proteolytic
enzyme) and secreted directly into the circulation
o In the circulation, renin cleaves angiotensin I from
angiotensinogen, which is converted into
angiotensin II with the presence of angiotensin
converting enzyme that in turn stimulates
aldosterone release
▪ Angiotensin II acts as a powerful
• Newly synthesized steroid hormones (corticosteroids) vasoconstrictor and stimulates aldosterone
are rapidly secreted from the cell. release
o Following secretion, all steroids bind to some extent

to plasma proteins: CBG and Albumin
THREE REGIONS OF THE ADRENAL CORTEX
Table 3. Three Distinct Layers of the Adrenal Cortex
Adrenal Cortex Classification Major Hormone/s
Zona Aldosterone,
Mineralocorticoids
Glomerulosa Corticosterone
Zona Fasciculata Glucocorticoids Cortisol
Dehydroepiandrosterone
Gonadocorticoids/
Zona Reticularis Sulfate (DHEA),
Sex Steroids
Androgens
• (2) Plasma Concentration of Sodium and Potassium
MINERALOCORTICOIDS o Directly influences the zona glomerulosa cells to
produce or secrete aldosterone
• Synthesized in Zona Glomerulosa
• Zona Glomerulosa ▪ E.g., low sodium and high potassium
o G-zone cells • (3) Adrenocorticotropic Hormone (ACTH)
o Outer portion (10%) o Causes small increases of aldosterone during stress
o Low cytoplasmic-to nuclear ratios
o Have small nuclei with dense • (4) Atrial natriuretic peptide (ANP)
chromatin with intermediate lipid
o Inhibits activity of the zona glomerulosa
inclusions
o Released by the muscle cells in the atria of the heart Actions of Aldosterone
▪ In response to high blood volume
• Helps control blood
 ANP acts to reduce water, sodium and pressure by holding on
adipose loads of the circulatory system to to salt and losing
reduce blood pressure potassium from the
o Different from the other three mechanisms which all blood.
influence the adrenal cortex to secrete aldosterone, • Normally, when there
while ANP has an exact opposite function is dehydration,
aldosterone turns on
▪ ANP inhibits the activity of the zona glomerulosa the sodium-potassium
to secrete aldosterone pumps.
Summary of the Four Mechanisms of Aldosterone o It pumps sodium
Secretion back into the blood
to maintain vascular volume.
• (1) Decreased blood o For each sodium retained, potassium is lost.
volume & decreased
sodium level → the • Stimulates sodium reabsorption by distal tubule and
kidney releases renin. collecting duct of the nephron and promotes potassium
and hydrogen ion excretion.
o Renin
• Increases transcription of Na/K pump
▪ Main regulator • Increases the expression of apical Na channels and an
▪ Initiates cascade Na/K/Cl cotransporter
that produces • Expands ECF volume
Angiotensin II
o Angiotensin II Table 4. Physiologic Effects of Steroids
▪ Stimulates the
release of Representative
Aldosterone Biological Effects
Hormone
• (2) The plasma
concentration of sodium • Protein nitrogen catabolism increased
• Gluconeogenesis
and potassium
• Increased blood glucose concentration
o ↓ sodium and ↑ potassium in the blood • Decreased glucose tolerance
• Increased liver glycogen
▪ direct stimulating effect in the adrenal cortex • Increased liver glycogenolysis
(zona glomerulosa) to produce aldosterone • Decreased peripheral uptake and
utilization of glucose
• (3) ACTH
• Decreased synthesis of acid-sulfated
o The hypothalamus secretes CRH in response to Cortisol mucopolysaccharides
long-term stress (as a • Fat synthesis and redistribution
representative of • Cellular or tissue effects
▪ CRH stimulates the anterior pituitary gland to glucocorticoid) • Anti-inflammatory
secrete ACTH. • Dissolution of lymphoid tissue
▪ ACTH facilitates the transport of cholesterol to • Lymphophenia
the adrenal mitochondria initiating aldosterone • Eosinopenia
production • Increased erythropoiesis
• Alteration of cellular permeability,
• The three mechanisms enhance the secretion of especially decreased membrane
aldosterone. permeability to water
• Increased Gastric (HCl and pepsin)
o Aldosterone – targets the kidney tubules
secretion
▪ Inside the kidney tubules, it increases the
absorption of sodium and water; and increases • Electrolyte regulation
the excretion of potassium. Aldosterone • Sodium retention
(as a • Potassium excretion
 In response to this, blood volume/pressure representative • Retention of water and expansion of
increases. mineralocorticoid) extracellular fluid volume
 Once this happens, the muscles in the atria of • Increases in blood pressure
the heart release ANP (Atrial natriuretic
peptide) • Protein nitrogen anabolism
Androgens
• Growth and maturation---osseous and
 ANP – has inhibitory effect that generates (as representative
muscular
sodium loss to reduce blood pressure sex hormones)
• Body hair (pubic and axillary)
Aldosterone: Clinical Significance (1) PAC/PRA Ratio
• Insufficiency • Most reliable screening test for primary

hyperaldosteronism
o Insufficient aldosterone secretion is usually seen • Most sensitive mean of differentiating primary from
with Adrenal gland destruction and G-zone enzyme secondary causes of hyperaldosteronism
deficiency.
o Complete failure to secrete aldosterone leads to • PAC (Plasma Aldosterone Concentration)
death due to dehydration and low blood volume
o Measures the aldosterone level
o Common in people with:
o This test alone CANNOT distinguish the primary
▪ Hypotension hyperaldosteronism from the other causes of
▪ Hyponatremia hyperaldosteronism
▪ Hyperkalemia
▪ To sort this out, renin requires to be measured.
▪ Metabolic Acidosis
▪ NOTE: Renin is an enzyme at the top of the
 Due to ↓ sodium = ↓ blood pressure/volume cascade that makes aldosterone
• Overproduction  Main regulator of aldosterone secretion
o Hyperaldosteronism can be caused by a condition • PRA (Plasma Renin Activity)
called Conn’s syndrome
o Measures renin activity
▪ Primary hyperaldosteronism and Secondary
▪ High Renin Level = not an adrenal tumor
hyperaldosteronism
▪ Low Renin Level = tumor in the adrenal gland is
 cirrhosis, ascites, nephrotic syndrome possible
▪ “Primary”  Since there is tumor, there is hyperactivity in
 The damage or abnormality is in the organ the adrenal gland. It consumes the renin.
itself where it is produced • Aldosterone Renin Ratio (ARR)
▪ “Secondary” o Simply the plasma aldosterone concentration
 Something else is causing the abnormality divided by the plasma renin activity (PAC/PRA)
o Common in people with: ▪ Instead of settling in the PRA test to distinguish
the primary from other causes of
▪ Hypertension (HTN) associated with increased
hyperaldosteronism, it was decided to compare
blood volume
the renin activity to how much of aldosterone is
▪ Hypernatremia
in the body
▪ Hypokalemia
▪ Metabolic alkalosis • Patients with primary hyperaldosteronism
o there PAC is high and PRA is low.
Conn’s Syndrome
• To complete the testing, we often try to suppress the
• Primary Hyperaldosteronism production by giving a large amount of sodium
• Rare condition caused by overproduction of
aldosterone o 2L of normal saline over 4
• Can be caused by: hours
o (1) Unilateral adenoma ▪ for volume expansion and

aldosterone suppression.
▪ Hyperactivity in one adrenal gland
▪ There is a unilateral adenoma (benign tumor) of o ACE inhibitor
the adrenal gland, causing a condition known as ▪ suppress aldosterone.
hyperaldosteronism.
o (2) Bilateral adrenal hyperplasia • Why we need suppressor
or inhibitor?
▪ When both adrenal glands are making too much
aldosterone o In healthy individuals,
aldosterone production
is inversely correlated
Laboratory Diagnosis with salt intake
o If the sodium
• (1) PAC/PRA Ratio decreases, it directly
• (2) Urinary Potassium Excretion influences the adrenal
• (3) Captopril Suppression gland to secrete more
• (4) 18-Hydroxycorticosterone aldosterone
• (5) Adrenal Imaging (CT or MRI) o If the sodium
increases, then there is
no need for it to signal
the adrenal gland.
o This test is based on the fact that aldosterone levels (3) Captopril Suppression
should be suppressed in individuals given a high salt
diet • Confirmatory test
o Since ACE (angiotensin-converting enzyme) is • Captopril
inhibited, then angiotensin I cannot be converted to o Used to treat high blood pressure, hypertension,
angiotensin II. congestive heart failure, kidney problems and to
▪ Angiotensin II – stimulate the release of improve survival after heart attack.
aldosterone ▪ Usually given to patients with hypertension, to
▪ Response: Less or cannot secrete aldosterone see if the cause is due to high aldosterone level.
o Test the adrenal gland if it is still functioning o Within 3 hours – 50 mg of captopril (1 mg/kg)
properly.
▪ High Aldosterone – aldosteronism
• PAC/PRA Criteria
 Plasma aldosterone remains high in primary
o PA/PRA greater than 25. hyperaldosteronism
o Low plasma renin that fails to increase with volume
▪ Suppressed – with other forms of HTN
depletion.
(Hypertension)
o High aldosterone that fails to decrease with saline or
angiotensin inhibition
(4) 18-Hydroxycorticosterone
Table 5. PAC/PRA Criteria • >100 ng/dL = Aldosterone-producing adenoma (APA)
• <100ng/dL = Idiopathic hyperaldosteronism (IHA)
Primary
Normal
Hyperaldosteronism (5) Adrenal Imaging (CT or MRI)
PAC/PRA >25 <25 • Used to visualize adrenal gland anatomy.
PRA Low High o To see whether there is a tumor.
o Sometimes, it can be misleading if the pathologists
PAC HIgh Low relay on the imaging studies alone
• It is important to complement it with other findings
• PAC/PRA ratio is a screening test; it is not before establishing a diagnosis.
confirmatory test.
• Physician usually suggest or request a CT scan to look GLUCOCORTICOIDS
for the adrenal tumor and surgery will be the option of
choice. • Synthesized in Zona Fasciculata
• Zona Fasciculata
o F-zone cells
o Middle portion (75%)
o Cords of clear cells,
with a high cytoplasmic-
to-nuclear ratio and
lipids laden with “foamy”
cytoplasm
• Function: Regulate blood pressure and glucose
homeostasis
Cortisol
• Most important glucocorticoid

• Maintain blood glucose by
inducing:
o Lipolysis
o Gluconeogenesis
▪ Muscle conversion into glucose causing amino
(2) Urinary Potassium Excretion acid release and for storage as liver glycogen
o Glycogenolysis
• Screening test for hyperaldosteronism
• In patients with hypertension and unprovoked • Most tissues are the target cells
hypokalemia.
o Most tissues need glucose or glycogen as a source
o > 30 mEq/day = hyperaldosteronism of potential energy
o < 30 mEq/day = increased renal K+ retention o Therefore, tissues also need glucocorticoids to
(diuretic or GI loss) regulate glucose homeostasis
• 90% = bound to CBG (corticosteroid binding globulin or o Zona fasciculata

transcortin) and albumin
▪ Progesterone converted into 17-OH
• 10% = free or unbound
Progesterone
• The only hormone that inhibits the secretion of ACTH
 Enzyme: 17α-hydroxylase and 17,20-lyase
o Mechanism: Negative feedback
▪ 17-OH Progesterone converted into 11-
deoxycortisol
Adrenal Cortex: Pathway for the Hormones Produced
 Enzyme: 21-hydroxylase
▪ 11-deoxycortisol converted into cortisol
 Enzyme: 11β-hydroxylase
• (4) Conversion of 17-OH pregnenolone and 17-OH
Progesterone has also two pathways:
o Zona fasciculata
o Zona reticularis
Negative Feedback Mechanism
• Cortisol
o has a very important role in providing negative
feedback to the brain, among all the hormones
• Steroid hormone synthesis regulated by ACTH produced in the adrenal cortex.
o ACTH
▪ main regulator for the production of
glucocorticoids
• (1) Once inside the adrenal mitochondria
o Cholesterol converted into pregnenolone
o Enzyme: cholesterol desmolase
• (2) Conversion of Pregnenolone has two pathways,
either converted inside the: • An increase glucocorticoid in the circulation → will send
a negative feedback signal to the hypothalamus
o Zona glomerulosa
o It will inhibit the release of corticotropin-releasing
▪ Pregnenolone converted into Progesterone hormone (CRH) from the hypothalamus and ACTH
▪ Enzyme: 3β-hydroxysteroid dehydrogenase release from the pituitary gland.
o Zona fasciculata
▪ Pregnenolone converted into 17-OH
Pregnenolone
 Enzyme: 17α-hydroxylase and 17,20-lyase
• (3) Conversion of Progesterone has two pathways:
o Zona glomerulosa
▪ Progesterone converted into
Deoxycorticosterone (DOC)
 Enzyme: 21-hydroxylase
▪ Deoxycorticosterone (DOC) converted into
corticosterone
 Enzyme: 11β-hydroxylase
• GR;
▪ Corticosterone converted into 18-OH glucocorticoid
corticosterone receptor
 Enzyme: 18-hydroxylase • HSP; heat
shock proteins
 18-OH corticosterone – marker for the
diagnosis of hyperaldosteronism
▪ 18-OH corticosterone converted into aldosterone
 Enzyme: 18-hydroxydehydrogenase
Metabolic Response to Fasting Table 6. Physiologic Effects of Cortisol
• Gluconeogenesis from amino acids (increased

Representative
expression of the enzymes). Hormone
Biologic Effects
o Enhancing the synthesis of gluconeogenic enzymes
such as glucose 6-phosphatase and • Protein nitrogen catabolism increased
phosphoenolpyruvate carboxykinase • Gluconeogenesis
• Increased blood glucose concentration
• Promotes redistribution of adipose tissue • Decrease glucose tolerance
• Mobilization of stored fat • Increased liver glycogen
• Increased liver glycogenolysis
o increased adipocyte differentiation lipogenesis in
• Decreased peripheral uptake and
tissues utilization of glucose
o use in β-oxidation and the production of ketone • Decreased synthesis of acid-sulfated
bodies. mucopolysaccharides
Cortisol • Fat synthesis and redistribution
Anti-inflammatory Effects (as a representative • Cellular or tissue effects
glucocorticoid) • Anti-inflammatory
• Dissolution of lymphoid tissue
• Lymphopenia
• Eosinopenia
• Increased erythropoiesis
• Alteration of cellular permeability,
especially decreased membrane
permeability to water
• Increased gastric (HCI and pepsin)
secretion
Clinical Significance of Cortisol
Addison’s Disease
• Used to alleviate inflammation
o Inhibition of production of prostaglandins and • Also called as adrenal insufficiency
leukotrienes (mediate inflammation). • Caused by:
o Primary adrenal
▪ occurs via stimulation of an inhibitor of
disorder
phospholipase A2, which is needed for
prostaglandins synthesis. ▪ destruction of
o Decrease the inflammation reaction by decreasing 90% of the
permeability of capillary membranes, reducing adrenal cortex
swelling. o Secondary disorder
o They also reduce effects of histamine.
▪ ACTH deficiency
▪ Glucocorticoids suppress hypersensitivity by due to
inhibiting the production of histamine by abnormality at the
basophils and mast cells hypothalamic-
pituitary level
Circadian Rhythm of Cortisol Secretion
Table 7. Signs and Symptoms
Frequency Symptoms Signs
Weakness
100% Fatigue Weight loss
Anorexia
90% Hyperpigmentation
Nausea
• Diurnal variation in the secretion of cortisol 50%
Diarrhea
• Morning around 8 to 9 – highest concentration
• Night – lowest concentration 10% Pain Adrenal calcification
Laboratory Diagnosis o 12:00 mn = 30 mg/kg orally given at midnight

o 8:00 am = Blood cortisol and 11-deoxycortisol
• (1) Serum Cortisol Level
o In normal or healthy
o Fluorometric Assay individual, when they orally
▪ Developed by Nelson and Samuels, which was administer Metyropone at
based on a technique first developed by Porter midnight, will block 11-β-
and Silber hydroxylase
o RIA and Chemiluminescent Assay ▪ Once 11-β-hydroxylase is
o HPLC with MS/MS blocked, 11- deoxycortisol
will increase and cortisol
▪ High-performance liquid chromatography (HPLC) is decreased
with tandem mass spectrometric (MS/MS)
o In secondary insufficiency, both are decreased
• (2) Basal Cortisol Level
o Interpretation:
o Extract blood in the morning
around 8:00-9:00 am, supine ▪ Normal:
▪ highest concentration  11-deoxycortisol >7 μg/dL (200 nmol/L)
o < 3 ug/dL (83 nmol/L) ▪ Abnormal:
o Normal individual: high  <7 μg/dL of 11-deoxycortisol, accompanied by
cortisol level <5 μg/dL cortisol
o Primary adrenal insufficiency
• (6) CRH Test
▪ Baseline cortisol is low
▪ ACTH is elevated o Used to diagnose hypocortisolism and to localize the
site of damage
• (3) Random Cortisol Level o Baseline ACTH and Cortisol
o IV: 100 ug of CRH
o Blood extracted anytime of the day
o Suggestive adrenal primary insufficiency: <10 ug/dL ▪ Given intravenously
o Useful in excluding the diagnosis if the result is ▪ Safe but expensive
elevated around >20 ug/dL
o Collect blood every 15 minutes for 60-90 minutes
• (4) ACTH Stimulation Assay o Plasma corticotropin usually peaks at 15-30 minutes
o Cortisol Peak:
o Standard diagnostic test for primary adrenal
insufficiency ▪ 30-40 minutes post-injection
▪ not diagnostic on secondary adrenal o Normal response:
insufficiency
▪ Cortisol greater than 20 μg/dL
o Useful tool to distinguish primary from secondary
o Blood is drawn anytime of the day for baseline Cushing’s Syndrome
cortisol, ACTH and aldosterone levels.
o Administer 250μg Cosyntropin • Overproduction of cortisol secretion
• Most common causes:
▪ Cosyntropin – Synthetic stimulator of cortisol and
aldosterone secretion o (1) ACTH-secreting pituitary adenoma aka
Cushing’s disease (68%)
o After 30 and 60 minutes collect serum for cortisol o (2) Autonomous cortisol production from an adrenal
o Normal response: > 18-20 μg/dL tumor (17%)
o (3) Ectopic ACTH or CRH production (15%)
• Primary and Secondary Hypersecretion of Cortisol
o Primary Hypersecretion of Cortisol is due to
problem within the adrenal cortex
o Secondary Hypersecretion of Cortisol is due to
hypothalamic problem or pituitary problem
• Cushing’s syndrome is a
group of clinical and
metabolic disorders
• (5) Overnight Metyrapone Test characterized by
adrenocortical
o Diagnostic test for secondary insufficiency hyperfunction
o Metyrapone is orally given at midnight
o Excess production of
▪ Inhibits 11-β-hydroxylase glucocorticoids, or
 preventing the conversion of 11-deoxycortisol glucocorticoids and
to cortisol androgens
o When serum cortisol exceeds the capacity of its

carrier protein binding, free cortisol levels rise
rapidly, increasing the free cortisol filtered into the
urine.
▪ Cortisol as a steroid hormone needs a carrier
protein to travel freely in the circulation
▪ In case of hypercortisolism, there will be
increase cortisol
▪ Free cortisol will be filtered out in the urine
▪ Disadvantage: values may be erroneous with
high urine volume or due to hydration
o Affected by hydration (3-5 L/day).
• (2) Urine 17-Hydroxycorticosteroid (17-OHCS)
o Break down products of cortisol and other
adrenocorticotropic hormones
o Not affected by volume changes, unlike urine free
Signs of Cushing’s Syndrome cortisol
o However, the sensitivity and the specificity of this
• Fat is deposited in the body trunk
method is poor
(central obesity or abdominal
obesity) • (3) 24-hour Urine Cortisol
o It is when excessive abdominal o Revised method of Urine Free Cortisol
fat around the stomach and
abdomen is built up ▪ It is also a reflection of unbound circulating
cortisol that is freely circulated by glomerulus
• Buffalo hump ▪ Unaffected by the level of circulating CBG or
• Moon facies (subcutaneous fat in circulating carrier protein
cheeks and submandibular)
o Measured by Tandem Mass Spectroscopy
• Purple striae o Most sensitive and specific for excess cortisol
• Blood-glucose levels rises o Collecting urine overnight (10 pm – 8 am) factored
chronically, causing adrenal by creatinine
diabetes
• May cause beta cells to die • (4) Dexamethasone Suppression Test
o Dexamethasone – a synthetic glucocorticoid
Cushing’s Disease medicine that acts as an exogenous cortisol
substitute
• Cushing’s Syndrome o Measure whether your ACTH secretion by the
o refers to the general state characterized by pituitary gland can be suppressed
excessive levels of cortisol in the blood
▪ As cortisol substitute, suppressing ACTH if the
• Cushing’s disease pituitary gland is normal and cortisol secretion if
the adrenal gland is normal.
o Type of Cushing’s syndrome caused by a benign
tumor in the pituitary gland o Overnight DST
▪ Because the tumor in the pituitary gland secretes ▪ Used to screen patients with autonomous
too much ACTH, resulting to hypercortisolism production of cortisol
▪ 1 mg at 11 pm – suppress the early morning
o Is a state of glucocorticoid excess resulting from an ACTH stimulated rise in cortisol
ACTH-secreting pituitary adenoma
o Most commonly iatrogenic in origin  Blood collection is done the next morning
▪ Hospital-induced or something that is caused by o In normal individual, once taken Dexamethasone

medical examination or treatment should suppress ACTH and cortisol suppression
o In cases of hypercortisolism, the ACTH and
o However, may be due to ectopic production of CRH cortisol remains high
or ACTH by a tumor or due to a primary adrenal
malignancy ▪ Suppressed free cortisol (<1.8 ug/dL) between 8-
9 am – negative test
Laboratory Diagnosis of Hypercortisolism • (5) CRH Stimulation Test
• (1) Urine Free Cortisol o Helpful in distinguishing types of Cushing’s

Syndrome (central disease versus primary adrenal
o Sensitive indicator of endogenous cortisol syndrome)
production/cortisolism o 8:00 am – serum cortisol and ACTH level is drawn
▪ Serum cortisol following CRH injection
Table 8. CRH Stimulation Test GONADOCORTICOIDS

• “Gonads” – sex organs; “corticoids” – adrenal cortex
ACTH CORTISOL • Sex steroid hormone that is produced by the adrenal
cortex.
ACTH-Independent
Cushing’s Syndrome Low High o There is a debate over the principal site of adrenal-
(adrenal cause) androgen synthesis.
ACTH-Dependent
Cushing’s Syndrome • Traditionally, it has been thought that adrenal
High High
(pituitary or ectopic androgens are synthesized exclusively in the zone
source) reticularis.
o The activity of 17, 20 lyase that converts to 17-OH
• ACTH-Independent Cushing’s syndrome pregnenolone to DHEA and 17--OH progesterone to
o Adrenal gland has abnormalities, hyperactive. A’Dione occurs within the zone fasciculata and zona
Therefore, cortisol level is high reticularis.
o Since only the adrenal gland has a problem, the o Final product happens inside the reticularis.
upper cascade – hypothalamus and pituitary gland
can still perform their function properly.
o ACTH is low due to the fact that:
▪ If there is an increase in glucocorticoid, there will
be negative feedback.
▪ The negative feedback mechanism inhibits your
hypothalamus and pituitary gland to secrete
CRH and ACTH therefore ACTH is low and
cortisol is high. Zona Reticularis
• ACTH-Dependent Cushing’s Syndrome • Zona Reticularis

o In the upper cascade, either the pituitary gland or o R-zone cells
the hypothalamus has problems. o Inner layer (15%)
o When either of them is hyperactive, when ACTH is o The zone is sharply demarcated with lipid-deficient
high- so is cortisol. cords of irregular, dense cells with lipofuscin
deposits.
▪ However, since adrenal gland belongs to the
lower cascade ACTH is then lowered. • R-cells produce:
• ACTH – the main regulator of cortisol secretion. o DHEA
o Androstenedione
o Hence why adrenal gland is affected.
Produces various types of Gonadocorticoids:
• DHEA and its sulfated form DHEAS (by

sulfotransferase)
o Both are precursors to more active androgens and
estrogens
o Though with minimal androgenic activity, it’s
adverse effects or cause by conversion to active
androgens in the adrenal and peripheral tissues
▪ such as your sebaceous glands, genitalia,
ovaries and testis.
• Androgens
o Male hormone
o Most important or major gonadocorticoids
▪ Androstenedione
▪ Testosterone – most important androgen
▪ 5-dihydrotestosterone (5-DHT)
• Estrogens
o Female hormone
▪ Estradiol – most common type estrogen Sex Characteristics
measured for pregnant women
• Steroid hormone, particularly gonadocorticoids, helps
▪ Estriol – often measured during pregnancy
for the development of sex characteristics for both male
▪ Estrone – measured for women who have gone
and female.
to menopause
• Both Androgens and Estrogens: Table 9. Male Secondary Sex Characteristics
o Secreted in minimal amounts in both sexes (females
and males) by the adrenal cortex Characteristics Usual Age Range
o In comparison, estrogen is secreted very minimally
by the adrenal cortex. The testicles begin to enlarge, and the 10 to 13
▪ Hence, androgens are most considered as the scrotum turns darker and coarser
important gonadocorticoids. Pubic hair begins to grow 10 to 15
▪ Their effects are masked by the hormones of the
testis and ovaries. The body grows taller and heavier 10 to 16
Androgens The penis begins to grow longer and 11 to 15

fuller
The voice begins to deepen 11 to 15

• Androgens contribute to:
Boys become fertile, meaning they are
o The onset of puberty capable of ejaculating semen
11 to 17
o The appearance of
Hair begins to grow under the arms
secondary sex 12 to 17
and on the face
characteristics
Glands in the skin and scalp begin to
o Sex drive in females. produce more oil, which can cause 12 to 17
skin blemishes
• Men
• In male, there is:
o less than 5% of testosterone from adrenal cortex.
o axillary hair, pubic hair, facial hair growth
• Women o sebaceous or oil secretions
o sex drive
o 40% to 65% of testosterone from adrenal cortex.
Table 10. Female Secondary Sex Characteristics
Characteristics Usual Age Range
The breasts begin to develop 7 to 13
Pubic hair begins to grow 8 to 14
The vagina grows longer, and its

outer lips (labia) become more 8 to 15
pronounced
The body grows taller and heavier 9 to 14
Menstruation begins 9 to 16
Hair begins to grow under the arms 11 to 16
Glands in the skin and scalp begin

to produce more oil, which can 11 to 16
cause skin blemishes
• In female, it helps for the development for the:

o Mammary glands
o Breast
o Pubic hair
o Axillary hair growth
o Sex drive
Table 11. Physiologic Effects of Androgens
Representative
Biological Effects
Hormone
• Protein nitrogen anabolism

Androgens
• Growth and maturation---osseous and
(as representative
muscular
sex hormones)
• Body hair (pubic and axillary)
Clinical Significance of Androgens
(1) Adrenal Androgens Excess in Males
• Excess adrenal androgens will be converted into

estrogen. Causes:
o Infertility
o Feminizing effects by inhibiting pituitary
gonadotropins
o Lower testosterone
▪ Hypogonadal symptoms
 Loss muscle mass
 Decreased hair growth
 Decrease testes size
 Decrease spermatogenesis
(2) Adrenal Androgens Excess in Females
• Can cause:
o Infertility
o Masculinizing effects
▪ Hirsutism (excessive hair)
▪ Acne
▪ Male pattern baldness
▪ Menstrual irregularities
▪ Virility
Laboratory Diagnosis
• Plasma DHEA,
• Plasma DHEAS
• Urine 17-ketosteroids
o These 3 can be used to identify patients with
adrenal causes or adrenal pathologic
masculinization in females and pathologic
feminization in males.
NOTE: Less than 10 percent of your DHEA and DHEAS
are produced by the gonads
o Therefore, elevated DHEA and DHEAS production
indicates adrenal hyperandrogenism.
[TRANS] LABORATORY UNIT 12: ADRENAL MEDULLA
ADRENAL MEDULLA CATECHOLAMINES

Anatomy and Histology • Any of a class of aromatic amines that includes a
number of neurotransmitters
• Embedded in the o DOPA
center of the cortex o Dopamine (D)
of each adrenal gland. o Norepinephrine (NE)
• 10% of the total o Epinephrine (E)
adrenal weight
• Norepinephrine (NE)
o 4-6 grams
o It is small. o Derived from the sympathetic nerve endings
• Pyramidal in structure. • Epinephrine (E)

o Principal product of adrenal medulla
o Made ONLY in the medulla
▪ Almost exclusively produced and secreted by the
adrenal medulla.
• The ratio of NE and E is 1:4.
• The ratio of NE and E in serum is 9:1.
o Since this catecholamine is synthesized and is
essential in the sympathetic nervous system, the
• The adrenal gland is divided into two distinct parts: peripheral ratio is more like 9:1.
o (1) Adrenal medulla Biosynthesis of Catecholamines
▪ The inner layer.
o (2) Adrenal cortex
▪ The outer layer.
• The biosynthesis begins with a sequential conversion of

phenylalanine substrates in a tightly regulated
compartmentalized manner.
o Cytoplasm
▪ All of the reactions take place in the cytoplasm
• Chromaffin cells EXCEPT for the production of norepinephrine
(NE).
o Produce catecholamines which includes:
o Lipid Vesicles or Outer Mitochondrial
▪ Epinephrine (E) Membranes
▪ Norepinephrine (NE)
▪ The production of NE occurs here.
ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO. LAMOSTE. MAHINAY. NAVARRO. CORTEZ. CAGAS BSMLS 3 1
LABORATORY UNIT 12: ADRENAL MEDULLA
• In the sympathetic neurons, the cytoplasmic • (4) Dopamine beta-hydroxylase (DBH)

dopamine is sequestered into the vesicles then it is
o Found in the terminal of post-ganglionic sympathetic
converted into NE.
neurons
o It is stored until the nerve stimulation causes its
release. • (5) Phenylethanolamine N-methyltransferase
(PNMT)
• In medulla chromaffin cells, the norepinephrine can
o When norepinephrine moves out, PNMT will be
possibly diffuse into the cytosol.
activated
o Phenylethanolamine N-methyltransferase
(PNMT) ▪ PNMT transfers the methyl group from
adenosylmethionine to norepinephrine. Thus,
▪ In the cytosol, the norepinephrine can be creating epinephrine
converted into epinephrine by a cortisol- ▪ PNMT enzyme is present only in the adrenal
dependent enzyme, PNMT. medulla
• Any form of stress that increases cortisol levels will also • (6) Epinephrine
stimulate epinephrine production
o Enters the VMAT-1
• Dopamine (free cytosolic epinephrine-like) (7)
o actively transported into secretory vesicles by VMAT • (7) Catecholamines, Calcium (4
(vesicle monoamine transporters) and ATP bind to granular
)
proteins called,
• Norepinephrine to Epinephrine Ratio is 9:1 chromogranins D
o
o 98% from post-ganglionic neuron o Catecholamines are (8) p
o 2% from medulla present in high amounts
(4 a
in the secretory vesicles
of adrenal medulla ) m
D in
• (8) Chromogranin o e
o Chromogranin B - most p
dominant chromogranin a
m
• (9) Release of catecholamines are inhibited by CNS
in
control
•
e
(10) ACh released form the preganglionic neurons in
Biosynthetic Pathway of the Catecholamines the splanchnic nerves acts on the:
o nicotinic ACh receptors (nAChRs)
• Rate-Limiting Step – conversion of L-tyrosine to L-3,4-
o postganglionic chromaffin cells
dihydroxyphenylalanine (L-DOPA)
o Converted by the action of the enzyme, tyrosine • (11) With this depolarization, it triggers the opening of
hydroxylase voltage gated 𝐶𝑎2+ channels
o a process that triggers the exocytotic release of
(4)
epinephrine
(4
) • Secretion of adrenal catecholamines
(2) D o accompanied by the release of ATP and the granule
o proteins
(3)
p
• Types of Chromogranin
a (5)
(1)
mi o Chromogranin A
(4
n (6) ) ▪ The release of chromogranin A has been used
e (4 D as a marker for adrenal medullary activity.
) o ▪ In the circulation, the catecholamines dissociate
D p from the binding complex and it is free to act on
• (1) Tyrosine Hydroxylase o the target tissues
a
o Cytosol of adrenal medullary cell,p sympathetic
m nerve o Chromogranin B
terminals, and specific cell within
a the CNS
in (central
nervous system) Adrenal Medulla: Physiologic Actions
m e
• in
(2) Aromatic l-amino acid Decarboxylase (AADC) • Adrenergic Receptors
e
o Numerous tissues including adrenal medulla o coupled with G Protein (termed as GPCR)
• (3) Vesicle Monoamine Transporter 1(VMAT-1) ▪ Alpha

▪ Beta – Both norepinephrine and epinephrine
o Moves the dopamine into the membrane enclosed in binds to alpha and beta
the vesicles
Table 1. Adrenergic Receptors o Dilation of bronchioles

o Changes in blood flow patterns leading to increased
Effect of Ligand alertness, decreased digestive system activity, and
Receptors Effectively binds reduced urine output
Binding
o Increased metabolic rate
Epinephrine, Increased free
𝑨𝒍𝒑𝒉𝒂𝟏
Norepinephrine calcium • Long-term Stress Response
Epinephrine, Decreased cyclic
𝑨𝒍𝒑𝒉𝒂𝟐
Norepinephrine AMP o Retention of sodium and water by kidneys
o Increased blood volume and blood pressure
Epinephrine, Increased cyclic
𝑩𝒆𝒕𝒂𝟏
Norepinephrine AMP o Proteins and fats converted to glucose or broken
down for energy
Increased cyclic
𝑩𝒆𝒕𝒂𝟐 Epinephrine
AMP
o Increased blood sugar
o Suppression of immune system
(3) Specific Physiologic Actions

(1) Receptors
• Increased rate and force of contraction of the heart
• Adrenergic Receptors muscle
o α1 is a post-synaptic receptor o This is predominantly an effect of epinephrine acting
▪ three subtypes: 1A, 1B, 1D through beta receptors
o In exercise there is relaxation of bronchial smooth
o α2 is both post- and pre-synaptic receptor muscles that will lead to the degradation of glycogen
▪ three subtypes: 2A, 2B, 2C for increased fuel and for lipolysis
o Alpha - inhibits secretion of insulin
• Andrenergic Receptors o Beta - secretes insulin
o β1 – located mainly in the heart and cortex o Hypoglycemia - triggers sympathetic response
o β2 – predominates in the lung and cerebellum • Constriction of blood vessels
o β3
o Norepinephrine, in particular, causes widespread
▪ in the adipose tissue vasoconstriction, resulting in increased resistance
▪ significance in obesity and hence arterial blood pressure
(2) Fight-or-flight response • Dilation of bronchioles
• a response when faced with severe external threat o Assists in pulmonary ventilation
• centrally driven release of adrenaline, as well as • Stimulation of lipolysis in fat cells
activation of other aspects of sympathetic division of
ANS o This provides fatty acids for energy production in
• activated in seconds (briefly around 10 seconds) many tissues and aids in conservation of dwindling
reserves of blood glucose
Short-term and Long-term Stress Response
• Increased metabolic rate
o Oxygen consumption and heat production increase
throughout the body in response to epinephrine
o Medullary hormones also promote breakdown of
glycogen in skeletal muscle to provide glucose for
energy production
• Dilation of the pupils
o Particularly important in situations where you are
surrounded by velociraptors under conditions of low
ambient light
• Inhibition of certain “non-essential” processes
o An example is inhibition of gastrointestinal secretion
and motor activity
• Common stimuli
o Exercise
o Hypoglycemia
• Short-term stress response o Hemorrhage
o Emotional distress
o Increased heart rate
o Increased blood pressure
o Liver converts glycogen to glucose and release
glucose to blood
Degradation and Elimination of Catecholamines • METABOLITES
• Reuptake into secretory vesicles o Free Catecholamines

o Metanephrines
• Uptake in nonneuronal cells (mostly liver)
o Vanillylmandelic acid
• Degradation
▪ Excreted via the kidneys
Degradation
Diseases
• COMT – catechol-methyltransferase (nonneuronal
tissues) Pheochromocytoma
o Degrades catecholamine, catechol estrogens, and • Rare catecholamine-producing tumors
other various drugs and substances that has
catechol component o Triad
o Is encoded by the COMT gene and has 2 iso forms ▪ Diaphoresis
▪ Soluble form ▪ Tachycardia
▪ Membrane bound long form ▪ Headache
o As the regulation of catecholamine is impaired in a • 24-Hour Urine
number of medical conditions, several o Creatinine is measured to verify the adequacy of the
pharmaceutical drugs target the catechol collection
methyltransferase to alter the activity and therefore o 25 mL of 6Eq/HCl – preservative
the availability of the catecholamine
o Substrate used in this process: Levodopa • Plasma
• MAO – monoamine oxidase (neurons) o Overnight fast
o (1) Patient in a reclining position in a quiet
o Has 2: environment, and a heparin lock is inserted IV
▪ MAO-A – breakdowns serotonin, melatonin, o (2) After 20-30 minutes, collect blood in a pre-chilled
norepinephrine, and epinephrine EDTA
▪ MAO-B – breakdowns phenethylamine, and o (3) Whole blood be kept in ice water until centrifuged
benzylamine o (4) Plasma should be separated within 2 hours of
▪ Both (MAO-A, MAO-B) breakdown dopamine, phlebotomy
tyramine, and tryptamine o Sample should be frozen immediately
Excretion • Urine Free NE, VMA, or HVA

• Plasma Catecholamines
o Combined resting plasma catecholamines greater
than 200 pg/mL = have a positive predictive value
of 97% when glomerular filtration rate is normal.
• Chromogranin A
o > 20 pg/mL = have a positive predictive value of
97% when glomerular filtration rate is normal.
• Urine and Plasma
• All catecholamines are rapidly eliminated from the o HPLC with Tandem Mass Spectrometry
target cells and the circulation by 3 mechanisms:
• CT
o (1) Re-uptake into the secretory vesicles • MRI
o (2) Uptake in the non-neuronal cells (mostly liver) • Clonidine Suppression Test
o (3) Degradation
o Alpha-adrenergic agonist
▪ Degradation relies on two enzymes such as the o Suppresses catecholamine release from the
COMT and MOA to produce metabolites from nervous system but has no effect on its release by
free catecholamines. the tumor
▪ Metabolites and free catecholamines are
eliminated by direct filtration into the urine and • Glucagon Stimulation Test
excreted as:
 5% - Free Catecholamines
 8% - Conjugated NE
 20% - Metanephrines
 30% - Vanillylmandelic acid (VMA)
▪ Urine epinephrine which is 50%. It is converted
from norepinephrine by renal and not adrenal
before excretion.
Table 2. Pharmacologic Tests for Diagnosing

Pheochromocytoma
Indications Interpretation
Normal:
Decrease in NE to below
normal or a -50%
decline from baseline. A
Patients with
decrease in
hypertension and
normetanephrines to
clinical findings or
below normal or a 40%
family history that is
decline from baseline.
highly suggestive of
Clonidine
pheochromocytoma
Suppression Pheochromocytoma:
and the
Test Failure of NE to drop
catecholamines are
below normal or
elevated but not to the
decrease by more than
extent that is
50% from baseline.
diagnostic of
Failure of
pheochromocytoma.
normetanephrines to
drop below normal or
decrease by more than
40% from baseline.
When clinical findings

or family history is
Pheochromocytoma: A
highly suggestive of
Glucagon threefold or greater
pheochromocytoma
Stimulation increase in plasma NE,
but blood pressure is
Test or a rise in the level to
normal and
>2000 pg/ml.
catecholamines are
only modestly
elevated.
Neuroblastoma
• Neural crest origin

o It originates in the ectoderm at the margin of neural
tube
• Occurring before age of 3
• Indications:
o High urine HVA (Homovanillic acid)
o High urinary VMA (Vanillylmandelic acid)
[TRANS] LABORATORY UNIT 13: CLINICAL TOXICOLOGY
TERMINOLOGIES further consideration and to identify presumptively

positive specimens that then require confirmatory
• Clinical toxicology testing.
o A subdivision of toxicology that involves the analysis • Toxins
of drugs. heavy metals, and other chemical agents
in body fluids and tissues for the purpose of patient o Poisonous substances that are produced by living
care cells or organisms
o Capable of causing disease when introduced into
• Toxidrome the body tissues
o A syndrome caused by a dangerous concentration o Often capable of inducing neutralizing antibodies or
of toxins in the body antitoxins
• Cholinergic toxidrome • Dependence
o A toxidrome that represents the acute phase of o Psychologic dependence

cholinesterase inhibitor poisoning ▪ Emotional and mental preoccupation with drug
acquisition
▪ Can be seen in neuromuscular junctions
▪ Use to receive positive reinforcement
• Confirmatory testing
o Physical dependence
o Used to confirm a positive or sometimes negative
result that had been presumptively classified as ▪ An altered physiologic condition caused by
positive for a specific drug. chronic exposure to a drug
o e.g., a sample that tested positive for cocaine is not  E.g., When taking cocaine, the body will be
reported right away programmed to function properly with cocaine
▪ Instead, it is subjected for confirmatory test  The body cannot function properly with the
▪ To establish that the sample is indeed has the withdrawal of abstinence of cocaine
presence of the drug (cocaine) ▪ Withdrawal syndrome
• Drugs of abuse  “discontinuation syndrome”
o Drugs that are repeatedly and deliberately used in a  Common in patients who are taking alcohol
way other than prescribed or socially sanctioned ▪ Causes abstinence or withdrawal symptoms
o i.e., a drug that is taken for nonmedicinal reasons
• Tolerance
▪ Cocaine, cannabinoids, and etc.
o Prior exposure to a drug decreases the response to
• Drug half-life (t1/2) a given dose
o The length of time it takes for one-half of an ▪ E.g., Only 1 tablet is needed to benefit for its
administered drug to decrease due to a biological effect when taking a drug for the first time
processes ▪ But, more tablets are needed to feel its effect
o Biological processes – mostly enzymatic when taking the drug more frequently
metabolism happening in the serum and liver ▪ Chronic drinkers can withstand many shots of
• Intoxication alcohol and it takes them long to get drunk than
occasional drinkers
o A state of impaired mental or physical functioning
resulting from ingestion of alcohol or drug o Dispositional or Metabolic tolerance
• Poison ▪ Consequence of enhanced elimination of a drug
o Substance that when relatively small amounts are o Functional or Cellular tolerance
ingested, inhaled, or absorbed, or when it is applied ▪ Develops as a result of adaptive processes
to, injected into, or developed within the body, has within cells
chemical action that may cause damage to structure
or disturbance of function, producing symptoms, o Behavioral tolerance
illness, or death ▪ Drug user learns to modify behavior while under
• Screening test the influence of the drug
o An initial test, that is used to "screen" urine

specimens to eliminate "negative" ones from
ALESNA. ASOY. BAGAMANO. DUMAGONOT. JUNIO. MAHINAY. SARGADO. AQUINO. CATAPANG. CAGAS.DACALOS BSMLS 3 1
LABORATORY UNIT 13: CLINICAL TOXICOLOGY
SCREENING PROCEDURES FOR DETECTION OF Theophylline

Multiple-dose oral activated charcoal:
DRUGS hemodialysis
Tricyclic
• Rapid and qualitative detection of drugs or other toxic Bicarbonate; Benzodiazepines
antidepressants
substances
• Sensitive but lack specificity
• Other screening tests such as Anion gap, ECG, and
o Hallmark or characteristic of screening procedures osmol gap are not a direct measure of the drugs that
• Screening tests include simple visual color tests (spot the patient took
test) and immunoassays o But are useful in narrowing down the possible
• Useful in emergency situations causes of toxicity
o To know immediately the drug that the patient took Determination of Anion Gap
to administer an appropriate antidote
• Initial screening test for all poisoned patients
• When low concentration of serum bicarbonate is
Table 1. Antidote or Specific Treatment for Intoxication
detected, the clinician should determine whether an
elevated anion gap exists
Toxin Antidote/Treatment • Normal value: 8-16mmol/L
• Formula used for anion gap (AG) calculation is as
follows:
Acetaminophen N-Acetylcysteine
Aluminum, iron Deferoxamine
Anticholinergic o Subtract the anions in the plasma (chloride and

Physostigmine
agents bicarbonate) to the cations in the plasma (sodium)
Dimecaprol; 2,3-dimercaptosuccinic acid;
Arsenic
d-penicillamine • Common causes of Increased Anion Gap
• MNEMONIC: MUDPILES
Multiple-dose oral activated charcoal;
Barbiturates
alkaline diuresis (phenobarbital only) o Methanol
o Uremia
Benzodiazepines Flumazemil o Diabetic ketoacidosis
o Paraldehyde
B-Blockers Gucagon o Iron, inhalants (i.e., carbon monoxide, cyanide,
toluene), isoniazid, ibuprofen
Calcium channel Calcium; glucagon; high-dose insulin
blockers infusion o Lactic acidosis
o Ethylene glycol, ethanol ketoacidosis
Multiple-dose oral activated charcoal; o Salicylates, starvation ketoacidosis,
Carbamazepine
charcoal hemoperfusion
sympathomimetics
Carbon monoxide Oxygen ▪ These are the agents/toxins to suspect if the
Amyl nitrite, sodium nitrate, sodium anion gap is above 16mmol/L
Cyamide
thiosulfate; hydroxocobalamin
Electrocardiogram
Digoxin Anti-digoxin Fab fragments
• The cardiovascular system is involved in acute toxicity
Ethylene glycol, Fomepizole (4-methylpyrazol) or ethanol; o The ECG pattern is helpful in the diagnosis and
methanol hemodialysis
initial management
Isoniazid Pyridoxine • Facility appropriate:
Calcium disodium edetate; dimercaprol; o Laboratory testing
Lead
2,3-dimercaptosuccinic acid o Diagnosis
Lithium Hemodialysis o Management of care
▪ For acutely poisoned patient or acute toxicity
Dimercaprol; 2,3-dimercaptosuccinic acid
Mercury
d-penicillamine Determination of Osmol Gap
Fomepizole (4-methylpyrazol) or ethanol;
Methanol • Main osmotically active constituents of serum are:
hemodiaysis; folate
Nitrites, nitrates Methylene blue o Sodium

o Chloride
Opioids Naloxone
o Bicarbonate
o Glucose
Organophosphate Atropine; pralidoxime (controversial for o Urea
or carbamate carbamates)
Bicarbonate; hemodialysis, activated
Salicylates
charcoal, alkaline diuresis
• Formula of serum osmolality in conventional and SI unit Gas Chromatography

o OSMc (mOsm/kg) = 2 Na (mmol/L) • Rapid
+ glucose (mg/dL)/18
• Capable of resolving a broad spectrum of drugs
+ urea (mg/dL) /2.8
• Widely used for qualitative and quantitative drug
analysis
o OSMc (mOsm/kg) = 2 Na (mmol/L)
• Gas chromatography coupled with Mass Spectrometer
+ glucose (mmol/L)
+ urea (mmol/L) o Provides analytical system with the greatest
Or accuracy for identification
o Used as a confirmatory test
o OSMc (mOsm/kg) = 1.86 Na (mmol/L)
+ glucose (mg/dL) /18 High-Performance Liquid Chromatography (HPLC)
+ urea (mg/dL) /2.8+ 9
• Analyses polar compounds without derivatization
o OSMc (mOsm/kg) = 1.86 Na (mmol/L) o Derivatization – conversion of chemical compound
+ glucose (mmol/L) into a by-product.
+ urea (mmol/L) +9
• Used for thermally labile drugs.
• To solve for the osmol gap:
Point-of-Care Devices
o Subtract the calculated osmolality of the patient from
the measured osmolality • Easy, rugged, and portable
• Used for at the site collection
• Normal value: <10mOsm/kg
• Results are available within minutes
• Elevated osmol gap
o Implies the presence of unmeasured osmotically BASIC TECHNIQUES FOR DETECTING DRUGS IN
active substances SERUM AND URINE
▪ such as volatile alcohols (Isopropanol, methanol,
Immunochemical Methods
ethylene glycols)
Spot Test • Common techniques used:
o Enzyme-Mediated (or Multiplied) Immunologic
• Screening test Technique
• Qualitative, rapid, easily performed, and non-instrument o Fluorescence Polarization Immunoassay
based
• Rule out the presence of drugs or to suggest the • Homogenous Immunoassay
presence of a drug o Used to perform most drug testing today.
o e.g., Ferric chloride and Trinder tests o These assays are all performed in a solution without
any requirement for the phase separation,
▪ Test for rapid identification of the patient with traditionally used to separate bound from free
salicylate toxicity ligand.
Immunoassays o Revolutionized toxicology
• Easy to perform Enzyme-Mediated Immunologic Technique (EMIT)

• Available for use on automated instrumentation
• A ‘marker’ drug (drug-enzyme
• Can provide semi-quantitative results complex) is used
• Methods of choice for initial screening
o Drug covalently attached to an
o Compared to Spot test enzyme (e.g., G-6-PD)
• Disadvantage: Cross-reactivity for similar drugs o Many enzymes can be used but
it depends on specific drug of interest or what
Planar Chromatography enzyme is ideal for the reaction.
• A.K.A. thin-layer chromatography • Exogenous drug (serum) competes with the D-E
• Advantages complex for the antidrug antibody
o No instrumentation o More exogenous drugs mean more free D-E, thus
o Simple and inexpensive an increase in enzymatic activity
• Specimen: Serum, Gastric Contents, and Urine

o Urine
▪ Specimen of choice
▪ Most drugs and drug metabolites are present in
the urine in high concentrations.
▪ Kidneys are the major excretory organ in the
body.
EMIT Scheme FPIA Scheme
• Concentration of the drug is directly proportional to

the free drug-enzyme complex and the product
• The drug-enzyme complex and antidrug antibody
specific for the particular drug of abuse or toxin.
o There is an active site for the binding of substance
in the drug-enzyme complex.
• (1) Sample without Drug
o The drug-enzyme
complex will bind to • (1) No drug present
the antidrug o The drug-probe complex will bind to the antidrug
antibody (highlighted antibody immobilizing it so that when polarized it will
within the red box) be stable.
o The substrate o It has the same quality of light when measured by
cannot bind to the the detector.
drug enzyme
complex producing • (2) Presence of drug (exogeneous drug)
no reaction. o Drug-probe complex is not bound; thus, it can move
• (2) Sample with an increased concentration of drug freely in the solution.
of abuse o It does not have the same quality of polarized light
when measured because it is mobile.
o Competitive o The drug-probe complex is tumbling
Binding
Summary: Drug-Binding to Antibodies
▪ It follows this
principle • Concentration of free marker* drug is a measure of D-
wherein the Ab (drug-antibody) formed
drug
competes for o Markers may be enzyme or fluorescent strobe.
the drug
• The concentration of D-Ab is related to D (drug)
antibody.
o The more D is present, the more D-Ab is formed
o The substrate binds to the active site of the drug-
o However, because Ab concentration is constant At
enzyme complex producing a product that can be
high concentrations of D, all Ab will be saturated,
measured if the drug is present.
and no further D-Ab could form
o With the presence of free drug
• In the use of antibody-antigen complex formation:
▪ It competes for the antidrug antibody vs. your
enzyme-drug complex. o Antigen – drug of interest
▪ The enzyme-drug complex will be overwhelmed
by the free drug because it has more chance of • Principle: Competitive binding
binding due to its increased amount in the
serum. NOTE: Recall the different types of inhibition (e.g., competitive,
▪ Thus, the substrate can freely bind to the drug non-competitive binding). Specifically, when using enzyme-
enzyme, releasing a product plus the free mediated immunoassay.
enzyme. Langmuir Expression
Fluorescence Polarization Immunoassay (FPIA)
𝑫𝑨𝒃𝒄𝒐𝒎𝒑𝒍𝒆𝒙 𝒏𝒌𝑫
𝒓= =
• A ‘marker’ drug (drug-probe 𝑨𝒃𝟎 𝒌𝑫 + 𝟏
complex) is used • Where:
o Drug covalently attached to a o Ab0 = total conc. of Ab
fluorescent probe molecule o k = equilibrium constant (D-Ab complex)
(fluorophore) o n = number of Ab-binding sites/molecule of Ab
o D = drug concentration
• Uses polarized light to detect if the probe is:
o Immobilized by the antidrug antibody
▪ emitted light with same polarization as exciting
light
o Exogeneous drug competes with the Drug-Probe
complex, and D-P “tumbles” in the solution
▪ decreased polarization
• The number expression can be linearized using • For a given solvent system, this ratio
Scatchard plot. is constant for the compound and
can be used to identify the
o It must be linear so that there is a fixed, measurable
compound in the mixture.
concentration of product
Chromatographic Techniques
• Separation and qualitative detection of drugs of

abuse and toxins, and to a lesser extent, determination
of therapeutic drug levels
o Must be coupled with spectrophotometry
• Used for confirmatory tests
• General operation and system components of thin-layer
o Confirmation of a positive immunoassay test result chromatography.
• Gold Standard: Gas Chromatography–Mass o In this example, the mobile phase moves up the
Spectrometry glass plate and the thin layer of adsorbent by means
o Although considered as the gold-standard, for of capillary action.
detection and quantitation of volatile and poisons, Toxi-lab® Drug Identification (ID) System
there are newer analytic techniques available.
• New Chromatographic Techniques
o Thin-Layer Chromatography
o High-Performance Liquid Chromatography
o Capillary Electrophoresis
o Liquid Chromatography–Mass Spectrometry (LCMS)
o Mass Spectrometry–Mass Spectrometry
Thin-Layer Chromatography (TLC)
• Qualitative detection of drugs • DRG Drug Screening

• Compounds separated based on their relative
o Drug Identification Systems-Detection and
affinities for:
Identification of more than 700 drugs from urine,
o a polar solid stationary phase serum or gastric specimens in less than 1 hour
▪ usually, hydrated silicate Procedures for Toxi-Lab® Drug ID System
o a nonpolar liquid mobile phase
• Specimen of choice: Urine
▪ such as 10% CH3OH in chloroform
o Large quantities may be collected and non-invasive.
TLC Scheme
• (1) Extraction Procedure
• Different compounds adsorb to the stationary phase at o acidic drugs are separated from basic
different positions as the mobile phase migrates up drugs
▪ Acidic drugs – barbiturates
 e.g., phenobarbital
▪ Basic drugs – amine derivatives
 e.g., methamphetamine or
ecstasy
o urine is treated
with base to isolate
the basic drugs
▪ Ammonium ion - soluble in water but not in
non-polar organic solvent.
▪ Amine-free base - soluble in non-polar organic
solvents.
o a nonpolar organic phase is used to extract the
compound
• A – more polar than B, so it has a higher affinity to polar ▪ This form can then be extracted into a non-polar
stationary phase (silica gel strip) organic phase and applied to a silicate strip.
• B – has higher affinity to the mobile phase (methanol in
chloroform)
• (2) Evaporation Procedure Reliability of the Method

o a small paper disk is added to the organic extraction
mixture • Sensitivity of the method is limited by the ability of the
o solvent is then evaporated so that all basic drugs naked eye to detect color changes and/or the presence
adsorb onto the paper disk of fluorescence
• (3) Separation Procedure o subjective, based on the person’s eyesight

o level of detection is on the order of 1 μg/mL of
o Perform thin-layer chromatography compound present on the strip
▪ paper disk is placed at the “origin” of the silicate • Extraction procedures are occasionally inefficient*
strip
▪ strip is placed in the migrating nonpolar solvent o one of the few disadvantages of using
chromatography
• (4) Identification of Specific Drugs
• Extraction and evaporation procedures are time-
o Standard color reaction consuming (30 min.)
▪ dipped successively in different solvents → color o Not helpful in cases of acute poisoning.
reaction
• Cocaine has a number of polar metabolites that barely
o Ultraviolet (UV) light migrate from the origin
▪ excites fluorescence in some compounds • Some difficulty in distinguishing among various
opiates may occur
o Reference Patterns - each drug can be identified
by its: o Opiates: One of the common drugs of abuse
o rf values can be close to one another
▪ retention pattern
▪ color and characteristic color change • Nontoxic drugs may give cross reaction (cx) color
▪ different reagents changes and rf values that are similar to those for
▪ presence or absence of fluorescence drugs of abuse
o e.g., antihistamines appear on the A strip very similar
to amphetamines
▪ Antihistamines are not drugs of abuse.
High-Performance Liquid Chromatography (HPLC)
• Quantitative detection and sharper separation of

therapeutic drugs and drugs of abuse
o Sensitivity: Nanomolar to micromolar range
Morphine • Stationary phase: Uniform, ultrafine particles packed

into a column
o Polar: Silicic acid
o Nonpolar: C-18 columns
▪ Reverse-phase chromatography
• Resistance to flow in the column is high
• A metabolite of heroin o Formerly known as the High-Pressure Liquid
o Retention Factor (RF): 0-14 Chromatography
o Color Characteristic: Dark red or purple ▪ High pressures are needed to deliver constant
o Diminishes in intensity and changes color on the 2nd flow of mobile phase.
solvent (water)
o Non-fluorescent • Internal standard
• If 1 or more characteristic differs from the pattern → o A compound similar in structure to the drug of
strong doubt interest
o Added to the specimen to be analyzed in a known
o about identification of the spot as morphine would concentration
exist
Uses: HPLC
• If all criteria are met → sensitivity and specificity of
the method is increased • Therapeutic drug monitoring (TDM)
o There must be no difference in the pattern when • Detection of cocaine and heroine in urine
using chromatography pattern for it to be reliable. • Separation and quantitation of tricyclic antidepressants
Capillary Electrophoresis (CE)
• A variant of TLC that has the advantages of HPLC

• A capillary tube lined with silicate is used as the solid
support in an electrophoresis apparatus
o Driving force for separation is the voltage (on the
order of 25 kV) rather than pressure
▪ This is in contrast with the HPLC which uses the
pressure as the driving force.
• Highly versatile
• The sketch of a typical separation of the major tricyclic o Can be used to separate serum proteins and small
antidepressants on high-performance liquid molecules.
chromatography (HPLC).
o A complete separation can be affected in 12 Advantages: CE
minutes.
o The concentration of each drug is on the order of • Analyte selectivity is based on different
100 mcg/mL. physicochemical principles of separation without the
need to change instrumental hardware
o Capillary zone electrophoresis
o Micellar electrokinetic capillary chromatography
(MECK)
o Capillary isotachophoresis
o Capillary isoelectric focusing
o Capillary electrochromatography
o Capillary gel electrophoresis
Uses: CE
Schematic Diagram: HPLC
• Not (yet) used as widespread as immunoassay
techniques in clinical toxicology
• Commonly used in analytic forensic toxicologic
studies and molecular dx studies.
General Design of a Liquid Chromatography as Used in a HPLC

Schematic Diagram: CE Instrumentation
Sample Machine: CE
• Parts:
o Fluorescence detector Gas Chromatography–Mass Spectrometry (GC-MS)
o Column chamber
o Chromatogram • “Gold standard” for drug testing
o Sample manager o Best confirmatory testing procedure
o Solvent manager o Highly sensitive and reliable
o Best for identifying volatile compounds
• Two techniques:
o (1) Gas-liquid chromatography
o (2) Mass spectrometry
(1) Gas-liquid chromatography
• Compounds are directly heated into the gas phase or

are derivatized to make them labile to facilitate heating
them into the gas phase
• Stationary phase: Liquid
o Usually, hydrocarbon or silicone oil that coats a solid
support in the column. Schematic Diagram: Gas Chromatography
o Different from TLC because the stationary phase in
TLC is solid.
▪ They differ in chemicals used to separate
byproducts.
• Mobile phase: Gas (carrier gas)
o Typically, an inert gas such as nitrogen, helium, or
argon or a low mass gas such as hydrogen.
• Separation is based on the ability of each compound to
adsorb to the stationary phase
o Which partially depends on the relative solubilities of
the compound in the gas versus the liquid phase
• The eluate will be detected and quantitated by mass
spectrometer.
Fermentograph of Cocaine Ions Using Gas Chromatography Mass
(2) Mass spectrometry Spectrometry
• Molecular ionization of the eluate • The specimen used is urine.

o Eluate is bombarded with electrons, turning them o Allows the detection of drugs past the ingestion.
(mostly) into cations with charge 1+
• Some molecules may undergo fragmentation
o Some bonds may break, forming unconnected
chemical species
• Molecule-ions are then passed thru an electric field
o Generated by four rods that are subjected to rapidly
alternating currents (quadrupole detector)
• Depending on the frequency of the AC, certain
molecule-ions with specific m/z can pass through the Sample Machine: Gas Chromatography Mass Spectrometry
field to the detector
• Separation of molecule-ions on the basis of their m/z Liquid Chromatography-Mass Spectrometry (LC-MS)
(mass/charge ratio)
• Can be utilized for non-volatile compounds
• Has limitations compared to GC-MS, but has become a
complementary method to GC-MS
o There are methods coupling GC-MS with LC-MS.
• Interface between LC and MS…
o Must volatilize non-volatile compounds separated in
LC
o Must remove liquid solvent from LC
o Must correct flow-rate incompatibility between LC
and MS
Interface methods:
General Design: Gas Chromatography • Electrospray ionization (ESI)
• Atmospheric pressure chemical ionization (APCI)
Electrospray ionization (ESI) o uses light of high λ (low f) that excites vibrionic
states of molecules involved in bond stretching and
bond angle bending
• each compound has a characteristic IR absorption
pattern (“fingerprint”)
o even closely related compounds can be
distinguished readily from one another - advantage
• For ionizable analytes of high MW and/or high polarity.

• A technique used in mass spectrometry to produce ions
using electrospray.
• A high voltage is applied to liquid to create a result. Infrared spectrum of Cocaine
Atmospheric pressure chemical ionization (APCI)
Morphine (right); Heroine (left)
• Morphine and Heroine have similar structure since

morphine is a derivative of heroine
• For analytes of lower MW and less polarity.
• Utilizes gas phase ion molecule reactions at
atmospheric pressure.
Uses: LC-MS
• Confirm positive test results from screening assays

o Drugs of abuses assays
o Poisoning detection in acute or chronic intoxication
o Therapeutic drug identification and quantitation
(TDM)
o Pharmacokinetic and drug metabolism studies
Infrared Spectrum of Morphine
GC/IR Machine
PHARMACOLOGY AND ANALYSIS OF SPECIFIC

Sample Machine: Liquid Chromatography-Mass Spectrophotometry
DRUGS
• Agents that cause cellular hypoxia
• Alcohols
Gas Chromatography Couples with IR Spectroscopy • Analgesics
(GC/IR) • Agents related to anticholinergic toxidrome
• Agents related to cholinergic toxidrome
• uses IR (or FTIR) spectroscopy • Drugs of abuse
o Infrared or transform infrared • Drugs used in sexual assault
•
Agents that cause cellular hypoxia • Treatment
• Carbon Monoxide o Since it’s a competitive inhibitor, just increase the

concentration of oxygen for it to displace the cyanide
• Cyanide
in the system
• Methemoglobin forming agents
o Get to fresh air
Carbon Monoxide o Rapidly wash body with soap and water
▪ Decontaminating the patient
• Colorless, odorless, tasteless gas
• CO are produced endogenously in the metabolic o Seek medical care
conversion of heme to biliverdin o Remove clothing
• Toxic effect • Analytical methods:
o CO combines tightly with the heme Fe2+ of o Spectrophotometry
hemoglobin to form carboxyhemoglobin. o Headspace gas chromatography
▪ Unable to deliver oxygen in the body
Ethanol
• 250 times greater than that for oxygen - avid or tightly
bound than oxygen • the most widely used and often abused chemical
• Causes hemoglobin-oxygen dissociation curve shift to • central nervous system (CNS) depression
the left. • blood alcohol concentration of 80 mg/dL. (0.08%) has
• Subunit increases the oxygen affinity for the remaining been established as the per se limit (North America)
subunits in the hemoglobin tetramer. • Philippines: 0.05 % will be penalized and for
professional drivers and motorcycle riders, 0.01%.
o In CO poisoning, oxygen cannot be released in the
heme o 0.05% for non-professional driver
o even with 250x affinity, CO locks the O2 in the o According to the RA 1086 or the Anti-Drug Driving
RBCs, decreasing tissue oxygenation Law
• Decreases the oxygen content of blood, it also
decreases oxygen availability to tissue.
• Analytical methods:
o Gas Chromatography (GC)
▪ Gas chromatographic methods measure the
carbon monoxide content of blood and
▪ considered to be reference procedures.
o Spectrophotometry
Cyanide
• One atom of carbon triple-bonded to one atom of • Metabolism of Ethanol - occurs in the hepatocytes of
nitrogen (C=N). the liver
• Toxic effects o (1) In the cytosol, ethanol is converted to
o serum readily crosses all biological membranes and acetaldehyde by the enzyme alcohol
avidly binds to heme iron (Fe3+) in the cytochrome a- dehydrogenase, which is inhibited by fomepizole
a3 complex within mitochondria. o (2) In the mitochondria, acetaldehyde is further
converted to acetate by acetaldehyde
▪ competitive inhibitor that causes decoupling of dehydrogenase which is inhibited by Disulfiram
oxidative phosphorylation.
▪ Result to hypoxia or low concentration to oxygen • Acetaldehyde is responsible for the signs and
in the blood symptoms of ethanol toxicity
• Immediate Symptoms o Disulfiram is a drug used in chronic ethanol abuse

– to prolong the effects of ethanol for them to avoid
o Headache ingesting the agents
o Nausea/vomiting
o Dizziness • Ethanol is a teratogen, and
o Rapid heart rate alcohol consumption
during pregnancy can
• Symptoms of Longer Exposure result in the birth of a baby
o Unconsciousness with Fetal Alcohol
o Convulsions Spectrum Disorder
o Respiratory failure (FASD)
o Coma o Specifically, this
o Death happens when a
mother ingests ethanol in the first trimester (most
crucial part, where major organs are developing)
o FASD is an umbrella term that describes the variety o Recommended to allow for clearance of residual
of effects of an individual whose mother drunk alcohol that may have been present in the mouth
alcohol during the pregnancy from recent drinking, use of alcohol containing
o These effects include physical, mental, behavioral, mouthwash or vomiting of alcohol in which gastric
and learning disabilities with a life-long implication fluid will duplicate the test and perform them 3-10
o It is the characteristic feature of the patients or minutes
babies with drunk mothers.
o Microcephaly – small skull ▪ Must be 20 mg/dL or 0.02% as an additional
safe cord against alcohol contamination
Analysis of Ethanol
Urine Ethanol
• Collection
• Alternative and less invasive for determination of
o Venipuncture site should be cleansed with alcohol alcohol
free and the analyte of interest is ethanol. There • Concentration of alcohol in urine is roughly 1.3 times
should be no contamination. that in blood
• Urine alcohol concentration reflects an average of the
▪ Hence, the use of alcohol-free disinfectant such
blood alcohol concentration during the period in which
as benzene chromium chloride.
urine is collected in the bladder
• Serum, plasma, and whole blood are suitable blood- • Represents ingestion of alcohol within the previous 8-12
related specimens for the determination of ethanol. hours
• Volatile nature of alcohols, specimens should be kept
o Not real time reflection of how much alcohol has
capped to avoid evaporative loss.
been taken
• Enzymatic assay is the method of choice in many o It’s a reflection of the excreted ethanol in the body
clinical laboratories.
• Ethanol is measured by oxidation to acetaldehyde with Analgesics
NAD, a reaction catalysed by Alcohol Dehydrogenase
(ADH). • substances that relieve pain without causing loss of
consciousness because there are agents that are
o Formation of NADH, measured at 340 nm analgesics as well as sedatives
o Same enzymatic reaction happening in the cytosol
of the hepatocytes. o Acetaminophen
o Salicylates
𝐴𝐷𝐻
𝐸𝑡ℎ𝑎𝑛𝑜𝑙 + 𝑁𝐴𝐷 → 𝐴𝑐𝑒𝑡𝑎𝑙𝑑𝑒ℎ𝑦𝑑𝑒 + 𝑁𝐴𝐷𝐻
Acetaminophen
Alcohol Breath Test
• Also known as Paracetamol
• Analgesic and antipyretic actions.
• Another way to measure Ethanol
• Principle o Most people use this for antipyretic or those for
increased temperature
o Alcohol capillary alveolar blood equilibrates with o It’s less used for analgesic or inhibition of pain
alveolar in air with a ratio of 2100:1 (blood to breath o Paracetamol is usually used for pediatric pain
ratio) because it is less toxic compared to other
• Understanding How Alcohol Breath Tests Work: analgesics like the non-steroidal inflammatory
agents
o (1) Alcohol moves from the mouth down the throat to
the stomach • In normal doses, acetaminophen is safe and effective,
o (2) In the stomach and small intestine, alcohol is but it may cause severe hepatic and renal toxicity when
absorbed into the blood that has already been consumed in overdose quantities.
exposed to the lung’s oxygen. o Acetaminophen overdose is common in the ER
o (3) This oxygenated blood carries the alcohol
throughout the body to the brain and lungs. Table 2. Clinical Stages of Acetaminophen Toxicity
o (4) Blood passes in front of the tiny air sacs in the
lungs called alveoli, where the alcohol is transferred
to the lungs and exhaled through the breath. Stage Symptoms Laboratory Findings
o (5) An alcohol breath test measures how much
alcohol is in a person’s breath. 12 hours after
ingestion, LFTs show
▪ The testing device uses that measurement to subclinical rise in
approximate how much alcohol is in a person’s 1 (1-24 hours
Patient asymptomatic or serum transaminase
oxygenated blood (blood alcohol content). reports anorexia, nausea concentrations (ALT,
after ingestion)
▪ An exact measurement is obtained by drawing or vomiting, malaise AST)
blood. • Acetaminophen is
hepatotoxic
• Before testing the presence of alcohol in our breath, the
Anorexia, nausea, and Elevated serum ALT
probation period is 15 minutes. 2 (18-72 hours omitting and right upper and AST, PT, and
after ingestion) quadrant abdominal pain. bilirubin. Renal
Tachycardia and function
hypotension may indicate abnormalities may o Uncontrollable shaking

volume losses. also be present, o Seizures
indicating o Burning throat pain
nephrotoxicity of o Vomiting pain
acetaminophen
Continued nausea and
o Decreased urination
vomiting, abdominal pain, • Trinder spot test
and tender/painful liver.
Hepatic problems may Severe hepatoxicity o Qualitative
3 (72-96 hours
manifest as jaundice, evident on serum o Rapid
coagulopathy, studies, and hepatic o Reaction between salicylate and Fe3+ to form a
after ingestion)
hypoglycaemia, and necrosis is diagnosed colored complex measured at 540 nm
hepatic encephalopathy. on liver biopsy
ARF develops in some • High performance liquid chromatography
critically ill patients. Death
may occur o The reference method to measure salicylate
Upon survival of critical because gas and liquid chromatograph makes most
4 (4 days- 3 illness in stage 3: specific method for these toxins.
weeks after complete resolution of N/A
ingestion) symptoms sand complete Agents Related to Anticholinergic Syndrome
resolution of organ failure.
ALT: alanine aminotransferase; ARF: acute renal failure; AST: aspartate • Tricyclic antidepressants
aminotransferase; LFT: liver-function test; NA: not applicable; PT: prothrombin
time
• Antipsychotics
• Antihistamines
Metabolism of Acetaminophen in the Liver Tricyclic Antidepressants
• (1) Acetaminophen
is conjugated to
glucuronide moiety
and sulfate moiety
o these are
nontoxic
byproducts of
acetaminophen
• (2) P450
• Tricyclic antidepressants (CAs) are named because of
metabolism forms a toxic by product known as N-
their three-ring structure
acetyl-p-benzo-quinone-imine (NAPQI)
• Prescribed for the treatment of depression
• (3) But in the presence of N-acetyl cysteine (NAC),
• The therapeutic mechanism of tricyclic antidepressants
this serves as a glutathione reservoir
is the blockade of neural reuptake of serotonin and/or
• (4) Glutathione antioxidant converts the NAPQI toxic by-
norepinephrine.
product into cysteine and mercapturic acid conjugates
(non-toxic) metabolite o These are the excitatory neurotransmitters, so in
depression, there is a need to increase the
o NAC is the antidote in acetaminophen toxicity or
excitatory neurotransmitters to inhibit or for the
overdose
person to feel not depressed.
NOTE: Acetaminophen or paracetamol is readily available.
• Analytical Methods
This is a very common cause of hepatotoxicity in patients who
are attempting suicide. o Chromatographic methods
o Immunoassay
Salicylates
Antipsychotics
• Acetylsalicylic acid (aspirin) is a salicylate that has
analgesic, antipyretic, and anti-inflammatory properties. • Drugs are generally used for numerous psychiatric
• Symptoms of Aspirin overdose disorders (E.g. Schizophrenia
• Analytical Methods
o Restlessness
o Irritability o Chromatography
o Excessive and unorganized talking o Immunoassays
o Fear or nervousness
o Dizziness Antihistamines
o Confusion
o Abnormally excited mood • Treatment of allergic reactions and as common sleep
o Hallucinations aids
o Drowsiness o Usually, the first-generation antihistamines are in the
o Loss of consciousness form of diphenhydramine
o Double vision o Benadryl (recommended) or Diphenhydramine
▪ has sedative effect, aside from antihistamine • Treatment

property
o Atropine – bronchial secretion
o A lesser sedative effect is observed in cetirizine o Pralidoxime – regenerate cholinesterase
▪ This also used in allergic reaction • Analytical Methods
• Methods of choice for their measurement include o The presence of urinary organophosphate and
o GC-MS/MS carbamate metabolites is generally assessed by
o LC-MS/MS GC-MS and GC-MS/ MS.
Agents Related to Cholinergic Syndrome
• Organophosphate
• Carbamate compounds
Organophosphates
• Organophosphate insecticides are toxic because they

inactivate acetylcholinesterase that are required for
hydrolyzing acetylcholine at nerve junctions
o As a result, this increases the acetylcholine in nerve
junctions
• The autonomic nervous system, which has 3 branches:
o sympathetic (flight or fight)
o parasympathetic (rest and digest), and
o enteric nervous system.
Detection of Drugs of Abuse Using Other Specimen
• It is not provided or easy to obtain serum or even urine

(case-to-case basis)
o Meconium
o Oral fluid
o Hair
o Sweat
Meconium
• The first discharge from newborns, which is a viscous

dark substance contains intestinal secretions, crenated
squamous cells, mucosal pigments, etc.
• Document of proof of the infant’s exposure to illicit
• For sympathetic and parasympathetic, due to increase
drugs
concentration of acetylcholine, the following symptoms
are manifested: • Limitation: Missed collection in low-birth-weight infants
o MNEMONIC: “DUMBBEELLS” o Too long to passed out meconium
▪ Diarrhea • Analytical techniques

▪ Urination o Radioimmunoassay (RIA)
▪ Miosis o Enzyme Immunoassay (EIA)
▪ Bradycardia o Fluorescence Immunoassay (FIA)
▪ Bronchospasm
▪ Emesis • Confirmatory
▪ Lacrimation o Mass Spectrometry
▪ Lethargy
▪ Salivation Oral Fluid (Saliva)
▪ Seizures
• Non-invasive, direct observation minimizing risk of
o MNEMONIC: “SLUDGE “
adulteration
▪ Salivation o By collecting the oral fluid, it is used to observe if it
▪ Lacrimation has been manipulated or if there are agents added
▪ Urination to it compared to other samples or specimens
▪ Diarrhea o Urine can easily be manipulated or adulterated
▪ GI cramps
▪ Emesis
• Disadvantages Ataxia
Blurred vision
o Short window of detection Confusion
o Small volume of sample Diplopia
Dysesthesias
▪ If the machine requires specific (larger) volume Sedative-hypnotic Hypotension
of sample, it is not easy to obtain or it can’t easily Lethargy/ coma
meet the sample volume Nystagmus
Respiratory depression
• Analytical methods: Sedation
o Gas Chromatography-Mass Spectrometry Slurred speech
Agitation
o Liquid Chromatography-Mass Spectrometry
Diaphoresis
o LC-MS/MS Excessive motor activity
Excessive speech
Hair Hallucinations
Sympathomimetic Hypertension
• Use for trace metals (lead, arsenic, mercury) Hyperthermia
o It is useful because melanin contributes to locking Insomnia
Restlessness
trace metals or drugs. Tachycardia
o This reflects chronic toxicity rather than acute Tremor
• Easily obtain, non-invasive, not easily altered
• Analytical method • Sympathomimetic
o Atomic Absorption Spectrometry o It mimics the activity of the sympathetic nervous
system
• Confirmatory
o Anything under the activation of the alpha or beta is
o Gas Chromatography–Mass Spectrometry included in the sympathomimetic toxidrome
o Amphetamine and Methamphetamine – most
Sweat common drug
• Use of sweat patch
• Offers possibility of monitoring drug use over extended
periods without need for frequent collection time
Table 3. Symptoms of the Important Toxidromes
Toxidrome Symptom
Agitation
Blurred vision
Decreased bowel sounds
Dry skin
Fever
Flushing
Hallucinations
Anticholinergic Ileus
Lethargy/ coma
Mydriasis
Myoclonus
Psychosis
Seizures
Tachycardia
Urinary retention
Diarrhea
Miosis
Bradycardia
Bronchorrhea
Cholinergic
Emesis
Lacrimation
Salivation
Urination
Bradycardia
Decreased bowel sounds
Hypotension
Hypothermia
Opioid
Lethargy/ com
Miosis
Shallow respirations
Slow respiratory rate

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[TRANS] LABORATORY UNIT 01: INTRODUCTION TO ENZYMOLOGY

Enzyme Location Nomenclature of Enzymes

• Found in all body tissues • 3 ways of naming enzymes:

• Appear in serum/plasma: • Depending on the substrate upon which the enzyme

▪ Enzymes are needed in the serum or plasma • Dehydrogenase

• Where the substance on which the enzyme acts • Deaminase

CLASSIFICATION OF ENZYMES • Forward Reaction

• Product o Lactate dehydrogenase oxidizes lactate.

o At the right side (2) Transferases

• Addition of a group to a double bond to become a single

• Amylose has a glycosidic bond (α-1,4 glycosidic bond)

o Has 3 isoenzymes, which are composed of 2

• Accompanied by an ATP-ADP conversion or a similar ▪ Electrophoresis – serum is added in the agarose

Isoenzyme vs. Isoforms

• Central Dogma of Life

• Enzymes are proteins

• Apoenzyme + Substrate + presence of cofactor =

• Adenosine triphosphate (ATP) Michaelis – Menten Theory

• Basic tenet of enzyme kinetics Bond Specificity

• The enzyme will only act upon a specific isomer of

o ES Complex [ES] - the physical binding of the  “dehydrogenase” – removal of hydrogen

• How the enzyme work?

• The enzyme can only form a complex to a specific

▪ No matter what molecule, for as long as it o Y axis

Michaelis-Menten Equation • The formula of Michaelis-Menten was reciprocated to

Enzyme Concentration vs Rate of Reaction

• The graph above describes the kinetics of the enzymes

• A mathematical transformation of the Michaelis-Menten

Lock and Key Theory (1) Substrate Concentration

• Are those which proceed at a rate exactly proportional

• The reactions are zero-order with respect to the

(2) Enzyme Concentration

• The higher the enzyme level,

• Proper substrate binding

• Second substrates of enzymatic reactions

• Interfere with enzyme reactions

Competitive Inhibition of Enzymes

• Competitive inhibitor competes with the substrate for

• When in high concentrations, the allosteric inhibitor o Vmax = 1 (unchanged)

o This changes the shape of the enzyme, thus • Purple curve –

• The more substrates added: o Vmax = 1 (unchanged)

o Vmax = 1 (unchanged) o If the serum of the patient is added to the reagent,

• Black line – uninhibited reaction o The rate of the increase in concentration

MEASUREMENT AND ASSAYS

• Ways to measure the enzyme activity in the lab:

Continuous-Monitoring Method (Kinetic Assay)

Common Cause of Deviation from Linearity

• Very high concentration of enzymes

Calculation of Enzyme Activity

International Unit (IU)

• Amount of enzyme that will catalyse the reaction of 1

International Unit per Liter (IU/L)

Katal Unite (mole/s)

• Amount of enzyme that will catalyse the reaction f 1

▪ Substrates are bigger molecules.

Specimen Collection, Storage, and Sources of Error • Reaction Principle

Table 4. Procedure Table 6. Reference Values

Pipette into cuvettes 25°C 37°C Values

Sample 20 µl 10 µl Temperature 25OC 37OC IFCC

Serum, plasma 120 U/I 220 U/I 28-100 U/I

U/I (25°C) = ∆𝐴/ min 𝑥 9864 Properties

o Lipase arises early and persist for up to 8 days as • Optimum pH:

Reference Range Diagnostic Significance