Subject: Medical Technology Assessment Program

Lecturer: Sir Roderick Balce

Lecture #1: Clinical Enzymology

Enzyme Classification and Nomenclature

Enzyme Classes Reaction Catalyzed Examples Additional Comments

Oxidoreductases Oxidation-Reduction between two Ceruloplasmin • Transfers Electrons, Hydrogens or Oxygens

substrates Lactate Dehydrogenase from a reductant molecule (Donor) to an
acceptor (oxidant)
• Usually use Cofactors, NADP, or NAD+
Transferases Transfer of Functional Groups Aspartate Transaminase • Catalyzes the transfer of a functional group
Alanine Aminotransferase other than hydrogen from one substrate to
another
Hydrolases Hydrolysis Peptidases • Catalyzes various bonds due to reaction with
Phosphatases WATER (hydrolysis)
Lipases like LPL • Natural Function: To break down
Nucleosidases Nutrients into smaller units for Digestion
Glycosidases
Lyases Removal of Groups from substrates Aldolase • Enzymes that catalyzes the breaking of a
to form Double Bonds Amylase chemical bond between two parts of a
molecule through biochemical means other
than hydrolysis and oxidation
• They usually form a DOUBLE BOND or
add a NEW RING STRUCTURE
Isomerases Interconversion of Geometric, Triose Phosphate Isomerase • Catalyzes the isomerization changes in a
Optical, and Positional Isomers molecule
• Aids in the conversion of a chemical
compound from one isomer to another
Ligases Bond formation coupled with ATP Glutathione synthetase • Catalyzes the joining of two substrate
hydrolysis molecules
• Coupled with the breaking of the
PYROPHOSPHATE BOND in ATP or a
similar compound

NOTE
Enzyme Kinetics
Most of the enzymes’
Enzymatic Reaction/Catalytic Mechanism names end in “-ase.”

- Since there are some chemical reactions that may Like: Lactate
occur at a slow rate, they needed enough kinetic Dehydrogrenase
energy to drive the reaction of an uncatalyzed
reaction Michaelis-Menten Equation
- Enzymes then intend to catalyze physiologic reactions
by lowering the activation energy (EXCESS
ENERGY)
- The lower the activation energy, the faster the
reaction
o Activation Energy – The excess energy that
was required to raise all molecules in 1 mole
of a compound at a certain temperature to its
transition state at the peak of the energy
barrier
 o The final reaction of an enzyme reaction
o Process: The substrate concentration from the
- Performed under the conditions of zero-order reactions first-order kinetics will soon be high enough to be
- Related to the Relationship of the substrate able to saturate all of the available enzymes
concentration with the velocity of the reaction ▪ The Maximum Velocity is reached
o Michaelis-Menten Constant (𝑲𝒎 ) o Product: The resultant free enzyme will
 derived from the theory of the immediately combine with excess free substrate
Michaelis-Menten Equation o The reaction rate depends only on enzyme
 a Constant for a specific enzyme concentration
and substrate under defined
reaction conditions
Expressions of Enzyme Activity
 indicates the amount of substrate
needed for a particular enzymatic IU (U)
reaction
• Also known as International Unit
Lineweaver-Burka Plot • 1 micromole of substrate per minute

Katal Unit (KU)

• 1 Mole of substrate/second

Other Factors that Influence Enzymatic Reactions

pH

• Changes in this factor will cause a denaturation in the

enzyme or influence its ionic state, which will result in:
- A double reciprocal plot of the Michaelis-Menten o multiple structural changes
constant o change in the charge of an amino acid residue in
- A Plot that yields a straight line the active site
- Taken both of the substrate concentration and the • Each enzyme operates in a specific pH range and maximally
velocity of an enzymatic reaction at a specific pH
• NOTE: Most physiologic enzymatic reactions occur in the
The Equation of Michaelis-Menten along with the pH range of 7.0-8.0
Lineweaver-Burke Plot becomes: • The pH for a reaction in the laboratory is carefully controlled
at an optimal pH by means of buffer solutions

Temperature
Effect of Substrate and Enzyme Concentration
• Increasing temperature usually increases the rate of a
• First Order Kinetics
chemical reaction by increasing the movement of molecules
o Initial or First Reaction of an Enzyme Reaction
• 37 degrees Celsius is the optimum temperature for
o Process: The substrate readily binds to the free
enzymatic activity.
enzyme at a low substrate concentration
• For each 10 degrees Celsius in temperature, the rate of
o Result: As the reaction rate increases steadily
the reaction doubles
due to the addition of more substrate, the amount
• Enzymes tend to be active at 25,30, or 36 degrees Celsius
of enzyme exceeds the amount of enzyme
o The reaction rate is directly proportional to the • The denaturation increases as temperature increases and is
substrate concentration significant at 40 to 50 degrees Celsius
• 60 to 65 degrees Celsius – results to inactivation of the
• Zero Order Kinetics enzyme
 o The inhibition may be REVERSIBLE because
some naturally present metabolic substances
combine reversibly with other enzymes
Cofactors ▪ it may also be IRREVERSIBLE if the
inhibitor destroys part of the enzyme
• Nonprotein entities that bind to particular enzymes before a
that is involved in catalytic activity
reaction occurs
o It causes a decrease in enzyme activity due to the
Activators configuration of the enzyme in a way to reduce or
abolish excess substrate to the active site
o Inorganic ions that alters the spatial configuration o Competes with the Regulator Molecules like the
of the enzyme for proper substrate binding cofactors
o Common Activators o Addition of substrate has no effect since it cannot
▪ Metallic (Calcium, Iron, Magnesium, reverse the alteration caused by inhibitor
Manganese, Zinc, and Potassium)
▪ Nonmetallic (Bromine and Chloride)

Coenzymes

o Organic cofactors that serve as second

substrates for enzymatic reactions
o They are Nucleotide Phosphates and Vitamins
▪ Prosthetic Groups – when a coenzyme
is tightly bound to an enzyme

Inhibitors
Uncompetitive Inhibitor
• An ion or enzyme that interferes with a reaction
o It binds straight to the enzyme-substrate
Competitive Inhibitors complex
o Physically binds to the active site of an enzyme o E + S = ES = E+P
and competes with the substrate for the same site o When we increase the substrate concentration, it
o With a substrate concentration that is significantly will result into more ES complexes where the
higher than the concentration of an inhibitor, this inhibitor binds and will increase the Inhibition
inhibition is REVERSIBLE ▪ This meant that the more substrate
▪ This is because the substrate is more concentration is increased, the more
likely than the inhibitor itself to bind to its inhibition will increase
the active site and the enzyme will not o The complex doesn’t yield any product
be destroyed

Noncompetitive Inhibitors
o Binds the enzyme at the allosteric site of the
enzyme
Enzymes of Clinical Significance ▪ It is negligible in the Normal
Serum/Plasma (not found in significant
amounts because it is found or
abundant in the CNS)
Creatine Kinase ▪ It is elevated in CNS damage, tumors
and childbirth
• Tissue Sources: Brain, Heart (Cardiac Muscle) & Skeletal ▪ Associated with the presence of Macro-
Muscles CK
o It is important to keep the tissue sources in mind
 atypical isoform of CK
because they vary with the diagnostic significance
 Migrates cathodal
• One of the enzyme markers used to detect myocardial
 A CK-BB complex with IgG
infarction
• Diagnostic Significance o CK-MB (CK2)
o Note: All are mostly related to the heart or the ▪ One of the specific enzyme markers to
skeletal muscles detect myocardial infarction
o Pronounced Elevation: > or = 5x ULN (Upper ▪ Present in significant amounts in the
Limit or Normal) myocardium or cardiac tissues
▪ Diseases under the Pronounced ▪ It is comprised in the Normal
Elevation of CK: Serum/Plasma with < 6% of the total CK
 Duchenne’s Muscular
Dystrophy -Highest o CK-MM (CK3)
associated source of CK ▪ Most Abundant isoenzyme in Normal
elevation Serum/Plasma
 Polymyositis ▪ Major isoenzyme found in straited
 Dermatositis muscle
 Myocardial Infarction ▪ In Normal Serum, it is comprised of 94-
o Increase: 4-6 hours 100% of the total CK
o Peak: 12-24 Hours ▪ Its abundance is the reason why its
o Normalizes: 48-72 elevation is significant for skeletal
Hours (2-3 days) muscle disorders

o Mild to Moderate Elevation: 2-4x ULN (Upper o CK-Mi

Limit or Normal) ▪ Mi = Mitochondrial CK
▪ Elevation is consistent with: ▪ Abnormal Minor isoenzyme that
 Acute Agitated Psychosis migrates cathodal to MM in
 Alcoholic Myopathy electrophoresis
 Severe/Intensive Exercise ▪ Detectable and Associated with
 Delirium Tremens extensive tissue damage
 Pulmonary Infarction • Electrophoretic Mobility of CK Isoenzymes
 Severe Ischemic Injury
 Intravascular Injections
 Muscular Trauma
 Hypothyroidism

• Isoenzymes/Major or Normal Isoenzymes of CK

o CK1, 2, 3 are based on their electrophoretic
mobility (CK1 – Most Anodal, CK2 – Middle, CK 3 o - is Cathode and + is Anode
– Most Cathodal) o If CK is normal, CK-MM migrates cathodal, CK-
MB migrates in between, while CK-BB migrates
o CK-BB (CK1) anodal
▪ Isoenzyme localized in the brain
 o If CK Electrophoresis includes abnormal
isoenzymes, then Macro-CK will travel in between
CK-MB and CK-MM since it becomes cathodal
when in combination with IgG while CK-Mi
migrates towards the cathode and beyond the
point of origin

• CK Determination
o Specific Considerations
▪ Avoid use of chelating agents because it
will remove magnesium, which is its ▪ Oliver-Rosalki
activator. Don’t use EDTA • Reverse CK reaction
▪ Use Serum or Heparinized Plasma • Its process acts in reverse of
▪ Avoid prolonged storage because CK is the Tanzer-Gilvarg
labile if stored 1. Creatine Phosphate + ADP
 CK-1 is most labile will have a major reaction in
▪ Avoid hemolysis even if CK is not the presence of Creatine
affected by hemolysis, but because Kinase
reverse CK will be falsely increased if o This produces
the hemolysis gross or heavy Creatine and ATP
2. ATP will react with Glucose
o Methods for Total CK determination (The Hexokinase Method from
▪ Tanzer-Gilvarg Assay Carbohydrate Determination)
 Forward/Direct CK reaction o They will react in the
1. Creatine is phosphorylated presence of
and is majorly reacted with Hexokinase
ATP in the presence of CK o These will produce
(Creatinine Kinase) ADP and glucose-6
o This produces phosphate
Creatine phosphate 3. G6P will react then with NADP
and ADP in the presence of Glucose-6
2. Then, ADP will be majorly Phosphate Dehydrogenase
reacting with o This will produce 6-
phosphoenolpyruvate in the phosphogluconolact
presence of PK (Pyruvate one and NADPH
Kinase) o NADPH – The
o This produces reduced form of
Pyruvate and ATP NAD which
3. Pyruvate then reacts with maximally absorbs
NADH in the presence of LDH at 340 nm
(Lactate Dehydrogenase)
o This produces
Lactate and NAD
o NAD – The product
that must be
detected to monitor ▪ Total CK Determination – its
the CK level at determination is dependent on the
340nm method either measuring the minimum
or maximum absorption. Its
measurement is based on the oxidized
or the reduced form of NAD or NADP
 o Reference Values it’s indicative of damage in any of the organs it’s
▪ Male: 15-160 IU/L localized at
▪ Female: 15-130 IU/L o Conditions indicative of the “Flipped Pattern:”
▪ Myocardial Infarction
Lactate Dehydrogenase ▪ Hemolytic disorders
 including Megaloblastic
• Least tissue-specific enzyme; least useful clinically because
Anemia and Pernicious
low tissue specificity decreases its utility clinically
Anemia which are the source
• The enzyme may be used for isoenzyme analysis but not for
of highest LD elevation
total enzyme level measurement since it is not reliable due
because of RBCs and its
to there are other tissues that secrete LD
precursors are all destroyed in
• Consists of 4 polypeptide chains and filled with mixture of H
the bone marrow
& M polypeptides
(Intramedullary Destruction
• Tissue Sources: Heart, RBCs, Renal Cortex, Lungs, Liver, of RBC precursor)
Skeletal Muscle ▪ Renal Infarction
• Isoenzymes: (Named by its migration in electrophoretic
mobility from Cathodal to Anodal) • Diagnostic Significance of LD
o LD-1 o Pronounced elevation
▪ Most cathodal or the fastest in ▪ 5x or more than the Upper Limit or
Electrophoresis Normal
▪ Also known as H4 ▪ Conditions Consistent with Elevation:
▪ Localized and is much abundant in
 Megaloblastic Anemia
Heart, RBCs and Renal Cortex
 Pernicious Anemia
o LD-2
 Renal Infarction
▪ Also known as H3M
 Systemic Shock and
▪ localized in Heart, RBCs and Renal
Hypoxia
Cortex with LD-1 being much more
 Hepatic Metastases
dominant
o LD-3  Hepatitis
▪ Also known as H2M2 o Moderate Elevation
▪ Localized in Lungs and are much more ▪ 3-4x than the Upper Limit or Normal
abundant ▪ Conditions Consistent with Elevation:
o LD-4  Myocardial Infarction –
▪ Also known as HM3 Elevation is not Pronounced:
▪ Most Abundant in the Liver and Skeletal o Increases at 12-24h
Muscles o Peaks at 48-72h
o LD-5 o Remains elevated
▪ Most anodal or slowest in up to 10 days
Electrophoresis  Hemolytic Conditions
▪ Also known as M4  Pulmonary Infarction
▪ Most Abundant in the Liver and Skeletal  Muscular Dystrophy
Muscles with LD-4  Delirium Tremens
• In a normal serum, LD2 is most abundant, followed by LD1  Leukemia
and smaller amounts of other isoenzymes  IM
o The Pattern in Normal Serum is always the
opposite with the intracellular environment of the o Slight Elevation
localized tissue areas or sources ▪ Up to 3x than the Upper Limit or Normal
o This pattern is known as Flipped Pattern in Serum ▪ Conditions Consistent with Elevation:
o It meant that the normal pattern is reversed  Most Liver Diseases
o The flipped pattern is known as LD1 > LD2, if it’s  Nephrotic Syndrome
in the intracellular, it is normal, but if it’s in serum,  Hypothyroidism
 Cholangitis
• LD Determination Aminotransferases/Transaminases
o Specimen Considerations
▪ Avoid Hemolysis because LD will be Aspartate Aminotransferases (AST)
greatly affected by it and cause a flipped
• Also known as: Serum glutamic-oxaloacetic transaminase
pattern
(the old name of AST)
▪ Recommended Storage: 25 degrees
• Along with ALT, it is not usually determined in the laboratory
Celsius (Room Temperature)
but it is useful to clarify discrepancies in terms of AST/ALT
 Do not freeze the samples for
ratio in cases of Alcoholic Liver Disease which is associated
LD because LD is cold-labile
in the elevation of one of the isoenzymes: Mitochondrial
with LD-5 being most-cold
AST
labile
• Tissue Sources: Liver, heart, and Skeletal Muscle
▪ Do not use EDTA because it inhibits LD
o The tissue sources are varying on the diagnostic
activity
significance
o Methods
▪ Wacker • Diagnostic Significance
o Pronounced Elevation
 Forward method
▪ Greater than or Equal to 5x of Upper
 Lactate is made to react with
Limit or Normal
NAD+ in the presence of
▪ Elevation is Consistent with
Lactate Dehydrogenase to
Conditions:
produce Pyruvate, NADH and
 Acute Hepatocellular
Hydrogen
Disorders
o Like CK, it measures
 Circulatory Collapse
the increase in
absorbance at  Myocardial Infarction
340nm o Increases at 6-8h
o Peaks at 18-24h
 pH: 7.1-7.4
o Normalizes within 4-
5 days
o Order of Elevation in
Plasma or Serum:
▪ Wroblewski-Ladeu
Myoglobin,
 Reverse Method
Troponin Iron/T,
 Pyruvate is made to react with
CK-MB, AST, LD
NADH and Hydrogen in the
o Moderate Elevation
presence of Lactate and
▪ 3-5x greater than the Upper Limit or
NAD+
Normal
o Like CK again, it
▪ Elevation is Consistent with
measures the
Conditions:
decrease in
 Muscular Dystrophy
absorbance at
340nm  Hepatic Tumor
 Biliary obstruction
 pH: 8.3-8.9
 Congestive Heart Failure
 Cardiac Arrhythmia
o Reference Values: 100-225 U/L o Slight Elevation
▪ Up to 3x greater than the Upper Limit or
Normal
▪ Elevation is Consistent with
Conditions:
 Cirrhosis
 Pericarditis
 Pulmonary Infarction
 Cerebrovascular Accident
• Isoenzymes ▪ Avoid Hemolysis because it will falsely
o Cytoplasmic AST elevate levels
▪ The typical isoform of AST that is ▪ Recommended Storage time:
normally determined in plasma/serum Refrigerated for 3-4 days
▪ First isoenzyme to be released after o Methods
tissue damage ▪ Reitman-Frankel
▪ For most of the conditions that are  Colorimetric
consistent with the elevation of AST, it is Spectrophotometry
mostly referred to this isoenzyme  Uses 2,4-DNPH (Color
Developer) and 0.4 N NaOH
o Mitochondrial AST (Color Intensifier)
▪ The isoform that is only released in
association with extensive tissue ▪ Coupled Enzymatic Reaction
damage o More commonly employed
▪ Same significance as Mitochondrial AST technique in ALT/AST
▪ For most Acute Hepatocellular determination
disorders, the De Ritis Ratio (the ratio o Remember the old names of
of AST/ALT) is always less than 1, ALT and AST to memorize
which meant that ALT level is higher in the process
AHCDs o SGOT is AST since O means
 But in Alcoholic Liver Oxaloacetate, it meant that
Disease, it is characterized by Aspartate’s reaction with a-
the elevation of mitochondrial ketoglutarate will produce
AST with a very long half-life oxaloacetate and glutamate
 It causes the De Ritis Ratio to o SGPT is ALT since P means
be greater than 3 Pyruvate, it meant that
Alanine’s reaction with the
Alanine Aminotransferase same enzyme as Asparate’s
(a-ketoglutarate) will produce
• Also known as Serum Glutamic Pyruvic Transaminase
Pyruvate and Glutamate
• Tissue Source: Predominant in the Liver o The new names tell us about
• Diagnostic Significance the reactants, while the old
o Acute Hepatocellular Disorders names tell us about the
• More liver & tissue-specific than AST products
• A better marker for AHDs because there are no other o Either Oxaloactate or Pyruvate
available marker and is characterized by higher and more will react with NADH and
sustained elevation Hydrogen in the presence of
o Elevated levels may persist for a long time Malate Dehydrogenase (AST)
o It is because ALT has a longer half-life of 47 or Lactate Dehydrogenase
hours compared to Cytoplasmic AST but not (ALT)
longer than Mitochondrial AST o It will produce Malate (AST) or
o The longer the half-life, the longer it will stay in the Lactate (ALT), but NAD will be
circulation the one to be measured
o It causes the De Ritis Ratio to be always less than because it will be detected
1 because ALT is always higher than AST in spectrophotometrically at
ACHDs except Alcoholic Liver Disease wherein 340nm
the opposite result happens due to the elevation of
Mitochondrial AST, which has an even longer half-
life than ALT

• AST/ALT Determination o Reference Values

o Specimen Considerations ▪ AST: 5-35 U/L
 ▪ ALT: 7-45 U/L ▪ NOTE: If abnormal isoenzyme is
included, Reagan ALP is much more
Alkaline Phosphatase heat stable than Placental ALP
o Chemical Inhibition
• Tissue Sources (Most Notable): Liver, Bone, Placenta,
▪ Urea: inhibits Bone ALP
Intestines
▪ Phenylalanine: inhibits Placental and
o Liver and Bone – most diseases indicative of
Intestinal ALP
elevation of Alkaline Phosphatase
▪ Levamisole: inhibits both Bone ALP and
• Diagnostic Significance Liver ALP
o ALP is elevated in Hepatobiliary Disorders
▪ Leucine: inhibits the abnormal
characterized by intra/extra-hepatic cholestasis
isoenzyme: Nagao ALP
o Pronounced Elevation
o Abnormal Isoenzymes: associated with
▪ Equal to or Greater than 5x the Upper
Carcinomas/Tumors; not cancer-specific
limit or Normal
▪ Regan
▪ Elevation is consistent with:
 more heat stable than P-ALP &
 Bile Duct Obstruction
inhibited by phenylalanine
 Biliary cirrhosis
 associated with Ovarian, Lung,
 Paget’s Disease Breast and Gynecologic cancers
 Osteogenic Sarcoma ▪ Nagao
 Hyperparathyroidism  Variant of Regan isoenzyme
o Moderate Elevation  Associated with Metastatic
▪ 3-5x higher than the Upper Limit or Carcinoma of the Pleural Surfaces,
Normal Adenocarcinoma of the Pancreas
▪ Elevation is consistent with: and Bile Duct
 Granulomatous or Infiltrative ▪ Kasahara
diseases of the liver  Associated with Hepatoma and
 IM (Infectious Mononucleosis) some G.I. tumors
 Metastatic bone tumors • ALP Determination
 Rickets o Specimen Considerations
 Osteomalacia ▪ Avoid Hemolysis (It will falsely increase
ALP levels)
o Slight Elevation ▪ Avoid prolonged storage time or
▪ Up to 3x higher than the Upper Limit or standing time (the pH will be falsely
Normal increase especially if tube is uncapped
▪ Elevation is consistent with: and will cause the escape of CO2; the
 Viral hepatitis pH increase will encourage of ALP
 Cirrhosis activity and falsely increase)
 Healing Bone Fractures o Reference Method: Bowers-McComb
 Growing Children ▪ Uses para-nitrophenyl phosphate as
 Pregnancy (because of substrate and same as Bessy, Lowry
placental isoenzyme of ALP) and Brock
• Isoenzyme Analysis ▪ End Color is YELLOW (para-nitrophenol
o Isoenzymes are named after the tissue sources from the hydrolysis of substrate)
o Analysis is done to be more tissue specific ▪ Optimal pH is 9-10
o Electrophoresis: Migration is from Anode to ▪ Assay requirements: Includes addition
Cathode: Liver ALP, Bone ALP, Placental ALP, of magnesium ions (co-factors), 2-
Intestinal ALP amino-2-methylpropanol buffer (Subject
o Heat stability: From most heat stable to heat to end product inhibition)
labile: Placental ALP, Intestinal ALP, Liver ALP, o Other Methods: same principle as Bowers-
Bone ALP McComb but employ different substrates;
Colorimetric/Spectrophotometric Methods
 ▪ Bessy, Lowry, and Brock – uses Para- ▪ Patient must be instructed not to take pain
nitrophenyl phosphate; relivers due to it being the cause of
▪ King-Armstrong – uses phenyl inappropriate of release of salivary amylase
phosphate; product is (Falsely Increase)
▪ Bodansky, Sinowara, Jones, Reinhart – o Methods
uses beta-glycerophosphate ▪ Amyloclastic
▪ Moss – uses alpha-naphthyl phosphate • Involves the measurement of the
▪ Huggins-Talalay – uses phenolphthalein decrease in the intensity of the
diphosphate color of starch iodine complex
▪ Klein, Babson and Reed – uses buffered • Proportional disappearance of blue-
phenolphthalein phosphate black color of the complex due to
o Reference Values: 30-90 U/L amylase acts in the starch
▪ Sacchrogenic
Pancreatic Enzymes
• Classic reference method
Amylase Lipase • Reported in Somogyi units
Tissue Sources Pancreas & Salivary Glands Pancreas o the amount of amylase
Diagnostic Acute Pancreatitis Acute Pancreatitis required to produce the
Significance Mumps (Elevation persists for equivalent of 1 milligram
Parotitis 8 days) of glucose
Macroamylasemia • Determines the amount of reducing
sugars as starch is hydrolyzed by
(Elevation persists for 2-3
days) AMS
Isoenzymes Pancreatic Amylase None • Measures the increase in
Salivary Amylase reducing agents’ form after
hydrolysis
Other • Macroamylasemia • Similar to ALT ▪ Chromogenic
Comments (Amylase bound to a and is more • Involves the measurement of the
protein/globulin) tissue (Pancreas)
increase in color intensity of soluble
o Decreases specific
Amylase’s dye-substrate solution
half-life • Higher and More • As amylase acts on the substrate,
o Becomes too Sustained the dye-substrate solution becomes
large enough Elevation for soluble and is associated as the
for glomeruli Pancreas increase in color intenstiy
to filter out
▪ Coupled-Enzymatic/Continuous-
• Supposedly, the
Monitoring Method
smallest enzyme
• Normally excreted in • Involves the measurement of
urine increase in absorbance at 340nm
• Earliest Pancreatic • Coenzyme in interest: NADH
Marker but Earliest to o Reference Values:
Normalize ▪ Serum: 25-130 U/L
• To differentiate ▪ Urine: 1-15 U/L
isoenzymes, use
inhibition assay
o Salivary • Lipase Determination
Amylase is o Specimen Considerations
inhibited by ▪ Avoid use of hemolyzed sample (Falsely
wheat germ: decreased because Hgb interferes with lipase
lectin activity)
• Amylase Determination ▪ Recommended Storage Time: 3 weeks
o Specimen Considerations ▪ Refrigerate the sample if it will be analyzed
▪ Do not use lipemic sample because late
triglycerides inhibit Amylase (Falsely
decrease)
 o Methods the bone is the source of
▪ Cherry-Crandall Method elevation
• uses olive oil as substrate as the
Best Indicator of Alcoholic Liver
source of Triglyceride
Disease (GGT is elevated 5x than
• Triglyceride hydrolyzes in the Upper Limit Unit)
presence of Lipase to Glycerol and 5’ Nucleotidase Indicative of: Intrahepatic
Fatty Acids Cholestasis (More Commonly
• The fatty acids are titrated in the associated)
original, but in the new method, it is
Used to identify the source of
in the detection of glycerol
increased ALP
• Same reaction as those tests in the • If 5’NT and GGT is increased,
triglyceride part of lipid profile but then the liver is the source of
lipase is used as the enzyme not as the elevation
the reagent in this determination • If Normal GGT and 5’NT, then
• You can couple lipase with the bone is the source of
peroxidase, which will perform elevation
coupled peroxidase reactions
Acid Phosphatase Tissue Source: bone, liver, spleen,
• You can do Trinder assay or kidney, erythrocytes, and platelets
absorb the chain of reactions at
340nm Elevation is associated with:
• Prostatic
carcinoma/hyperplasia
• Resolution of Rape Cases
▪ Turbidimetric Method • Paget’s disease
• Involves in the measurement of the • Gaucher’s disease
rate of clearing as fats are • Idiopathic Thrombocytopenic
hydrolyzed by LPS Purpura
▪ Colorimetric Methods
Though elevation is indicative of
• Based on the coupled reactions Prostate CA, Prostatic-Specific
with peroxidase or glycerol kinase Antigen is better because it is
• Modification of the Cherry-Crandall specific to prostate
Method wherein glycerol is
detected/determined using glycerol Specimen Considerations:
kinase and peroxidase coupled • Avoid Hemolysis (ACP is
present in RBCs)
reaction
• Maintain Anaerobic Collection
(Keep the tube capped at all
o Reference Value: 0-1.0 U/mL times) If not, pH will be
increased and ACP will lose its
Miscellaneous Enzymes activity
• Freeze the Sample if delayed
Gamma Glutamyl Transferase Indicative of: analyzation
• Hepatobiliary disorders,
• Alcoholic Liver Disease Laboratory Diagnosis:
(Source of Highest Elevation of • Other methods under ALP may
GGT), also be used but Roy Method
• Microsomal induction by drugs is better
and alcohol • Roy Method
- Substrate is
Used to Identify the source of thymolphthalein
Increased ALP monophosphate;
• If 5’NT and GGT is increased, - most specific for ACP
then the liver is the source of • Chemical Inhibition Assay
the elevation - Use tartrate (Inhibit
• If Normal GGT and 5’NT, then Prostate ACP),
 formaldehyde and
copper (Inhibit RBC
ACP) Aldolase Elevations are associated with the
Glucose-6 Phosphate Associated with: indication of skeletal muscle
Dehydrogenase • Drug-Induced Hemolytic disorders
Anemia Glutamate Dehydrogenase Elevations are associated with the
o usually associated indication of hepatic disorders
with the decreased
levels or deficiency
of the enzyme Lecture #2: Electrolytes and Blood Gases
o episodes of
intravascular Functions of Electrolytes
hemolysis following
intake of certain • Volume and Osmotic Regulation
drugs, food, or o Function of Sodium, Potassium and Chloride Ions
substances
• Myocardial Rhythm, Contractility and Neuromuscular Excitability
containing oxidants
o o Function of Potassium, Calcium and Magnesium
• Cofactors in Enzyme Activation
Deficiency of the enzyme is usually o Function of Calcium, Magnesium, Zinc, and Chloride
screened in the Newborn Screening • Regulation of ATPase-ion pumps
Profile o Function of Magnesium
Most Common Deficiency in the • Blood Coagulation
Philippines (Incidence is highest) o Function of Calcium and Magnesium
• Production and use of ATP from glucose
Function is related to pentose o Function of Magnesium, Phosphate
phosphate pathway/hexose • Maintenance of Acid-Base Balance
monophosphate pathway
o Function of Bicarbonate, Potassium, Chloride
If you are G6PD deficient, the
Sodium
reducing potential of RBCs is
reduced, it causes to them not
being able to counter oxidative • Most abundant and Major extracellular cation
stresses and the RBCs become • It represents 90% of all the extracellular cations in the plasma
prone to hemolysis • Largely determines the osmolality of plasma; Major contributor to
plasma osmolality
Significant in RBCs especially when
decreased Methods of Laboratory Determination

Specimen: • Ion-Specific Electrode: Component is Glass

• EDTA whole blood (For o Direct
confirmation)
 There is no sample dilution
• in Newborn: Capillary Blood
Pseudocholinesterase Indicative of: o Indirect
• Pesticide Poisoning  Requires sample dilution
- Chronic Exposure to  Prone to pseudohyponatremia when the levels of
Organophosphates lipids and proteins are high in plasma and sodium
• Liver Disease is low
• Genetic Variants of • Flame Photometry
Pseudocholinesterases
o Sodium atoms follow excitation, which produces or emits
Significant in Serum when yellow light
decreased • Albanese-Lein
o Traditional Colorimetric method or of Sodium Determination
Deficiency makes you sensitive to
certain muscle relaxants:
succinylcholine or mivacarium
(used in anesthesia)
Reference Values: • Dehydration
• Profuse Sweating
• Normal: 136-145mmol/L or mEq/L • Diabetes Insipidus
• Panic: < or equal to 120 mmol/L for Hyponatremia; > or equal to 160 • Renal Tubular Disorder
mmol/L for Hypernatremia
• Conversion factor from mmol/L to mEq/L: 1 Potassium
• Panic values must be taken note of in terms of electrolytes and blood
gases because the slight decrease or increase from the normal may • Major Intracellular Cation
be detrimental • Concentration: 20x higher inside the cells

Clinical Significance Methods

• Hyponatremia • Ion-Selective Electrode: Component is Valinomycin

o Increased Sodium loss • Flame photometry: Potassium ions following excitation emits Violet
 Meant that Sodium level is low because it is Light
excreted in urine or gastrointestinal tract • Lockhead-Purcell: Colorimetric Assay
 Conditions Associated:
Reference Values:
• Addison’s disease
• Salt-losing nephropathy • Normal: 3.5-5.1 mmol/L (serum); 3.5-4.5 mmol/L (Plasma)
• Ketonuria (Diabetic Ketoacidosis) o NOTE: The level of Potassium in Serum is higher than
• Prolonged Vomiting or Diarrhea plasma because of the release of potassium in platelets
• Severe Burns when the blood clots
• Diuretics • Panic: < or Equal to 2.8 mmol/L; > or equal to 6.2 mmol/L
• Conversion factor from mmol/L to mEq/L is: 1
o Increased Water Retention
 Dilutional hyponatremia Clinical Significance
 Appears if sodium level is low because plasma is
diluted as a result of increased water retention • Hypokalemia
 Conditions Associated: o Cellular Shift
• Renal Failure  Influx of Potassium
• Congestive Heart Failure  Potassium outside the cell enters and causes a
decrease in potassium level
• Nephrotic Syndrome
 Conditions Associated:
• Hepatic Cirrhosis
• Alkalosis
o Water imbalance • Insulin Overdose
 Conditions Associated: o Gastrointestinal Loss
• Increased Water Intake  Loss of Potassium in Gastrointestinal Tract
• SIADH  Conditions Associated:
• Pseudohyponatremia • Vomiting
• Diarrhea
• Hypernatremia • Gastric Suction
o Increased Sodium intake or retention • Laxatives
 Absolute Hypernatremia • Malabsorption
 Conditions Associated: o Renal Loss
• Overdose of Sodium Bicarbonate  Loss of Potassium in Urine
(intake)  Conditions Associated:
• Hyperaldosteronism (retention) – • Renal Tubular Acidosis
opposite of Addison’s disease • Hyperaldosteronism
o Increased water loss • Thiazide Diuretics
 Relative Hypernatremia o One class that leads to
 Conditions Associated: hyperkalemia
• Hyperkalemia
o Cellular Shift • Cotlove Chloridometry
 Efflux of Potassium o uses the principle of coulometry
 Release of Potassium from the Cells, which o involves the use of amperometric coulometric titration
increases the plasma potassium level  Coulometric Determination, Coulometric Titration,
 Usually characterized by the damage, injury, and and Amperometric detection
destruction of cells • Meter in Amperes serves as the
 Conditions Associated: detector system
• Acidosis: decrease in pH level by 0.1 • Schales-Schales method
causes an increase in potassium by 0.2- o Also known as mercurimetric titration method
1.7 mmol/L o Uses as diphenyl carbozone as indicator
• Chemotherapy o End Product is blue violet color
• Muscle Injury
• Leukemia • Gibson and Cooke’s Pilocarpine iontophoresis
• Hemolysis o Gold Standard for Sweat Chloride Determination

Reference Values
o Increased Potassium Intake
 Conditions Associated: • Normal: 98-107 mmol/L (serum)
• Oral or IV infusion/ potassium • Panic: < or Equal to 80 mmol/L for Hypochloremia; > or Equal to 120
replacement mmol/L for Hyperchloremia
o Decreased Renal Excretion • Conversion factor to mEq/L: 1
 If the kidney fails, then potassium will be excreted
too much and not be regulated by aldosterone Clinical Significance
 Aldosterone:
• Promotes sodium reabsorption that • Hypochloremia
increases plasma sodium o Results from Chloride Loss in Urine, which results to low
• Excretes Potassium that decreases plasma chloride levels
plasma potassium  Aldosterone deficiency (Addison’s Disease)
 Conditions Associated:  Salt-losing Nephropathy
• Renal Failure  Diabetes Ketoacidosis
• Hypoaldosteronism  Prolonged Vomiting
• Potassium Sparring Duties: Potassium
is spared due to urinary loss o Results from High bicarbonate levels (due to Chloride Shift)
 Metabolic Alkalosis
Chloride  Compensated Respiratory Acidosis
• Hyperchloremia
• Major extracellular anion (Most abundant and is followed by o Low Bicarbonate levels due high chloride levels
Bicarbonate)  GI loss of Bicarbonate
• Passively follow Na+ (Counterion of Sodium)  Renal Tubular Acidosis: loss of HCO3 in urine
o Loss of sodium in urine will lead to loss of chloride as well  Metabolic Acidosis
• Inverse relationship with Bicarbonate because of the “Chloride Shift” • Cystic Fibrosis
o As chloride leaves the cell, the equivalent amount of o Multi-systemic disorder Characterized by high serum and
bicarbonate enters the cell to maintain electric neutrality sweat chloride levels
 They must shift in concentrations to balance the
charges inside and outside the cells Magnesium
o Bicarbonate and Chloride are both major anions
• Essential cofactor of >300 enzymes
Methods • 2nd most abundant intracellular anion
• 10x higher in RBCs
• ISE: Component is Anion exchange membrane
• Distribution of total plasma Magnesium:
o Octyl propyl Ammonium Chloride Decanol??
o 55% Ionized/Free (physiologically active form)
 o 30% Bound to proteins (primarily albumin) • Drug-Induced
o 15% Bound to ions • Hypermagnesemia
o Increased Magnesium Intake
Methods
 Through drugs
• AAS  Conditions Associated:
o Gold Standard for divalent ions • Antacids
• Enemas
• Dye-Binding • Cathartics
o More commonly used technique for Magnesium
determination o Decreased Magnesium Excretion
o Uses Calmagite dye  Conditions Associated: (same as hyperkalemia)
o Magnesium binds to Calmagite dye, which produces a color • Renal Failure
complex that may be measured spectrophotometrically • Hypoaldosteronism: Aldosterone has the
o End Product is Red-Violet same effect on Potassium and
Magnesium
• Dye-Lake Method
o Uses titan/Clayton thiazole yellow (Reagent) Calcium
o Positive Result is Red-Lake • Regulatory factors (Hormones and Organs for the regulation of
plasma calcium levels)
Reference Values o Parathyroid Hormone: From the Parathyroid Glands
o Vitamin D: activated in the kidneys
• Normal: 1.26-2.1 mEq/L (1.51-2.52 mg/dL)
 Both PTH and Vit. D promote increase of plasma
• Panic: for Hypomagnesemia, < or Equal to 1 mg/dL; for calcium levels by renal reabsorption, intestinal
Hypermagnesmia, > or Equal to 4.7 absorption, and bone resorption
• Conversion factor of mEq/L to mmol/L: 0.50 o Calcitonin: From the Thyroid Glands
• Conversion factor of mg/dL to mmol/L: 0.417  Decreases plasma calcium levels by inhibiting the
• Valence: 2 functions of PTH and Vitamin D
• Distribution of total plasma Calcium
Clinical Significance
o 50% ionized (free; physiologically active)
• Hypomagnesemia o 40% bound to proteins (primarily albumin)
o Decreased Magnesium Intake o 10% bound to ions
 Conditions Associated: • Specimen for ionized calcium determination must be avoided
• Poor Diet exposure to air, maintain anaerobic collection, and keep specimen
tube capped
• Starvation
• Prolonged Magnesium Deficient IV- Methods
therapy
• Chronic Alcoholism • AAS
o Also known as Atomic Absorption spectrometry
o Decreased Magnesium Absorption o Gold standard
 Conditions Associated: (same with Hypokalemia) • ISE
• Malabsorption syndrome o For ionized calcium determination
• Intestinal resection o Component: Liquid Membrane Impregnated with Special
• Nasogastric suction Molecules that selectively bind calcium
• Prolonged vomiting • Dye-Binding
• Diarrhea o Colorimetric method
• Laxatives o Uses CPC (cresolphthalein complexone) or asenazo
o Increased Excretion
Reference Values
 Conditions Associated:
• Renal • Normal (Total Calcium): 4.3-5.0 mEq/L (8.6-10 mg/dL)
• Endocrine
• Panic: < or equal to 6 for hypocalcemia; > or equal to 13 for • 85% in the bones, 15% soft tissues, < 1% in serum/plasma
hypercalcemia (TOTAL CALCIUM) (Distribution of phosphate in the body)
• Conversion factor of mg/dL to mmol/L: 0.25 (in terms of atomic weight)
• Conversion factor mEq/L to mmol/L: 0.50 Method
• Valence: 2
• Fiske-Subbarow
• Derive 50% if you will find the normal and panic values for ionized o Only notable method for phosphate determination
calcium o Also called: Ammonium Molybdate method
o Phosphate reacts with Ammonium Molybdate, forming
Clinical Significance
Ammonium Phosphomolybdate complex
o Product: Ammonium phosphomolybdate complex
(Colorless)
o Product is monitored through UV light
 More accurate way to estimate phosphorus
 its variation of the technique wherein the
ammonium phosphomolybdate complex is
reduced to phosphomolybdenum blue using
appropriate reducing reagent like ascorbic acid
and measured spectrophotometrically

Reference Values
• TIP from sir Balce: Not all conditions in PTH are associated with the
• Normal (Pi): 2.4-4.4 mg/dL
relationship of calcium levels and PTH. Sometimes, it is low and high
• Panic: for Hypophosphatemia, < or equal to 1; for
or high and low, which is why the four gradients above is used to
Hyperphosphatemia, > or equal to 8.9 mg/dL
differentiate the diseases under the abnormal calcium levels
• Conversion factor of mg/dL to mmol/L: 0.323
• Hypocalcemia
• Valence: 3
o Calcium is low, PTH is low:
 Primary Hypoparathyroidism Clinical Significance
o Calcium is low, PTH is normal to high:
 Secondary Hypoparathyroidism, • Hypophosphatemia
 Chronic renal failure, o May result from increased loss of phosphate in urine
 Pseudohypoparathyroidism, o Conditions Associated:
 Hypoalbuminemia,  Diabetes Ketoacidosis
 Hypomagnesemia,  Long term TPN
 Vitamin D Deficiency  IBD
• Hypercalcemia  Anorexia Nervosa
o Calcium is high, PTH is low:  Alcoholism
 Hypercalcemia of Malignancy o May result from
 Multiple Myeloma  Hyperparathyroidism: if PTH increases plasma
 Increased Vitamin D calcium primarily through renal reabsorption and
 Prolonged Immobilization: bone wasting decreases plasma phosphate through excretion
o Calcium is high, PTH is normal to high:  Vitamin D Deficiency (rickets, osteomalacia) due
 Primary Hyperparathyroidism to decreased renal reabsorption
• Hyperphosphatemia
Phosphate o May result from increased intake of phosphate or decreased
• 3rd Major intracellular anion excretion of phosphate
• Component of several essential biomolecules (nucleic acids, ATP,  Conditions associated
etc.) • Renal Failure
• Usually measured as inorganic phosphate/phosphorus • Milk
• 55% of phosphate is ionized, 10% bound to proteins, 35% bound to • Laxatives
ions o May result from phosphate’s release from cells
 o It may be caused by technical factors like hemolyzed  High Osmolal Gap (>12 mOsm/kg)
samples • Renal Failure
 Conditions Associated: • Diabeteic Ketoacidosis
• Neoplastic disorders • Lactate Acidosis
• Intravascular hemolysis • Salicylic (Aspirin) Intoxication/Poisoning
• Lymphoblastic Leukemia • Alcohol Poisoning
• Ethanol/Methanol/Ethylene Glycol
Osmolality and Osmolal Gap
Poisoning
Osmolality

• Expressed in mOsm/kg
• Measured using clinical osmometers

Methods

• Osmometry (Measured Osmolality)

o based on the measurement of a decrease in freezing point
or vapor pressure (principle in osmolality determination)
o Most osmometers measure the freezing point as osmolality
increases
o It’s Called measured because we are using a machine,
which meant that all osmotic particles in plasma are
detected
• Calculated Osmolality
o Based on the Sodium, Glucose and BUN levels
o Estimation of Plasma Osmolality based on three analytes
o Not all osmotic particles will be detected
o Expressed in mmol/L

Formula

• Osmolal Gap
o Difference between calculated osmolality and measured
osmolality
o Gap is only 5-10 mOsm/L under normal conditions

Formula: Measured Osmolality – Calculated Osmolality

Reference Values

• Serum Osmolality: 275-295 mOsm/kg

• Osmolal Gap: 5-10 mOsm/kg

Clinical Significance

• OG: > 12 mOsm/kg

o There are other analytes/ osmotic particles that increases
osmolality other than Na, Glucose and BUN
o It is important to determine Osmolality to diagnose diseases
such as:
 ANION GAP

• the difference between measured cations and measured anions

✓ Used as form of quality control (QC) for electrolyte determinations
• Formulas:
o AG= (Na+ + K+)- (HCO3-+ Cl-) or AG+ Na+- (HCO3-+ Cl-)→ use lower reference values for this formula
o These four analytes constitutes the electrolyte panel
• Reference Values:
o 10-20 mmol/L or 7-16 mmol/L
• Clinical Significance:
o AG
▪ Causes: increased unmeasured anions (renal failure, lactic acidosis, diabetic ketoacidosis, intoxication with salicylate alcohol
ethylene glycol ), decreased unmeasured cations (hypocalcemia) and lab error (Falsely increased Na+ or Falsely decreased Cl,
bicarbonate)
o AG
▪ Causes: Decreased unmeasured anions (hypoalbuminemia), increased unmeasured cations (hypercalcemia, multiple myeloma) ,
lab error (Falsely decreased Na+ or Falsely increased Cl, bicarbonate)

ACID-BASE HOMEOSTASIS

✓ Buffer Systems
o Hemoglobin- 38 histidine residues bind H+
o Plasma Proteins- free carboxyl and amino groups bind H+
o Bicarbonate/ Carbonic Acid (most important/ major)
o Henderson-Hasselbalch equation:

Carbonic anhydrase
CO2 + H2O→ H2CO3→ H2CO3- +H+

➔ shift to the left (carbon dioxide is low), shirt to the right (carbon dioxide is high)
➔ pH = pKa (of buffer system which is 6.1) + log HCO3-/ H2CO3 --> derived to pCO2 x 0.03
▪ When the ratio is 20:1, the pH is 7.4
▪ pH may be increased by HCO3 or pCO2
▪ pH may be decreased by pCO2 HCO3

ARTERIAL BLOOD GAS PARAMETERS

Parameter Method/ Derivation Reference Range

pH pH electrode 7.35 -7.45 (PV: ≤ 7.2; ≥ 7.6)
pCO2 pCO2 electrode 35-45 mmHg (PV: ≤ 20; ≥ 60)
pO2 pO2 electrode 80-110 mmHg (PV: <40)
HCO3- Total CO2 -1 22-26 mmol/L (PV: ≤ 10; ≥ 40)
Total CO2 HCO3- + H2CO3 + CO2 23-27 mmol/L
Base excess Calculated -2 to + 2
O2 saturation Co-oximetry or differential 95%-100%
spectrophotometry

✓ pH and pCO2 are both based on potentiometry

✓ pO2 is based on amperometry
✓ HCO3- represents most of the carbon dioxide in plasma and can be calculated by Total CO2 -1

FACTORS THAT AFFECT ABG VALUES

• Temperature
o For every 1C increased
▪ pCO2 increases by 3%
▪ pO2 decreases by 7%
▪ pH decreases by 0.015
• Specimen exposure to air (air bubbles)
o pO2
o pCO2
o pH
• Prolonged storage of specimen (bacterial contamination)
o pO2
o pCO2
o pH
• Excess anticoagulant (heparin)
o This is acidic pH

✓ Lactic acid would decrease the pH

✓ Collection must be anaerobic, and must be transported on ice and analyzed immediately.

ACID-BASE IMBALANCES

Condition Major Cause/s pH HCO3 pCO2 Compensation

Metabolic acidosis DKA, Lactic
acidosis/ Hypoxic
acidosis N/ Hyperventilation
(increased anion
gap, increased
osmolal gap)
Metabolic alkalosis Vomiting, sodium
bicarbonate
overdose,
corticosteroid N/ Hypoventilation
overdose,
hypokalemia, GI
suction
Respiratory COPDs, acute
acidosis airway obstruction, Reabsorption of
circulatory failure, N/ HCO3-
hypoventilation
Respiratory Anxiety, acute
alkalosis pain, pulmonary
embolism, hypoxia
induce N/ Excretion of HCO3-
hyperventilation,
pulmonary edema,
certain drugs and
CNN disorders

✓ HCO3- metabolic component, maintained by kidneys

✓ pCO2- respiratory component, maintained by lungs
✓ pH and pCO2 is inversely proportional
✓ pH and HCO3 is directly proportional
✓ pCO2 is abnormal
✓ HCO3- abnormal in metabolic
✓ pCO2- abnormal in respiratory
 Lecture #3: CLINICAL ENDOCRINOLOGY

A. THE HYPOTHALAMIC-PITUITARY-TARGET ORGAN (HPT) AXIS

1. Feedback Loop
a. Positive feedback
• begins when the hypothalamus received input to produce a releasing factor that acts on the pituitary gland which in turn
releases tropic hormones that act on a specific target gland to promote hormone synthesis and release.

Shown are three types of HPT axis:

• hypothalamic, pituitary, thyroid axis

• hypothalamic, pituitary, adrenal axis
• hypothalamic, pituitary, gonadal axis

The hypothalamus stimulates the pituitary via secretion of thyrotropin releasing hormone which stimulates the anterior pituitary to secrete TSH (thyroid
stimulating hormone) which stimulates the thyroid to produce thyroxine or T4, when its level increases in the blood, increasing level may suppress further
secretion of pituitary and hypothalamic hormones that is called negative feedback. When the increasing level of the target organ hormone suppresses further
secretion of anterior pituitary and hypothalamic hormones. This is negative feedback.

b. Negative feedback- occurs when elevated levels of a hormone turn off the secretion of a releasing hormone from the hypothalamus
and a stimulating hormone from the pituitary gland.
• Long feedback loop
- involves negative feedback of the target cell hormone at the pituitary gland and hypothalamus.
- A hormone produced by the target gland feeds back negatively at the anterior pituitary and the hypothalamus,
prevents further secretion of anterior pituitary hormone or a tropic hormone and HRH (hypothalamic releasing
hormone), inhibits secretion of tropic hormone by the pituitary and inhibits secretion of hypothalamic releasing
hormone by the hypothalamus.
• Short feedback loop
- involves the anterior pituitary tropic hormone feeding back at the hypothalamus.
- Tropic hormone from the anterior pituitary feeds back at the hypothalamus inhibiting the further secretion of
hypothalamic releasing hormone.
• Ultra-short feedback loop
- involves the anterior pituitary hormone feeding back at the anterior pituitary.
- The tropic hormone produced by the anterior pituitary inhibits further secretion by this very organ that produced it.
Feeds back to the same organ that produced it.

2. Disorders of Hormone Secretion (2 major groups: hypersecretion/ hypo secretion)

a. Primary disorders refer to a defect in the target organ
b. Secondary/ Central disorders refer to a defect in the anterior pituitary gland
• (if hypersecreting, it may be due to adenoma (tumor) in pituitary responsible for uncontrolled hormone synthesis);
• (if hyposecreting, panhypopituitarism, all anterior pituitary hormones are low or undetectable).
c. Tertiary disorders refer to a defect in the hypothalamus (rare).
 B. HYPOTHALAMUS
• Consist of several nuclei including clusters of neurosecretory cells that are responsible for producing releasing hormones and inhibiting
hormones.
Hormone Action
Gonadotropin-releasing hormone Stimulates release of FSH, LH
Corticotropin-releasing hormone Stimulates release of ACTH
Thyrotropin-releasing hormone Stimulates release of TSH
Growth hormone-releasing hormone Stimulates release of GH or somatotropin
Somatostatin Stimulates release of GH, TSH
Dopamine (prolactin inhibitory factor) Stimulates release of Prolactin (PRL)
✓ Releasing Hormones
o Gonadotropin-releasing hormone
Tertiary disorder, because the lesion is localized
o Corticotropin-releasing hormone
in hypothalamus
o Thyrotropin-releasing hormone
o Growth hormone-releasing hormone
o Somatostatin

✓ Inhibiting hormones
o Somatostatin
o Dopamine (prolactin inhibitory factor)

C. PITUITARY GLAND/ HYPOPHYSIS

• Previously referred to as the master gland; connected to the hypothalamus by the infundibulum/ pituitary stalk/ hypophyseal stalk
o Infundibulum- refers to funnel shape opening to another organ.
o Pituitary stalk- houses the so called hypothalamic pituitary portal system (HPPS)→ clusters of blood vessels.
• Endocrine systems that operate independently of the pituitary gland:
o RAAS (Renin Angiotensin Aldosterone System)
o Calcium-Parathyroid Axis
o Glucose Insulin Axis.
• 2 Major Lobes
o Adenohypophysis- anterior pituitary lobe
▪ Pars tuberalis (enclosed by the pituitary stalk)
▪ Pars distalis (most glandular cells (hormone producing cells) are located)
▪ Pars intermedia (referred to as intermediate lobe; part of anterior pituitary)
o Neurohypophysis (pars nervosa)- posterior pituitary lobe

1. ANTERIOR PITUITARY GLAND

✓ 5 distinct populations of glandular cells: somatotrophs (secrete GH), lactotrophs (secrete PRL). thyrotrophs (secrete TSH),
gonadotrophs (secrete FSH and LH) and corticotrophs (secrete ACTH)

Hormone Target Tissue Action Clinical significance

FSH Follicle development; • Secondary Hyper/Hypogonadism
secretion of estrogen;
sperm production
Gonads (testes and (spermatogenesis)
ovaries)
LH Stimulates ovulation and
secretion of progesterone
(females)/ testosterone
(males)
ACTH Adrenal cortex Release of cortisol • Secondary Hyper/Hypocortisolism

• Cushing’s disease- lesion is in the anterior

pituitary that is hypersecreting. (secondary
hypercortisolism)
TSH Thyroid Release of thyroid • Secondary hyper/hypothyroidism
hormones
GH Whole body Growth of muscles and long • Hypersecretory state (child): Pituitary Giantism
bones; stimulates release of • Hypersecretory state (adult): Acromegaly
 hepatic IGF-1 (Insulin • Hyposecretory state: Pituitary Dwarfism
Growth Factor-1) • Hypersecretory: test if SUPPRESION (Oral
glucose loading/ OGTT)
• Hyposecretory: test if STIMULATION (Insulin
Stimulation Test)
No net increase in a patient suffering in pituitary dwarfism or
it could be related to pan hypopituitarism (all anterior pituitary
hormones are secreted I low or not at all levels)
PRL Breast/ mammary Production of milk • Most notable is Hyperprolactenemia due to
glands prolactinoma which is a type of pituitary adenoma.

Manifestations: Hypersecretion
• Females: Amenorrhea, unovulation, infertility,
galactorrhea, inappropriate milk production
• Males: impotence, galactorrhea

Manifestations: Hyposecretion
• Females: Lack of lactation in Post-partum females

✓ Tropic Hormones
o FSH (Follicle Stimulating Hormone)
o LH (Luteinizing Hormone)
o ACTH (Adenocorticotropic Hormone)
o TSH (Thyroid Stimulating Hormone)
✓ Direct Effectors (not part of HPT axis)
o GH (Growth Hormone)
o PRL
✓ Cushing’s disease vs Cushing syndrome
o Cushing’s disease- lesion is in the anterior pituitary that is hypersecreting. (secondary hypercortisolism)
o Cushing’s syndrome: Primary hyper cortisolism (problem is adrenal cortex)

2. POSTERIOR PITUITARY GLAND

✓ Serves as repository of hormones produced by the supraoptic and paraventricular nuclei of the hypothalamus and releases them on demand.

Hormone Target tissue Action Clinical Significance

ADH/AVP Renal tubules i(collecting duct Increases water • Hypersecretory state: SIADH
vasopressin of nephrons) and arterioles reabsorption and Blood (Syndrome of Inappropriate Anti-
pressure (BP) Diuretic Hormone Secretion)
• Hyposecretion state: DI
• Plasma Parameters: Plasma
Osmolality and Plasma Sodium
• Urine Parameters: Urine
Osmolality, Urine Specific Gravity
Oxytocin Uterus and breasts Uterine contraction,
ejection of milk

✓ SIADH:
o If water is excessively reabsorbed, plasma will become dilute, the osmolality will decrease, sodium will be diluted;
o its level will be low called relative hyponatremia.
o Urine is concentrated (High osmolality; high specific gravity)
✓ Diabetes Inspidus
o Two types:
▪ Neurogenic (TYPE 1 DI)-lack of production;
▪ Nephrogenic (TYPE 2 DI)- lack of response/ action; called resistance (there is ADH but to not response)
P. Osm/ Na+ U. Osm/ S.G.(spec. grav)
SIADH
DI
 Lecture #4: Therapeutic Drug Monitoring

A. Basic Pharmacologic Concepts

Peak • Highest point

• The amount of drug absorbed and distributed is greater than the
amount metabolized and excreted

Trough • Lowest Concentration achieved just before the next dose

Half-life • Time required for the concentration of the drug to decrease by half
• Affected by protein and binding

Steady State • amount of drug absorbed, and distributed equals amount

metabolized and excreted
• usually reached after 5-7 half-lives.

TD50 • drug dose that produces adverse effect in 50% of the population.

ED50 • drug dose that produces beneficial effect in 50% of the population

Therapeutic Index 𝑇𝐷50

FORMULA:
𝐸𝐷50

MTC (maximum toxic concentration) • lowest concentration of drug in the blood that will produce adverse
response

MEC (minimum effective concentration) • lowest concentration of drug in the blood that will produce desired
effect.

Therapeutic Range • range of values between the MTC and MEC that produce therapeutic
effect

NOTE:

• The wider the Therapeutic Index is, the safer the drug
• TDM is primarily indicated for drugs narrow therapeutic indices
• Drugs with narrow TI requires TDM because it could cause adverse effect and can lead to toxic effects.
• Goal of TDM: To maximize the efficacy and minimize the toxicity.

B. Instrumentation
• Thin Layer Chromatography
o used as semi-quantitative screening test
o used in urine drug screening
▪ Rf = distance migrated by a sample component /distance migrated by the solvent.
• Liquid Chromatography
o For insufficiently volatile and thermolabile compounds
▪ Retention time = time it takes for a compound to elute in GC or HPLC.
• Gas Chromatography
o useful for compounds that are naturally volatile or can easily be converted into a volatile form (various organic molecules including many
drugs)
• Mass Spectrometry
o common detector system in GC or HPLC

• Immunoassays
o Routinely used in the determination of drugs of abuse & tumor markers
▪ Enzyme Immunoassay
▪ Radioimmunoassay
 ▪ Chemiluminescent immunoassay
▪ Immunoturbidimetry (Particle Enhanced Turbidimetry Inhibition Immunoassay –PETINIA)
▪ Homogenous assays

Note:

• Difference between GC and HPLC is the application with regards to the nature of the compound
• Quantification is based on the peak size/height

C. Pharmacokinetics
• Study of what the drug does to a body
• Drug activity or fate of drugs in the body as influenced by:

1. Absorption
o Through the GI tract for orally administered drugs
o dependent on many factors and is related to the drug’s bioavailability (100% if the route is intravenous administration)

2. Distribution
o diffusion out of the vasculature into interstitial and intracellular spaces
o dependent on the lipid solubility of the drug

3. Metabolism
o hepatic uptake and enzymatic biotransformation during passage through the liver (first-pass metabolism)
4. Excretion or Elimination
o elimination through hepatic clearance, renal filtration, or a combination of the two

NOTE:

• There are two common routes of administration: ORAL and PARENTERAL

o Main difference between the two, parenteral administration leads to 100% bioavailability (intravenous administration).
o This is necessary for drugs that are susceptible for chemical modification by the liver. For orally administered drugs there is what we call
firstpass metabolism in the liver.
o While in the blood stream, most drugs are primarily bound to protein including albumin and alpha 1 acid glycoprotein. Albumin transports
acidic drugs while alpha 1 acid glycoprotein transports basic drugs.
• Free drug which equilibrate the intravascular systemic circulation and the extravascular compartment and into the target sites of action.
• Drugs are excreted to its metabolites through hepatic or renal mechanisms.
 D. Pharmacodynamics
• Study of the Biochemical and physiological effects of drugs and the mechanisms of their actions
• involves receptor binding, post-receptor effects, and chemical interactions.

E. Specimen Considerations
• Establish steady state before sampling.
• STAT sampling when toxicity is suspected
• Trough specimen before next dose is administered
• Peak specimen - Oral: 1 hour after administration (1 and 1/2 hour > 2 hrs)
o IV: 15-30 minutes after administration
o IM: 30-60 minutes after administration
• Appropriate specimens / Collection tubes
o Serum – preferred for most drug assays
o Heparinized plasma – suitable for most drugs except lithium and free drug assays.
o SST and PST – may falsely ↓ due to drug absorption by the gel.
o EDTA whole blood – appropriate for Immunosuppressants.
o Hirudin tube - recommended for the impedance method of aspirin determination.

NOTE:

• Gel separator tubes must NOT be used because the gel absorbs certain drugs such as, antiarrhythmic and TCAs, where it may lead to falsely ↓to
these therapeutic drugs.
• In heparinized plasma lithium and free drug assays are excluded since it may affect protein binding.

F. Drugs that Require TDM

Classification Representative Drugs

Analgesics • Acetylsalicylate (Aspirin): toxic effects include mixed acid-base disorder, Reye syndrome
• Acetaminophen (Tylenol): toxic effect include severe hepatotoxicity
Cardioactive Drugs / • Digoxin – Cardiac Glycosides
Cardiotropics • Antiarrhythmics – treatment of ventricular tachycardias or tachyarrhythmias
o Class I (Lidocaine, Quinidine, Procainamide) – blockage of sodium channels
o Class II (Propranolol) – block adrenergic receptors
o Class III (Amiodarone) – block potassium channels
o Class IV (Verapamil) – block calcium channels
Antieplileptics / • Gabaergic Drugs
Anticonvulsants / Anti-seizures o Phenobartial – slow-acting barbiturate; inactive form: primidone
o Benzodiazepines
• Glutamatergic Drugs
o Ethosuximide – treat petit mal/ absence seizure
o Phenytoion
o Carbamazepine
• Valporic Acid – treats petit mal/absence seizure; General and Partial Seizures
• New Generation Anticonvulsants – Tiagabine, Vigbatrin, Gabapentin, Topiramate, Felbamate
Psychoactive drugs/ • Neuroleptics / Antipsychotics – Chlorpromazine, Haloperidol, Clozapine, Olanzapine are used to treat
Antidepressants Schizophrenia
• Antidepressants and Mood Stabilizers
o Lithium - used to treat bipolar or manic depressive disorder
o TCAs (Amitriptyline, Imipramine, Doxepin) – used to treat clinical depression

Bronchodilators Theophylline – used to treat asthma or other COPD

Antibiotics • Aminoglycosides (Tobramycin, Amikacin, Gentamicin, Kanamycin)
 used to treat gram negative infections; toxic effects include ototoxicity & nephrotoxicity
 Mechanism of Action: disruption of protein synthesis
• Vancomysin
 used to treat gram positive bacterial infections
 may cause ototoxicity, nephrotoxicity and red man syndrome
 Mechanism of Action: inhibition of cell wall synthesis
Immunosuppressants • Calcineurin Inhibitors – reduce the production of IL-2 inhibiting the proliferation of T-lymphocytes
o Cyclosporine – used to prevent graft rejection and graft vs host disease
o Tacrolimus – same indication as cyclosporine but is 100x more potent
• Proliferation Signal Inhibitors – Sirolimus, Everolimus, Mycophenolate
Antineoplastics • Methotrexate
o inhibits DNA synthesis;
o requires “Leucovorin rescue”;
o folic acid antagonist
o Indications: Acute Lymphoblastic Leukemia and Hodgkin’s Lymphoma

• Busulfan
o Alkylating agent used to treat Leukemias and lymphomas (CML)

Other Comments:

• In Acetylsalicylate (ASPIRIN) has analgesic, antidiuretic, anti-inflammatory effects, and acts by inhibiting cyclooxygenase there by decreasing
thromboxane and prostaglandin formation.
o MIXED ACID-BASED disorder
▪ acute ingestion of aspirin may lead to metabolic acidosis since salicylate is acidic.
▪ But it could also lead to respiratory alkalosis due to hyperventilation since salicylate is also a direct stimulator of the respiratory.
• Digoxin
o indicated for the treatment of atrial fibrillation and congestive heart failure.
o It acts by inhibiting the sodium potassium ATPase

• When a drug metabolite exhibits the same effects as the parent drug or it enhances its toxic effects that metabolite must be determined just like N-
acetyl procainamide (NAPA) which the metabolite of Procainamide
• Seizures result from an uncontrolled brain activity. To treat seizures, it is either increase the inhibition using Gabaergic drugs or decrease the
stimulation of using glutamatergic drugs.
o Gabaergic drugs such as Phenobartial and Benzodiazepines are used to treat tonic clonic seizure/ grandmal, generalized and simple
seizures.
▪ Tonic clonic seizure/ grandma – muscle stiffening and twitching
▪ Generalized – involves both hemispheres
▪ Simple seizures – no loss of consciousness

• AMITRIPTYLINE AND IMIPRAMINE

o metabolites that exert the same effects as the parent drug.
▪ Metabolite of Amitriptyline = Nortrityline
▪ Metabolite of Imipramine = Desipramine
• For aminoglycosides peak and trough monitoring are necessary.
• Leucovorin Rescue
o administration of the leucovorin to off-set the effect of Methotrexate and spare the normal cells from its cytotoxic effects.
 Illicit Drugs

Classification Representative Drugs

Stimulants Cocaine
• local anesthetic;
• primary metabolite: Benzoylecgonine;
• short half life

Amphetamines (MDMA, Methamphetamine) - used to treat narcolepsy and attention deficit disorder
• MDMA - Methylenedioxymethamphetamine (ecstasy)
• Methamphetamine – shabu
Hallucinogens Marijuana (Cannabis sativa)
• primary cannabinoid component is Tetrahydrocannabinol (THC)

Phencyclidine (angel hair / angel dust)

• drug with lipophilic nature
• Effects: Auditory and Visual Hallucinations

Lysergic acid diethylamide

• most potent and most potent pharmacologic agents known
• Effects: Blurred vision, panic reactions (bad trip)
Opiates/Narcotics – used • Naturally occurring – opium, morphine, codeine (antitussive)
for sedation, analgesia, • Chemically modified – heroin, hydromorphone, oxycodone
anesthesia • Fully synthetic – meperidine, methadone, fentanyl, propoxyphene, pentazocine
o Fentanyl – 80x more potent than morphine
Sedative/Hypnotics – Barbiturates – Amobarbital, Secobarbital, Pentobarbital, Phenobarbital
CNS depressants • Amobarbital – intermediate acting
• Secobarbital & Pentobarbital – short acting
• Phenobarbital – long acting

Benzodiazepines – diazepam (Valium), Chloridiazepoxide, Lorazepam

Metaqualone

Other Substances and Poisons

• Acute toxicity – associated with a single, short-term exposure to a substance, the dose of which is sufficient to cause immediate toxic effects
• Chronic toxicity – results from repeated frequent exposure for extended periods at doses insufficient to cause an immediate response; may affect
different systems

1. Ethanol
o most substance abused
o chronic consumption of ethanol could lead to chronic hepatitis progressing to cirrhosis

Stages of Impairment
BAC LEVEL OF IMPAIRMENT
0.01 – 0.05% w/v No obvious impairment
0.03 – 0.12% w/v Mild Euphoria & Some Impairment of Motor Skills
>0.1% w/v Legal Intoxication (in U.S.)
0.09-0.25% w/v Loss of Critical Judgement and Memory Impairment
0.18-0.30% w/v Mental Confusion, Strongly Impaired Motor Skills, and Slurred Speech
0.27-0.40% w/v Impaired Consciousness
0.35-0.50% w/v Coma & Possibly Death
Methods

• Osmometry – alcohol will increase plasma osmolality

• Enzymatic Assay – measurement of ethanol is based on the increase of absorbance at 340nm
 • Gas Liquid Chromatography – reference method for ethanol determination
o can differentiate the various types of alcohol and quantify them

2. Methanol
o Causes acidosis, blindness and death due to formation of severe acidosis
o Metabolized by hepatic alcohol dehydrogenase to formaldehyde → Formic acid (responsible of optic neuropathy)
o Severe acidosis leading to death

3. Isopropanol
o produces severe, acute ethanol-like symptoms that persist for a long period of time
o Metabolized to acetone; long half -life
4. Ethylene Glycol
o ingestion produces severe metabolic acidosis and renal tubular damage
o Common component of hydraulic fluid and antifreeze.
o It may cause severe metabolic acidosis resulting in the formation of severe toxic products including oxalic and glycolic acid.
5. Carbon Monoxide
o 245x GREATER AFFINITY for Hb compared to oxygen; shifts the oxyhemoglobin dissociation curve to the LEFT causing hypoxia
o Methods
▪ Spot test (screening test) – positive result is cherry red appearance of the blood
▪ Spectrophotometry
▪ Gas Chromatography
▪ Co-oximetry
6. Cyanide
o commonly used as RODENTICIDE/ INSECTICIDE;
o characteristic odor bitter almond;
o binds to heme iron and mitochondrial cytochrome oxidase
7. Heavy Metals
o Arsenic
▪ component of insecticides, pesticides, and herbicides;
▪ high affinity for keratin;
▪ binds to sulfhydryl groups of proteins; garlic breath odor and metallic taste;
▪ “Mees lines”
o Cadmium
▪ metal food containers or industrial exposure;
▪ may cause Renal tubular dysfunction
o Lead
▪ inhibits many enzymes and affects vitamin D metabolism and heme synthesis pathway
• ALA dehydratase – early in the pathway; ↑ urine ALA
• Ferrochelatase/ heme synthase – late in the pathway; catalyzes the insertion of iron to the Protoporphyrin IX.
o ↑ Free Erythrocyte Protoporphyrin – tested in blood not in urine.
o Mercury
▪ acquired through inhalation and ingestion; may also cause renal tubular damage
▪ Toxic form: Methyl mercury; elimination rate is slow because it is protein bound and it inhibits many enzymes

Methods for Determination of Heavy Metals

• Reinsch test – screening test for arsenic and mercuric

o Arsenic – black
o Mercury – silver gray
• Atomic absorption spectrometry (AAS) – commonly used method
o Gold standard for Divalent cations and trace metals
• Anodic Stripping Voltammetry (ASV) – for lead; based on polarography