ENZYME CLASSIFICATION AND NOMENCLATURE

CLINICAL CHEMISTRY 322 1. OXIDOREDUCTASES

ENZYMES - Catalyze redox rxn.
PRELIM: WEEK 2 LEC (02/01/2023)
between 2 substrates
CHAPTER OUTLINE (PPT) - A - + B → A + B-
I. Introduction - E.g: Dehydrogenase (LD)
II. General Properties & Definitions
III. Enzyme Classification and Nomenclature 2. TRANSFERASE
IV. Enzyme Kinetics - Catalyze the transfer of a group (Methyl, Carboxyl, Phosphate)
V. Enzymes of Clinical Significance between 2 substrates (A-X + B → A + B-X)
- Eg: Transferase (ALT, AST, GGT), Kinase (CK)
INTRODUCTION
ENZYME
o hastens chemical reactions in organic matter
o catalyzes single or limited number of chemical
reactions
o large molecules confined in cells
o increase membrane permeability allows them
to enter the blood
o measured in terms of their activity and not of
their absolute values
o appear in serum after cellular injury,
degradation of cells or from storage areas
o used clinically as evidence of organ damage
o specific for substrate that it converts to a
defined product 3. HYDROLASES
- Catalyze hydrolysis of various bonds
FUNCTIONS - A–B + H2O → A–OH + B–H
1. Hydration of CO2 (respiration) – Carbonic Anhydrase - Eg:Amylase (AMY), Lipase (LPS), Phosphatase (ACP)
2. Nerve induction – Choline acetyltransferase (Cholinergic enzymes)
3. Muscle contraction – AchE
4. Nutrient degradation (digestion): Pepsin, Amylase, Lipase
5. Growth and reproduction: Helicase (Cell division)
6. Energy storage and use: LDH, PPK1 & PPK2 (Conversion and
conservation of ATP)

GENERAL PROPERTIES & DEFINITIONS

4. LYASES
COMPONENTS OF AN ENZYME
- Removal of groups from substrates without hydrolysis; product
o ACTIVE SITE – remain double bonds
A cavity of an - ATP → cAMP + PPi
enzyme where - Fructose biphosphate aldolase (ALS)
substrates bind
and undergo a
chemical reaction
o ALLOSTERIC SITE –
A cavity other than
the active site that
binds regulatory molecules

TERMS ASSOCIATED WITH AN ENZYME 5. ISOMERASES

1. Substrates - Interconversion of geometric, optical or positional isomers
- Substances acted upon by enzymes - A→B
- Specific for their particular enzyme - Triphosphate isomerase (TPI)
2. Cofactors
- Substance added in the enzyme substrate complex to
manifest the enzyme activity
a. Coenzyme or Prosthetic group
o Organic: Nucleotide (NADP) & Vitamins
b. Activator
o Inorganic: Metal ion (Cl, Mg, Cu)
3. Isoenzyme
- Similar enzymatic activity but differ in physical, biochemical 6. LIGASES
and immunologic characteristics - Joins 2 substrate molecules, coupled
4. Apoenzyme with breaking of pyrophosphate bond
- The protein portion of the enzyme in ATP
- Subject to denaturation, in which enzyme losses its activity - Ab + C → A–C + b
5. Holoenzymes - Glutathione Synthetase (GSH-S)
- An active substance formed by combination of a co-enzyme
and an apoenzyme. ENZYME NOMENCLATURE
6. Proenzyme or Zymogens o standardized by Enzyme Commission (EC) in 1961 and revised in
- An inactive enzyme precursor 1972 and 1978
- Eg. Coagulation factors and Digestive enzymes o classified according to:
- biochemical function
- substrate catalyzed
- class of reaction catalyzed
o individual identification numbers
- ex. AMS EC 3.2.1.1

   table   

[SARANILLO, KA.]
 ENZYME NOMENCLATURE
CLINICAL CHEMISTRY 322 o (Enzymes are named in terms of the reaction they catalyzed.)
BASICS OF ENZYMOLOGY
PRELIM: WEEK 2 LEC (02/01/2023) 1. TRIVIAL NAMES (e.g.,trypsin,pepsin) give no indication of the
ENZYMES function of the enzyme. In some cases, the trivial name is the
o Organic catalysts that are produced by the cell but are not name of the substrate with the suffix-ase added (e.g.., urease,
dependent on the intact cell for their activity. ribonuclease)
o Biologic proteins that catalyze biochemical reactions without 2. INTERNATIONAL UNION OF BIOCHEMISTS (IUB) SYSTEM.
altering the equilibrium point of the reaction or being consumed Major features:
or changed in composition a. There are six major classes of enzymes, each with 4 to 13
o hastens chemical reactions in organic matter subclasses.
o catalyzes single or limited number of chemical reactions b. Enzyme name has two parts:
o large molecules confined in cells ➢ 1st – name of substrate
o increase membrane permeability allows them to enter the blood ➢ 2nd –type of reaction (ends in –ase)
o measured in terms of their activity and not of their absolute values e.g., succinate dehydrogenase, methyl transferase
o appear in serum after cellular injury, degradation of cells or from 3. ENZYME NAME HAS SYSTEMATIC CODE NUMBER (E.C.)
storage areas EC 1. Oxidoreductases
o used clinically as evidence of organ damage 1st digit – class of enzyme EC 2. Transferases
o specific for substrate that it converts to a defined product 2nd digit – subclass EC 3. Hydrolases
3rd digit- subsubclass EC 4. Lyases
4th digit- particular name of the enzyme EC 5. Isomerases
FUNCTIONS EC 6. Ligases
o Hydration of CO2 (respiration) – Carbonic Anhydrase
o Nerve induction – Choline acetyltransferase (Cholinergic enzymes) CLASSIFICATIONS OF ENZYMES
o Muscle contraction – AchE 1. OXIDOREDUCTASE – removal or addition of electrons
o Nutrient degradation (digestion): Pepsin, Amylase, Lipase (reduction-oxidation ["redox"] reaction.)
o Growth and reproduction: Helicase (Cell division) - oxidase - cytochrome oxidase
o Energy storage and use: LDH, PPK1 & PPK2 (Conversion and - Dehydrogenase
conservation of ATP) ➢ lactate dehydrogenase (LDH)
➢ malate dehydrogenase (MDH)
COMPONENTS OF AN ENZYME ➢ isocitrate dehydrogenase (ICD)
o Active Site - A cavity of an enzyme where substrates bind and
undergo a chemical reaction 2. TRANSFERASES – catalyze the transfer of a functional group
o Allosteric Site - A cavity other than the active site that binds other than hydrogen from one substrate to another.
regulatory molecules - aspartate aminotransferase (AST)
- alanine aminotransferase (ALT)
TERMS ASSOCIATED WITH AN ENZYME - creatine kinase (CN) or creatine phosphokinase (CPK)
CATALYST ▪ Any substance that can increase the velocity of a - gamma glutamyl transferase (GGI)
chemical reaction. - ornithine carbamyl transferase (OCT)
SUBSTRATE ▪ Substances that are chemically transformed by the
enzyme 3. HYDROLASES – catalyze the hydrolysis of a bond by the addition of water.
▪ Reactants in a chemical reaction
Esterases ▪ acid phosphatase (ACP)
HOLOENZYME ▪ an active substance formed by combination of a
▪ Alkaline phosphatase (ALP)
coenzyme (cofactor) and apoenzyme
APOENZYME ▪ the protein portion subject to denaturation, in ▪ cholinesterase (CLS)
which the enzyme loses its activity. Catalytically ▪ lipase (LPS)
inactive protein when cofactor is removed. They are Peptidases ▪ trypsin (PTS)
heat labile and dialyzable. ▪ pepsin (PPS)
ISOENZYME ▪ enzymes present !it an individual with similar ▪ leucine aminopeptidase (LAP)
enzymatic activity but differ in their physical Glycosidase ▪ amylate, kAMS)
biochemical and immunologic characteristics ▪ amylo 1,6 glycosidase
METALLOENZYME ▪ enzymes whose metal ions are intrinsically part of
▪ galactosidase
the molecule such as catalase and cytochrome
oxidase.
PROENZYME ▪ inactive precursor of enzymes, also referred to as 4. LYASES – catalyze the removal of a group from a substrate
zymogens without hydrolysis forming products with double bond.
SUBSTRATES ▪ substances acted upon by the enzymes which are - Aldolases
specific for each of their particular enzymes - glutamate decarboxylase
COFACTORS ▪ These are non-protein substances/compounds - pyruvate decarboxylase
needed by an enzyme before enzymatic activity can - tryptophan decarboxylase
be manifested. Cofactors are thermostable and
dialyzable.
5. ISOMERASE – catalyse the intramolecular arrangement of the
A. ORGANIC MOLECULE - Coenzyme substrate compound.
o It hastens enzymatic reaction but undergoes a - glucose phosphate isomerase
change or is consumed to another product - ribose phosphate isomerase
o e.g.: NAD (Nicotinamide adenine dinucleotide)
NADP
6. LIGASES – catalyze the joining of two substrate molecules
B. INORGANIC SUBSTANCES – Activators coupled with breaking of the pyrophosphate bond in ATP or
o Metal ion – Activator similar compound
In such, the metal ion may serve as:
o A bridge to hold the substrate and enzyme GENERAL NATURE OF ENZYMES
together 1. M/ost enzymes are proteins.
o The primary catalytic center 2. Enzyme catalysis involves the formation of enzyme substrate
o Stabilizing agent in the conformation for (ES)complex.
catalytic activity 3. Enzymes are not consumed, destroyed or produce during the process
o e.g.: Ca, Cl, Br- Zn2 of catalysis.
4. Enzymes do not cause reactions to take place, but they greatly
enhance the rate of the reactions that would proceed much slower in
their absence.
5. Enzymes are highly specific, interacting with one or a few specific
substrates and catalyzing only one type of chemical reaction.
6. The substrate is bound to a specific region of the enzyme called active
site.

[SARANILLO, KA.]
 ENZYME KINETICS FACTORS THAT INFLUENCE ENZYMATIC RXNS.
CATALYTIC MECHANISM OF ENZYMES A. SUBSTRATE CONCENTRATION
o Enzymes catalyze physiologic reactions by lowering the activation energy o First order kinetics (michaelismenten)
level that the reactants must reach. - Reaction rate is proportional to
o Rate of reaction is proportional to the conc. Of molecules that have the substrate concentration. -
attained energy equal to the free energy of activation. An increase in the concentration
o Free energy – energy assoc. with a chemical reaction that is available of substrate produces also an
after the losses during chemical reaction; It is an energy readily available
increase in the rate of reaction,
to be used in a reaction
provided all other conditions
are kept constant However, the rate of the reaction reaches
a maximal value at a particular concentration of substrate,
and higher concentrations of substrate do not result in
increased rate of reaction (Saturation kinetics)

B. ENZYME CONCENTRATION
o ZERO ORDER KINETICS
- Only a fixed number of substrate (in excess) is converted to
product per sec.
- An increase in the concentration of enzymes produces an
increase in the rate of reaction, provided that the other
conditions remain the same and that a constant but excess
amount of substrate Is present. Meaning, if the amount of
o With the aid of an enzyme, the formation of a by-product is faster. This
enzyme is doubled, the reaction proceeds twice as fast
is by lowering the activation energy needed in a chemical reaction. By - Substrate conc. is in excess to ensure that no more than
lowering the activation energy, more substrates will be converted into 20% of the available substrate is converted to product.
products, which are useful to the different tissue functions.
C. PH
MICHAELIS-MENTEN CONSTANT - The rate of any chemical
o one of the most useful models in the systematic investigation of enzyme reaction is usually increased 2-3
rates was proposed by Leonor Michaelis and Maud Menten in 1913. times for every I0 degrees
o The equation relates the rate of reaction of an enzyme-catalyzed Celsius rise in temperature. - 7.0
reaction to substrate concentration, maximum velocity and Michaelis
- 8.0 (Optimal)
constant.
o Describes the relationship between enzyme, Substrate, and Product
- Controlled by buffers
o gives the means to determine total enzyme concentration in serum and
other body fluids D. TEMPERATURE
o An enzyme (E) catalyzes a reaction by combining with its substrate (S) to - Within ± 0.1°C (e.g: 25°C, 30°C, 37°C)
create an enzyme—substrate complex (ES). The enzyme-substrate
complex according to Michaelis and Menten can either dissociate back E. COFACTORS
to E + S or breakdown to product (P) and free enzyme (provided that the - Cofactors are essential in their functions to stabilize ES
product has a low affinity for the enzyme).
complex and aid in the chemical reaction.
- Nutritional availability of the different cofactors can
influence the rate of reaction of enzymes as enzymes rely
on their cofactors in most of the biochemical reactions they
Where: undergo.
V = Velocity of the reaction
Vmax = Maximum velocity a. Essential ions – include K, Ca, Mg, Na, Mn, Fe, Co, Se, Zn and Cu.
S = Substrate concentration - Activator ions
Km = Michaelis-Menten constant - Metal ions tightly bound to enzyme (metalloid proteins)
b. Coenzymes – include derivatives of B complex vitamins and the
TYPES OF SPECIFICITY: Fat soluble vitamins themselves.
o ABSOLUTE SPECIFICITY – enzymes combine with only one - Co-substrates
o ATP, S-adenosyl-methionin, Ascorbic acid, NAD, NADP,
substrate and catalyzes only one corresponding reaction Tetrahydrofolate
o GROUP SPECIFICITY – enzymes combining with all substrates o Coenzyme A, Retinal, Vitamin K b. Prosthetic groups
containing a particular chemical group o FAD, FMN, Pyridoxyl phosphate, Biotin, Adenosyl
o BOND SPECIFICITY – enzymes are specific to chemical bonds cobalamin, Lipoamide
o STEREOISOMERIC SPECIFICITY – enzymes that predominantly
F. INHIBITORS
combine with only one optical isomer of a certain compound
REVERSIBLE REACTIONS
o Competitive Inhibitor
THEORIES AND TYPES OF ENZYME SPECIFICITY - These are substances that compete with the substrate for
EMIL FISHER’S LOCK AND KEY THEORY enzyme binding because they are chemically analogous to the
o It is based on the rigid enzyme molecule into which the substrate substrate and bind to the active sites of enzymes
fits. The shape of the key (substrate) must conform into the lock - Infinity has higher affinity with the enzyme
(enzyme). - Reversible by increasing substrate concentration.
o Absolute Specificity – enzymes combine with only one substrate - ↑-Substrate, ↑ - Km, N – vmax
and catalyzes only one corresponding reaction - The effect of the inhibitor can be counteracted by adding
excess substrate to bind the enzyme. The amount of the
KOSHLAND’S INDUCED FIT THEORY inhibitor is then negligible by comparison, and the reaction will
o It is based on the attachment of a substrate to the active site of an proceed at a slower rate but to the same maximum velocity as
enzyme, which then causes conformational changes in the enzyme. This an uninhibited reaction.
theory Is more acceptable because the protein molecule Is flexible - e.g. Statins and ACE-inhibitors
enough to allow conformational changes and also allow some
explanation on the influence of hormones on enzymatic activity. o Non-Competitive Inhibitor
o Absolute Specificity – enzymes combine with only one substrate and
- These are substances that do not resemble the substrate and
catalyzes only one corresponding reaction
bind to the enzyme in areas other than the active site
o Group Specificity – enzymes combining with all substrates containing a
- Binding of the inhibitor in the allosteric site or other sites could
particular chemical group
o Bond Specificity – enzymes are specific to chemical bonds result to configurational changes in the active site.
o Stereoisomeric Specificity – enzymes that predominantly combine with - May be reversible in the respect that some naturally present
only one optical isomer of a certain compound metabolic substances combine reversibly with certain
enzymes.

[SARANILLO, KA.]
 - May be irreversible if the inhibitor destroys part of the enzyme UNITS FOR EXPRESSING ENZYME ACTIVITY
involved in catalytic activity. INTERNATIONAL UNIT (I.U. OR U)
- Even if the inhibitor binds reversibly and does not inactivate the o Equivalent to the amount of enzyme that catalyzes the
enzyme, the presence of the inhibitor when it is bound to the conversion of 1 micromole of substrate per minute under
enzyme slows the rate of the reaction. Thus, the maximum controlled conditions. (μmol /min)
reaction velocity cannot be achieved. Increasing substrate
levels has no influence on the binding of a noncompetitive KATAL UNIT (K.U.)
inhibitor, so the km is unchanged.
o Equivalent to the amount of enzyme that catalyzes the
- e.g. Monoamine Oxidase Inhibitors, Oxamic Acid
conversion of 1 mole of substrate per second under controlled
- ↑ - Substrate, N – Km, ↓ - Vmax
conditions. (mol/s)
IRREVERSIBLE REACTIONS
o Uncompetitive inhibition PITFALLS IN ENZYMES ASSAYS
- inhibits enzyme by binding to the enzyme substrate complex o Hemolysis case falsely evaluated values due to the release of
- inhibitor is attracted to the ES complex. enzymes from red blood cells.
- ↑ - Substrate, ↓ – Km, ↓ - Vmax o Serum rather than plasma is the preferred specimen due to the
- e.g. Arsenic, Lithium adverse effects of anticoagulants on enzyme activity
o Lactescent or milky serum causes variable absorption by the
o Irreversible Inhibitor spectrophotometer
- Inhibitor acts as substrate analog. o Storage
- Inhibitor binds with the active site but still allows for the
binding of substrate by forming covalent bonds with enzyme at STORAGE OF SAMPLES
the active site. o Most enzymes are stable at 6 degree celsius for at least 24 hrs
- A.k.a: suicide inhibitor e.g. Aspirin, Sarin Gas o Few enzymes are inactivated at refrigerator temp (LD 4 and 5)

G. SALTS QUALITY CONTROL PROGRAM FOR ENZYME

- The ionic strength of solutions affects enzyme activity o Strict adherence to zero-order kinetics
H. PROTEINS o Proportionality with increments of sample
- When enzyme activity is over the linear limits of the assay, o Use of pooled frozen serum or stable reference materials as controls
dilution of plasma typically requires the use of enzyme o Replicate measurements to evaluate precision of assays
diluents containing plasma proteins.
o Enzyme Induction WHY STUDY ENZYMES?
- This phenomenon states that a certain enzyme has the o Enzymes are essential for life.
ability to adapt to their biochemical systems o Mutation on the genes that code for enzymes can cause diseases.
o Enzymes are targets for the therapeutic drugs.
MEASUREMENT OF ENZYME ACTIVITY o Enzymes have several uses and applications.
MEASUREMENT OF CATALYTIC ACTIVITY - Confirmation of diagnosis of certain diseases.
1. ↑ In product concentration - As drugs for treatment of some clinical conditions.
2. ↓ in substrate concentration - Useful in the study of genes.
3. ↓ or ↑ in coenzyme concentration 4 - As markers in the identification of cell parts.
4. Dependent on enzyme concentration
5. Performed in zero-order kinetics *Mutation on the genes that code for enzymes may cause deficiency
of these enzyme thus causing diseases:
METHODS OF MEASURING ENZYMATIC REACTION INBORN ERRORS OF METABOLISM DEFICIENT ENZYMES
1. Fixed Time (Two Point) Assay 1. Phenylketonuria Phenylalanine hydroxylase
- reagents are combined and the amount of reaction is 2. Albinism Tyrosinase
measured (e.g. hydrolases) 3. Maple syrup urine disease alpha -keto acid dehydrogenase
- Also known as Endpoint analysis 4. Gaucher’s disease Glucocerebrosidase
o Reaction is initiated by addition of substrate 5. Niemann-Pick’s disease Sphingomyelinase
o Reaction is allowed to proceed for a period of 6. Tay Sach’s disease Hexosaminidase A
time
o Measurement is done at the end of the reaction *Enzymes are good targets for therapeutic drugs
o Disadvantage: underestimation of the “true”
enzyme activity and linearity of reaction cannot DRUGS ENZYME INHIBITED DISEASES
be observed Acyclovir DNA polymerase Herpes simplex
2. Continuous-Monitoring (Kinetic/Multipoint) Assay Zidovudine (AZT) Reverse transcriptase AIDS
- Change in concentration of the indicator substance at Rifampicin RNA polymerase Tuberculosis
several intervals Chloramphenicol Peptidyl transferase Bacterial infections
- Continuous measurement of change in concentration as
function of time *Enzymes in clinical diagnosis:
- Preferred than fixed-time; The linearity of the reaction is ELEVATED SERUM ENZYME DISEASES DIAGNOSED
readily observed and adequately verified for the presence
1. Creatine kinase Myocardial infarction
of deviations.
2. Amylase & lipase Acute pancreatitis
- preferred for concentrated samples
3. Alanine aminotransferase Liver damage
- Use of coupled reactions
- Enzymatic activity is measured by coupling the activity with 4. Acid phosphatase Cancer of the prostate
colorimetric reaction 5. Alkaline phosphatase Bone & liver disease
6. Lactate dehydrogenase Myocardial infarction,
o In principle, enzyme activity may be measured by: haemolytic anemias
- Rate of disappearance of Substrate
- Rate of appearance of product
o In actual practice, it is easier to measure the increases in the products REFERENCES:
and the decreases in the substrate. Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2022). Clinical Chemistry:
o Commonly, it is favorable to measure enzymes at a saturating Principles, Techniques, and Correlations. 9th ed. Philadelphia: Wolters
condition where zero-order kinetics with respect to substrate conc. Kluwer Health/Hippincott Williams & Wilkins.
occur. Under this condition, the reaction rate is directly proportional
to the total amount of enzymes present. Henry, J. B., McPherson, R. A., & Pincus, M. R. (2022). Henry's clinical
o At saturation, the rate of product formation with time is constant. diagnosis and management by laboratory methods. 24th ed. Philadelphia,
o Lag Phase – occurs upon mixing the reagent and the sample. PA: Elsevier/Saunders.
o Transition State – happens when the substrate is converted into
product and is released.

[SARANILLO, KA.]
 MEASUREMENT OF ENZYME ACTIVITY
CLINICAL CHEMISTRY 322 o ↑ in product conc.
CATALYTIC MECHANISM OF ENZYMES
PRELIM: WEEK 3 LEC (02/08/2023)
o ↓ in substrate conc.
o ↓ or ↑ in coenzyme conc. (NADH)
CATALYTIC MECHANISM
o ↑ in altered enzyme concentration
o Enzymes catalyze physiologic reactions by lowering the
o Dependent on enzyme concentration
activation energy level that the reactants must reach.
o Performed in zero-order kinetics

GENERAL METHODS OF MEASURING ENZYMATIC REACTION

a. Fixed time (Two point) Assay – Reagents are combined and the
amount of reaction is measured (e.g. Hydrolases)
b. Continuous-monitoring or kinetic assays – Measurements at
specific time intervals. Rate of change in cofactor, product, etc

CALCULATION OF ENZYMATIC ACTIVITY

o IU (EC) • μmol /min
o Kat (SI) • mol/s

MI PROFILE LIVER ENZ. PANCREATIC ENZ. OTHER ENZ.

- CK - ALT - AMS - ACP
- AST - ALP - LPS
o Relationship between - LDH - LDH
Enzyme, Substrate and - GGT
Product
MI PROFILE
CREATINE KINASE (CK)
a. Function and Tissue Source
- Storage of high-energy creatine phosphate in muscle cells
- Highest activities in skeletal muscle, heart (AMI), and brain
tissue
b. Total CK Determination
o ABSOLUTE SPECIFICITY – Combines with only 1 substrate and
catalyzes only one reaction (E.g. AMY, LPS) o FORWARD REACTION (TANZER-GIVARG)
o GROUP SPECIFICITY – Combine with all substrates containing a -Measure ↓ in
particular chemical group (E.g. ACP, ALP) abs. at 340 nm
o BOND SPECIFICITY – Specific to chemical bonds (E.g. AMY, LPS)
-pH: 9.0
o STERIOISOMETRIC SPECIFICITY – Combine with one optical
isomer(E.g. Glucose Oxidase)

FACTORS THAT INFLUENCE ENZYMATIC REACTIONS o REVERSE REACTION (OLIVER-ROSALKI)

a. Substrate concentration d. Temperature -Measure ↑
b. Enzyme concentration e. Cofactors abs. at 340 nm
-6x faster than
c. pH f. Inhibitors
forward rxn.
-pH: 6.8
SUBTRATE CONCENTRATION
o First order kinetics (Michaelis- Menten) – Reaction rate is
proportional to the substrate concentration. o SOURCES OF ERROR
- Source of Error
ENZYME CONCENTRATION - Hemolysis: ↑ CK due to ↑ AK activity
o Zero order kinetics – Only a fixed number of substrate (in excess) - Physical activity & IM injections: ↑ CK
is converted to product per sec. - Light: Inactivates CK
o a & b. Substrate and Enzyme concentration
o First and Zero order kinetics o TOTAL CK DETERMINATION
- M: 15-160 U/L
PH – 7 .0 - 8.0, Controlled by buffers Reference Range - F: 15-130 U/L
- CK-MB: <6% of total CK
TEMPERATURE – Within ± 0.1°C, E.g: 25°C, 30°C, 37°C
o CK ISOENZYMES
COFACTORS CK-3 / CK-MM CK-2 / CK-MB CK-1 / CK-BB
Muscle type Hybrid type Brain type
o Activators – Metallic (Ca2+) and NonMetallic (Cl-)
Slowest 2nd fastest Fastest
o Coenzymes (prosthetic groups) – 2nd substrates (NAD or NADH)
Striated muscle & Heart tissues CNS, GIT & uterus
normal serum (pregnancy)
INHIBITORS - After MI, CK-MB (>6%) begin to rise within 4-8 hrs, peak at
o Competitive: inhibitors bind to the active site of an enzyme 12-24 hrs, and return to normal in 48-72 hrs.
o Noncompetitive: inhibitors bind to the allosteric site of an
enzyme
o Uncompetitive: inhibitors bind to an enzyme substrate complex
o IRREVISIBLE INHIBITOR - The
Substrate becomes an analog of the
inhibitor to the substrate. INHIBITOR
o KM (MICHAELIS MENTEN
CONSTANT) - concentration of
substrate at half of Vmax

o REFERENCE VALUES
- CK-MM → 94-100%
- CK-MB → 0-6%

[SARANILLO, KA.]
 ASPARTATE AMINOTRANSFERASE (AST) PANCREATIC ENZYMES
a. Function and Tissue Source AMYLASE (AMS)
- Serum glutamic-oxaloacetic transaminase o Alpha-1-4 glucan-4-glucohydrolase
- Highest activities in cardiac, liver & skeletal muscle - smallest enzyme (MW: 50,000 – 55,000 daltons)
- filtered by renal glomerulus
- earliest pancreatic marker
- major tissue source: acinar cells of pancreas and salivary
- ↑ in hepatocellular & skeletal muscle dis. glands
- AST levels begin to rise in 6-8 hours, peak hours, and return - other tissue source: adipose tissue, fallopian tube, small
to normal in 5 days. intestine and skeletal muscles
- reference value: 60 – 180 SU/dL (Somogyi units)
95 – 290 U/L

o ISOENZYMES
1. S-type (ptyalin) inhibitor: wheat germ lectin
b. Total AST Determination
2. P-type (amylopsin)
- KARMEN METHOD
o Uses malate dehydrogenase
o METHODS OF ANALYSIS
o Measure ↓ in absorbance at 340 nm
- inhibitor: heparin, triglyceride, wheat germ
o Falsely ↑ in hemolyzed sample
- ↑ AMS activity (remedy: dilution)
- ↑ AMS levels: morphine and other opiates for blood
sampling

o GENERAL SUBSTRATE: STARCH

1. Saccharogenic reference method reported in Somogyi units
LACTATE DEHYDROGENASE (LDH)
a. Function and Tissue Source
- Interconversion of lactate and pyruvate
- Widely distributed, highest activities in heart, liver, skeletal 2. Amyloclastic – measures activity by decrease in substrate
muscle and RBC concentration
3. Chromogenic – measures activity by increase in color
TOTAL LDH DETERMINATION intensity of soluble dye-substrate solution
o Wrobleuski – Cabaud or Wacker method 4. Coupled enzyme
- Forward Reaction (Lactate 🡪 Pyruvate)
- ↑ in absorbance is monitored at 340 nm
- pH: 8.3 – 8.9

o Wrobleuski – La Due
- Reverse Reaction (Pyruvate 🡪 Lactate)
- ↓ in absorbance is monitored at 340 nm
- pH: 7.1–7.4, 3x faster
- LD begin to rise within 10-24 hrs, peak at 48-72 hrs, and o AMYLASE/CREATININE RATIO
remains elevated for 10 days. - reference value: 1% – 4% (0.01 – 0.04)
- Reference Range: 100-225 U/L - acute pancreatitis: > 4% (up to 15%)
- Tetramer containing two active subunits - formula:

Evaluation of acute pancreatitis and parotitis RENAL FAILURE

↑ AMS plasma ↑ AMS plasma
↑ AMS urine: up to 7 days ↓ AMS urine

o INCREASE SERUM AMYLASE

LDH ISOENZYMES DETERMINATION
- Acute pancreatitis - Alcoholism
o Relative conc. in normal serum:
- Ectopic pregnancy - Mumps
- LDH-2>LDH-1>LDH-3>LDH-4>LDH-5
- Peptic ulcer
o In AMI &
Intravascular ENZYMES RISE PEAK NORMALIZE
hemolysis, AMS 2-12 hours 24 hours 3-5 days
LDH-1 and LDH-
2 demonstrate
a Flipped pattern (LDH-1 > LDH-2)

[SARANILLO, KA.]
 LIPASE (LPS) OTHERS
o Triacylglycerol acylhydrolase hydrolyzes ester linkages of fats
PSEUDOCHOLINE ESTERASE
- most specific pancreatic marker
o reflects synthetic function of liver
- Late pancreatic marker
o antixenobiotic enzyme: remove benzyl group from cocaine
- major tissue source: pancreas
o marker for insecticide/pesticide poisoning (↓ PChE)
- reference value: 0 – 1 U/ml
o ↓ PChE: acute hepatitis, cirrhosis, metastatic hepatocarcinoma,
malnutrition, organophosphate poisoning
o involve in metabolism of anticholinergic drugs
o succinylcholine: monitor effect of muscle relaxants
- o tissue source: liver, myocardium, pancreas
o method: Ellman technique, potentiometric
METHODS OF ANALYSIS o reference value: 0.5-1.3 pH units (plasma)
o substrate: olive oil / triolein (most pure form of TAG) additional:
colipase (protein secreted by pancreas) + bile salts ANGIOTENSIN CONVERTING ENZYME (ACE)
o inhibitor: hemoglobin o aka: peptidyldipeptidase A / kininase II
1. Peroxidase coupling most commonly used does not use 50% o hydrolase enzyme
olive oil o converts angiotensin I → angiotensin II (lungs)
2. Tietz and Fiereck o indicator for neuronal dysfunction × target of drugs used to ↓
3. Cherry Crandal method blood pressure
o tissue source: lungs, testes, macrophage, epithelioid cells
REFERENCE METHOD o used in diagnosis and monitoring of sarcoidosis
o principle: hydrolysis of olive oil after 24 hours at 37°C and
titration of fatty acids using NaOH INCREASE ACE
o end product: fatty acids Sarcoidosis Acute and Chronic bronchitis
Multiple sclerosis HIV infection
Addison’s disease Leprosy

CERULOPLASMIN
o chronic pancreatitis = acinar cell degradation (↓ amylase + lipase o copper carrying protein / enzyme
production) o marker for hepatolenticular disease  Wilson’s disease
ENZYMES RISE PEAK NORMALIZE
LPS 6 hours 24 hours 8-14 days ORNITHINE CARBAMOYL TRANSFERASE (OCT)
o marker for hepatobiliary disease
OTHER ENZYMES
o 5’ nucleotidase (5’N) GLUCOSE 6-PHOSPHATE DEHYDROGENASE (G-6-PD)
o phosphoric monoester hydrolase o maintains NADPH in reduce form in the RBC
- major tissue source: liver o newborn screening marker
- marker for: o tissue source: adrenal cortex, RBC, lymph nodes
o hepatobiliary disease (secondary: post hepatic)  o primaquine: drug induced hemolytic anemia
o infiltrative lesions of liver o specimen: red cell hemolysate, serum
- method of analysis: o ↑ G-6-PD: myocardial infarction and megaloblastic anemia
o Dixon & Purdon, o reference value: 10-15 U/g hemoglobin 
o Campbell, 1200-2000 mU/ml packed RBC
o Belfield & Goldberg
- reference value: 0 – 1.6 units

LIVER ENZYMES
GAMMA GLUTAMYL transamine peptidase / TRANSFERASE (GGT)
o transfer of glutamyl groups between peptides / amino acid
- major tissue source: liver, biliary duct, kidney, prostate,
pancreas
- affects cell membrane and microsomal function:
o warfarin
o phenobarbital
o phenytoin therapy
- method: Szass, Rosalki & Tarrow, Orlowski
- substrate: ꝩ-glutamyl-p-nitroanilide
- reference value:
o Female : 5-30 U/L
o Male: 6-45 U/L
- differentiating ↑ ALP levels
- secondary: post hepatic
- sensitive indicator of alcoholism
o monitoring effects of abstention
- ↑ GGT is also seen in pancreatic and prostate disorder

[SARANILLO, KA.]
 CLINICAL CHEMISTRY 322
FLUIDS AND ELECTROLYTES
PRELIM: WEEK 4 LEC (02/15/2023)

WATER
o 40-75% of body weight
o Function:
a. Transport nutrients to
the cells
b. Remove waste
products
o Location:
a. ICF: 2/3
b. ECF: 1/3
- Intravascular DETERMINATION
(25%) and interstitial fluid (75%) o Osmolality (Serum or urine
o Any substance dissolve in a solvent will
DISTRIBUTION OF BODY WATER IN ADULT 1. ↓ freezing point by 1.858C
Compartment (%) of Body Weight (%) of Total Body H2O 2. ↑ boiling point by 0.52C
Extracellular 20 33 3. ↓ vapor pressure (dew point) by 0.3 mmHg
Plasma (Intavascular) 5 8 4. ↑ Osmotic pressure by 17,000 mmHg
Interstitial 15 25 o Main contributors are Na+, Cl-, Urea and Glucose
Intracellular 40 67
FLUIDS AND ELECTROLYTES
OSMOLALITY o Ions capable of carrying an electrion
o Formula: Osmolality = sol. In mol/kg solvent - Cations (positive)
o Concentration of ions is maintained by: - Anions (negative)
1. PASSIVE TRANSPORT o Electroneutrality: equal number of
- Passive movement of ions across a membrane cations an anions
2. ACTIVE TRANSPORT o Average water content: 40-75%
- Requires energy to move ions across a membrane - Extracellular fluid (ECF): 1/3 (16 liters)
- ATPase-dependent ion pumps - Intracellular fluid (ICF): 2/3 (24 liters)
o 30 liters fluid: passes from blood → tissue spaces
o Sweat: 50 mmol/L sodium + 5 mmol/L potassium
o Salt content: main determinant of ECF volume
o ↓ vasopressin: 10-20 liters fluid excretion daily
o Edema: 3-liter fluid retention
o Plasma: 12% higher fluid content than whole blood

PHYSIOLOGIC IMPORTANCE
o Concentration of solutes per kg of solvent (millimoles/kg)
1. Volume and osmotic regulation – Na, Cl, K
o Regulated by:
2. For myocardial rhythm and contractibility – K, Ca, Mg
a. Thirst sensation
3. Important cofactors in enzyme activation – Ca, Mg, Zn, Cl, K
o Response to consume more fluids
4. For the regulation of ATP ion pumps – Mg
o Prevents water deficit
5. For neuromuscular excitability – K, Ca, Mg
b. Arginine vasopressin hormone (AVP)
6. For the production and use of ATP from the glucose – Mg, PO4
o Antidiuretic Hormone (ADH)
7. Maintenance of acid base balance – HCO3, K, Cl, PO4
o ↑ reabsorption of water in kidneys
8. Replication of DNA and translation of mRNA - Mg
o Suppresses in excess H20 load
o Activated in H2O deficit

c. Renin-Angiotensin-aldosterone system (RAAS)

d. Atrial Natriuretic Peptide (ANP)

o ↑ Na+ and H2O exretion in the kidney

e. Glomerular Filtration Rate (GFR)

o ↑ with vol. expansion & ↓ with vol. depletion

[SARANILLO, KA.]
 POTASSIUM HYPOKALEMIA
o Aka: kalium Gastric suction Diuretics
and laxative abuse
o Major intracellular cation
Gastrointestinal Intestinal tumor Hyperaldosteronism
o Total plasma body potassium = 2% loss and malabsorption
o K in RBC: 105 mmol/L Cancer and Renal Cushing’s syndrome
o Filtered in glomeruli, reabsorbed by proximal tubules radiotherapy loss
o Ascending loop of henle: K, Na, Cl Alkalosis Leukemia
o Single most important analyte Intracellular Bartter’s syndrome
o Function: heart contraction, neuromuscular excitability, ICF shift Insulin overdose Gitelman’s syndrome
volume and hydrogen ion regulation Malignant
o Reference value: 3.5-5.2 mmol/L hypertension
o Critical value:
- 6.5 mmol/L: hyperkalemia PSEUDOHYPOKALEMIA
- 2.5 mmol/L: hypokalemia o Leukocytosis: ↓ K levels if left at room temperature

METHODS OF ANALYSIS EFFECTS TO CARDIAC MUSCLE

o Hemolysis of 0.5% RBC = ↑ 0.5 mmol/L HYPERKALEMIA HYPOKALEMIA
o Platelet release: plasma levels are ↓ 0.1-0.7 mmol/L compared ↓ resting membrane potential ↑ resting membrane potential
to serum (RMP) (RMP)
o Muscular activity ↑ cell excitability ↓ cell excitability
- ↑ 0.3-1.2 mmol/L: mild-moderate exercise Plasma levels: Arrhythmia and paralysis
- ↑ 2-3 mmol/L: vigorous exercise, fist clenching o 6-7 mmol/L: alter ECG
o Prolonged contact of serum to RBC o 8 mmol/L: lack of muscle
o Prolonged tourniquet application excitability
o Preferred sample: heparinized plasma o 10 mmol/L : fatal, cardiac
arrest
1. Emission flame photometry Cessation of contraction
2. Ion selective electrode PH IMBALANCE, DRUGS, HORMONES
- Vancomycin gel Acidosis = ↑ 0.2-1.7 mmol/L Alkalosis (0.1) = ↓ 0.4 mmol/L
3. Atomic absorption spectrophotometry ↓ insulin ↑ aldosterone
4. Colorimetry (Lock head and Purcell) Therapeutic K Insulin and catecholamines
- Na cobaltinitrite
Hyperkelemic drugs
- End color: BLUE VIOLET

HYPERKALEMIA
DIFFERENTIAL DIAGNOSIS OF HYPERKALEMIA
o Increase K concentration
o 3 major mechanisms of increase K
- Reduced aldosterone: hyporeninemic hypoaldosteronism
- Renal failure: most common, ↓ GFR & ↓ tubular secretion
o Reduced distal delivery of sodium

HYPERKALEMIA
Decrease renal Acute/Chronic Acidosis
excretion renal failure
Severe Muscle/cellular
dehydration injury
Addison’s Extracellular Chemotherapy
disease Shift
Increase intake Oral or IV Vigorous exercise
infusion
Use of immuno- Tacrolimus Digitalis
suppresive and intoxication
drugs Cyclosporine

PSEUDOHYPERKALEMIA
o ↑ plasma K levels with normal ECG
o Causes:
Hemolysis High blast counts (leukemias)
Thrombocytosis Recentrifugation of SST
Prolonged tourniquet application Blood stored on ice
Fist clenching IV fluid

HYPOKALEMIA
o Plasma K levels: 3.0 – 3.4 mmol/L (mild) DIFFERENTIAL DIAGNOSIS OF HYPOKALEMIA
o ↓ Mg = ↓ K
o ↓ plasma K (retained) to balance secrete another cation (NH4 ion)

Impaired renal function / Renal loss ↑ aldosterone

Extra renal loss Diarrhea

[SARANILLO, KA.]
 SODIUM HYPONATREMIA WITH NRF (estimate results)
Cause Serum Urine 24hr Urine Serum K
o Aka: Natrium
Na Na urine Na osmolarity
o Major extracellular cation Overhydration ↓ ↓ ↓ ↓ N-↓
o Principal osmotic particle outside the cell Diuretics ↓ ↓ ↑ ↓ ↓
o Major contributor of osmolarity: together with Cl and HCO3 SIADH ↓ ↑ ↑ ↑ N-↓
o Concentration depends on intake and water excretion Adrenal failure Mild ↑ N ↑ ↑
- Aldosterone (take action for sodium) Barterr’s ↓ ↓ ↑ ↓ ↓
o (+) serum abnormalities = urine Na & osmolarity syndrome
o Every 100 mg/dL ↑ in glucose = ↓ 1.6 mmol/L Na Diabetic ↓ N N N ↑
hyperosmolarity
Reference value Critical value
o 135-145 mmol/L (serum) o 160 mmol/L (hypernatremia) PSEUDOHYPONATREMIA
o 136-150 mmol/L (CSF) o 120 mmol/L (hyponatremia) o Systematic error in measurement
o Artifactual hyponatremia:
HORMONES - hyperlipidemia and
1. Aldosterone hyperproteinemia
- Na retention and K excretion
2. Atrial Natriuretic Factor (ANF)
- Antihypertensive agent
- Tissue source: cardia atria CHLORIDE
- Block aldosterone and renin secretion o major extracellular anion
- Inhibits action of angiotensin II and vasopressin o chief counter ion of sodium
- Promotes natriuresis o functions: water balance and osmotic pressure (Na & Cl), blood
volume and electroneutrality
METHODS OF ANALYSIS o enzyme activator: AMS
1. Emission Flame Photometry o excreted via: urine and sweat
2. Ion selective electrode o reference value: 98-107 mmol/L
- Glass aluminum silicate: most commonly used
3. Atomic absorption spectrophotometry METHODS OF ANALYSIS
4. Colorimetry (Albanese and Lein) o marked hemolysis: increased Cl levels (dilution)
- Na is precipitated as Zn uranyl acetate o slightly lower values: post prandial specimen
- End color: YELLOW o ↓ Cl = ↑ HCO3
o interference: bromide, cyanide, cysteine
HYPERATREMIA
o Increase Na concentration 1. Mercuric titration (Schales and Schales)
- >145 mmol/L: hyper - indicator: diphenylcarbazone
- 150-160 mmol/L: moderate water deficit - end product: HgCl2
- >165 mmol/L: severe water deficit - end color: blue violet
o Causes: loss of water, gain of Na or both 2. Spectrophotometric
o 1-2% water deficit = severe thirst - Mercuric Thiocyanate (Whitehorn titration method):
o Perspiration and breathing: 1L/day water loss reddish complex
o Chronic hypernatremia: indicative of hypothalamic disease - Ferric perchlorate: colored complex
3. Coulometric Amperometric titration
HYPERNATREMIA - Cotlove Chloridometer
Diabetes insipidus Hyperaldosteronism
4. Ion Selective Electrode
(Conn’s Disease)
Increase
- ion exchange membrane (tri-noctylpropylammonium
Renal tubular Sodium bicarbonate
disorder intake or infusion chloride decanol)
Excess Prolonged retention Increase oral or IV intake - most commonly used
water diarrhea of NaCl
loss Profuse sweating Ingestion of sea water HYPERCHLOREMIA HYPOCHLOREMIA
Severe burns Renal tubular acidosis Prolonged vomiting
Vomiting Decrease water intake Diabetes insipidus Aldosterone deficiency
Hyperventilation Salicylate intoxication Metabolic alkalosis
Primary hyperparathyroidism Salt-losing nephritis
HYPONATREMIA Metabolic acidosis
o Decrease Na concentration Prolonged diarrhea
- >135 mmol/L
- 125-130 mmol/L: (+) symptoms - - part 1 - -
- 130 mmol/L: clinical concern
- <125 mmol/L: severe neuropsychiatric symptoms
o (+) glucose or mannitol, glycine and ketones (seen in DM)
o SIADH: ↓ aldosterone, ↑ water retention
o Note: K and Na have inversely relationship in the kidney
reabsorption

HYPONATREMIA
Increase Diuretic use SIADH
sodium Saline infusion Hepatic cirrhosis
loss Increase
Renal failure water Primary polydipsia
Increase Nephrotic syndrome retention CNS abnormalities
water Aldosterone Myxedema
retention deficiency
Cancer Barterr’s syndrome

[SARANILLO, KA.]
 CALCIUM PHOSPHORUS
o 99% is the bone; 1% in blood and ECF o Counter ion of K
o Maximally absorbed in the duodenum o Omnipresent: 85% in bones, 15% in the ECF
o Absorption is best at acidic pH o Inverse relationship with calcium
o Urinary excretion: major net loss o Maximal absorbed in jejunum
o Function blood coagulation, neutral transmission, enzyme o Function: phosphorylation of glucose,
activity, excitability of skeletal and cardiac muscle. co-entry of K
o Reference value o Inorganic phosphate: most predominant in serum
- Total calcium: 8.6 to 10 mg/dl (adult) o Reference values: 2.7-4.5 mg/dl (Adult)
8.8 to 10.8 (child) 4.5-5.5 mg/dl (child)
- Ionized calcium: 4.6 to 5.3 mg/dl (adult) <1.0 g/dL or 0.3 mmol/L (severe)
4.8 to 5.5 mg/dl (child) INORGANIC PHOSPHORUS
FORMS OF CALCIUM 1. Organic phosphate: principal anion within the cells
o Ionized calcium – 50% Ionized calcium – sensitive and 2. Inorganic phosphate: part of the blood buffer
o Protein bound calcium – 40% specific for calcium disorders
o Complexed with anions – 10% ↓ 1 g/dl serum albumin = ↓ 0.8 FORMS OF PHOSPHORUS
mg/dL total Ca+2 1. Free or Unbound form: 55%
2. Complexed with ions: 35%
FACTORS AFFECTING CALCIUM LEVELS 3. Protein bound: 10%
1. 1,25-dihydroxycholecalciferol (Activated Vitamin D3)
- ↑ intestinal absorption, kidney reabsorption, mobilization HOMONES AFFECTING PHOSPHATE LEVEL
from bones 1. Parathyroid hormone
- Decreases phosphate by renal excretion
2. Parathyroid hormone (PTH) 2. Calcitonin
- ↑ kidney reabsorption, mobilization from bones, activates - Inhibits bone resorption
bone resorption 3. Growth hormone
- ↓ urinary Ca loss - Increases renal phosphate reabsorption
- Stimulates Vit D → Vit D3 (kidney)
METHODS OF ANALYSIS
3. Calcitonin o Fasting is required: ↑ carbohydrate diet = ↓ phosphorus
- Thyroid hormone o pH dependent
- Secreted by parafollicular C cells o separate pRBC and serum immediately
- Inhibits: PTH, Vitamin D3, bone resorption o PO4: form measure in the laboratory
- ↑ urinary Ca loss o mEq/L: unit of measurement
o circadian rhythm: ↑ in late morning, ↓ evening
METHODS OF ANALYSIS
o Specimen of choice: serum 1. FISKE SUBBAROW METHOD (Ammonium molybdate method)
o ↓ pH of reagent: ↑ liberation of Ca from albumin - Most commonly used
- Reducing agents:
1. Precipitation and redox titration a. Pictol (amino naphthol sulfonic acid)
Clark Collip Ferro Ham Chloranilic b. Elon (Methyl amino phenol)
precipitation Acid precipitation c. Ascorbic acid
d. Senidine (N-phenyl-p-phenylene diamine hydrochloride)
End product oxalic acid chloranilic acid
- End product: ammonium molybdate complex
End color PURPLE PURPLE
- End color: blue
2. Ortho-Cresolpthalein Complexone dyes (colorimetric) - Wavelength: 340 nm at alkaline pH
- Dye: arzeno III
- Mg inhibitor: 8-hydroxyquinolone (chelator)
HYPERPHOSPHATEMIA HYPOPHOSPHATEMIA
3. EDTA titration (Bachra, Dowel and Sobel)
Hypoparathyroidism Alcohol abuse
4. Ion selective electrode: liquid membrane
Renal failure Primary hyperthyroidism
5. Atomic Absorption Spectrophotometry: reference method
Lymphoblastic leukemia Avitaminosis D
6. Emission Flame photometry: orange-red
Hypervitaminosis D Myxedema
DIAGNOSTIC SIGNIFICANCE Renal tubular defects
o (+) symptoms: total calcium levels <7.5 mg/dL (1.88 mmol/L) (↑ phosphate, ↓ calcium, Transcellular shift (major)
↑ BUN and Creatinine)
↓ CALCIUM ↑ CALCIUM
Prolonged contact to pRBC Venous occlusion
Recumbent posture Acidosis
Alkalosis Dehydration
Tetany Hemoconcentration
Parathyroid disease Parathyroid hormone –
(primary hypocalcemia) related protein (PTHRP)
Renal failure
Estrogen Secondary hyperparathyroidism
(ionized calcium)

HYPERCALCEMIA HYPOCALCEMIA
Primary hyperthyroidism Alkalosis
Cancer (lung and mammary) Vitamin D deficiency
Acidosis Primary hypoparathyroidism
Increase Vitamin D Acute pancreatitis
Multiple Myeloma Hypomagnesemia
Sarcoidosis Malabsorption syndrome
Hyperthyroidism Renal tubular failure
Milk-alkali syndrome

[SARANILLO, KA.]
 MAGNESIUM BICARBONATE
o 2nd major intracellular cation o 2nd most abundant anion in the ECF
o 4th most abundant cation in body o HCO3 = undissociated NaHCO3, carbonate, carbamate
o Enzyme activator: CK and ALP o Accounts 90% of total CO2
o Stored in: 53% bones, o ↑ bicarbonate: renal failure
46% muscles and soft tissues, o Function: major component of the blood buffer system
1% serum and RBC o Specimen: blood anaerobically collected (serum/heparinized)
o ↓ Mg = ↓ K o Specimen left uncapped: ↓ 6 mmol/L
o Reference values: 1.2 – 2.1 mEq/L
5 mmol (life threatening) METHOD OF ANALYSIS
1. Ion selective electrode
o Function: vasodilator; - pCO2 electrode
↓ uterine hyperactivity in eclampsia;
↑ uterine blood flow; 2. Enzymatic
Maintaining structures of DNA, RNA, ribosomes - Phosphoenolpyruvate carboxylase
Synthesis of CHO, CHON, lipids; - Phosphoenolpyruvate dehydrogenase
Neuromuscular transmission;
Cofactor;
Regulate movement of K in the myocardium

FORMS OF PHOSPHORUS
1. Free or Unbound form: 55%
2. Complexed with ions: 35%
3. Protein bound: 10%

HORMONES AFFECTING MAGNESIUM LEVELS

1. Parathyroid hormone
- ↑ renal absorption of magnesium
- ↑ intestinal absorption of magnesium

2. Aldosterone and thyroxine

- ↑ renal excretion of magnesium

METHODS OF ANALYSIS
1. Colorimetric method
a. Calmagite method: reddish – violet complex
b. Formazen dye method: colored complex
c. Magnesium thymol blue method: colored complex

2. Atomic absorption spectrophotometry: reference method

3. Dye-Lake method
a. Titan yellow dye
o Clayton yellow
o Thiazole yellow

HYPERPMAGNESEMIA HYPOMAGNESEMIA
Diabetic coma Acute renal failure
Addison’s disease Malnutrition
Chronic renal failure Malabsorption syndrome
↑ intake of antacids, enemas, Chronic alcoholism
and cathartics Severe diarrhea

[SARANILLO, KA.]
 FREQUENTLY ORDERED TUMOR MARKERS
CLINICAL CHEMISTRY 322 1. ALPHA FETOPROTEIN
TUMOR MARKERS - Upper normal value: 15 ng/ml
PRELIM: WEEK 5 LEC (02/22/2023)
2. B2- MICROGLOBULIN (B2M)
MAJOR PROCESSES INVOLVED IN CELL GROWTH - Low molecular weight protein
1. Proliferation - Part of the normal L-chain of human histocompatibility locus
2. Differentiation antigen (HLA) that is expressed on the surface of most nucleated
o Cancer is a disease of abnormal growth cell
o Tumorigenesis is when the tumor begin to emerge when the - Non-specific TM: Elevated in solid tumors
o Lymphoproliferative disease
growth and development of a normal cell loses control
o Inflammatory disorders
- Assay: stable in serum
NEOPLASIA VS HYPERPLASIA - Urine <6.0 pH – degrades rapidly
HYPERPLASIA - Normal value: 0.9 – 2.5 mg/L
o Involves the multiplication of cells in an organ or tissue, which 3. CA 125
may consequently have increased in volume. - High molecular weight mucin like glycoprotein
o Serves a useful purpose and is controlled by stimuli - Expressed: caulomic epithelium (during embryonic development),
o Elevation of tumor markers is transient human ovarian carcinoma cell
- Indicator: Non-mucinous epithelial ovarian carcinoma (serous
cystadenocarcinoma of the ovary)
NEOPLASIA - Upper limit: 35 U/ml
o Unregulated and serves no purpose 4. CA 153
o Elevation of tumor markers will be a long-lasting phenomenon if - HMW mucin glycoprotein in expressed by various adenocarcinoma
not treated – especially those associated with the breast
- Indicator: Breast cancer (>25 U/ml)
BENIGN VS MALIGNANT o Other: chronic hepatitis, liver cirrhosis, sarcoidosis, TB,
BENIGN SLE
o Sensitive and specific for metastatic breast cancer than
o Tumor remain at the primary site and present a smaller risk to
CEA
the host 5. CA 19-9
o At this stage the patient stands a good chance of being - HMW mucin
successfully treated by the complete removal of the tumor - Related to lewis blood group substance
o Early detection is critical to cancer prevention in general to high- - Le (a-b+) or Le (a+b-)
risk families in particular - Indicator: not organ specific, elevated in various adenocarcinoma,
o Well differentiated and composed of cells resembling the nature pancreatic, lung, colorectal, gastric CA
of normal cells from the tissue of origin of the neoplasm - Upper limit: 37 U/ml
6. CEA
- It is a glycoprotein
MALIGNANT - First of the so called CE-protein
o Due to genetic instability of tumor cells - Most widely used TM for GI cancer
o Most tumor cells undergo a benign stage, gradually progress to - Upper limit: 5ng/MI
malignancy and eventually become metastasized if not treated - False positive: heavy smoker / radiation treatment /
o METASTASIS chemotherapy, liver damage – impair CEA clearance
- Most cancer death are associated with metastic disease. - >10ng//MI – definitive malignancy
- Due to multiple genetic changes that result to uncontrolled 7. CHROMOGRANIN A.
- Major soluble protein of the chromaffin granules -a
proliferation
catecholamine- storage vesicle
- Tumor cells at the primary site penetrate the surrounding - Released from adrenal medulla with catecholamine
cells, then invade blood or lymphatic vessels and are carried - INDICATOR of Pheochromocytoma
to distant sites - Peptide producing tumor - Carcinoid tumor
- Pancreatic tumor - Small cell lung cancer
OTHER RELATED TERMS 8. ESTROGEN RECEPTOR
ANGIOGENESIS - Localized in the nuclei of mammary & uterine tissue
o It is a vital and complex biological process, forming new - Used to identify those patient most like to benefit from endocrine
capillaries from pre-existing blood vessels and infusing tissue therapy
- Patient whom primary tumor are ER/PgR-RICh also experience
with supplies of oxygen and nutrients.
longer disease-free intervals after mastectomy
9. HCG (HUMAN CHORIONIC GONADOTROPHIN)
DIFFERENTIATION - A sialoglycoprotein – consisting of non-covalently linked alpha and
o It is the process by which specialized cells becomes more beta dissimilar subunits
specialized cells. - Highly elevated = trophoblastic tumor choriocarcinoma, germ cell
tumor (ovary/testis), seminomas Beta HcG
- Free beta – HcG sensitive and specific for aggressive neoplasm
10. HVA (HOMOVANILLIC ACID) & VMA (VANILLYL MANDELIC ACID)
- Excreted in larger than normal amount in patient with tumor
originating from neural crest
- Both for: pheochromocytoma
o Neuroblastoma for children
11. SCCS (SQUAMOUS CELL CARCINOMA ANTIGEN)
- Indicative of: squamous cell CA of the head, neck, lung, esophagus,
anal canal, renal failure & advance cervical CA
- LASA-P (Lipid associated Sialic Acid in Plasma)
- Increased: Leukemia, Lhymphoma, hodgkin’s disease, melanoma,
non-malignant disease
- Not specific to any specific types of tumor used in conjugation with
other tumor marker
12. NEURON SPECIFIC ENOLASE (NSE)
- Gamma subunit of an enolase isoenzymes in the glycolytic pathway
APOPTOSIS
- Elevated: tumors originating from the neuroendocrine cell system
o A programmed cell death or a physiologic death including glucagonoma and insulinomas
o It is natural self-destruct system present in all cells - Highest levels in Oat Cell
o Failure to cells to undergo apoptotic cell death may lead to cancer - Children: Neuroblastoma
o Natural process the body employ for the replacement of cells 13. PSA (PROSTATIC SPECIFIC ANTIGEN)
and the deletion of damage cells - Single antigen glycoprotein
o Eliminate cells – produced excess, developed improperly have - Kalikrein ike serine protease produced exclusively by the epithelial
sustained genetic damaged. lining the acini-cell of the prostate gland
- It is a major protein in seminal plasma
- Lack specificity -inc, BPH, PROSTATITIS, INFRACTION

[SARANILLO, KA.]
 STAGES OF CANCER A good tumor marker should have those properties:
STAGE 1 Usually means that a cancer is relatively small and 1. A tumor marker should be present in or produced by tumor itself
contained within the organ it started in 2. A tumor marker should not be present in healthy tissues
STAGE 2 Usually means that the tumor is larger than stage 1, 3. Plasma level of a tumor marker should be at a minimum level
but the cancer has not started to spread into the in healthy subject and in benign condition
surrounding tissues. Sometimes stage 2 means that 4. A tumor marker should be specific for a tissue. It should have
the cancer cells have spread into lymph nodes close different immunological properties when it synthesized in
to the tumor. This depends on the particular type of other tissues.
cancer. 5. Plasma level of the tumor marker should be in proportion to
STAGE 3 Usually means the cancer is larger. It may have he both size of tumor and cavity of tumor
started to spread into surrounding tissues and there 6. Half-life of a tumor should not be very long
are cancer cells in the lymph nodes in the area. 7. A tumor marker should be present in plasma at a detectable
STAGE 4 Means the cancer has spread from where it started level, even though tumor size is very small
to another body organ. This is also called secondary Tumor markers can be classified as respect with the type of the
or metastatic cancer. molecule:
1. Enzyme or isoenzyme (ALP, PAP)
Note: Many cancers are associated with the abnormal production of 2. Hormones (Calcitonin)
some molecules which can be measured in plasma. These molecules 3. Oncofetal antigens (AFP, CEA)
are known as tumor markers 4. Carbohydrate epitopes recognized by monoclonal antibodies
(Ca 15-3, CA 19-9, CA 125)
TUMOR MARKERS 5. Receptors (Estrogen, progesterone)
o Biological substance synthesized and released by cancer cells 6. Oncogenes and tumor suppressor genes (BRCA 1 and 2)
o Substance produced by the host in response to cancerous tissue Potential uses of tumor marker
o Present in: Circulation o Screening in general population
Cavity fluids o Differential diagnosis of symptomatic patients
Cell membrane o Clinical staging of cancer
Cytoplasm/nucleus of the cell o Estimating tumor volume
ENZYMES o As a prognostic indicator for disease progression
o Malignant tumors have rate of glycolytic activity o Evaluating the success of treatment
o Detecting the recurrence of cancer
ENZYME ASSOCIATED MALIGNANT DISEASE
o Monitoring response to therapy
Prostatic Acid Phosphatase
Lysozyme Colon cancer, monocytic &
ENZYMES
myelomonocytic leukemia ALKALINE PHOSPHATASE (ALP)
Lactate dehydrogenase Acute leukemia, malignant o Increase ALP activities are seen:
lymphoma, germ cell tumors, - Primary or secondary liver cancer
metastic colon, breast and lung - Metastatic cancer with bone or liver involvement
cancers. - Placental ALP, regan isoenzyme, elevates in a variety of
5’ nucleotide malignancies, including ovarian, lung, gastrointestinal
phosphodiesterase cancers and Hodgkin’s disease
Sialyltransferase Nonspecific
Fucosyltransferase Multiple malignant tumor PROSTATIC ACID PHOSPHASE (PAP)
Thymidine kinase Hodgkin’s lymphoma, small cell o It is used for staging prostate cancer and for monitoring therapy
carcinoma of the lung o Osteogenic sarcoma
o Multiple myeloma
ISOENZYME ASSOCIATED MALIGNANT DISEASE o Bone metastasis of other cancers and in some benign conditions
CK-BB Adenocarcinoma of the prostate, such as osteoporosis and hyperparathyroidism
lung and stomach
Type A macro CK Metastic liver cancer PROSTATE SPECIFIC ANTIGEN (PSA)
Type 1 macro CK Neoplastic disease o The clinical use of PAP has been replaced by PSA.
Mitochondrial CK-IgA Prognosticator for patients with o PSA is much more specific for screening or for detection early
Complex advance tumor cancer. It is found in mainly prostatic tissue
Galactosyl transferase II o Urinary bladder catheterization and digital rectal examination
Placental ALP may lead an increased PSA level in serum
Placental like ALP (REGAN) Germ cell tumor, ovarian cancer o The ratio between free and total PSA is an reliable marker for
Liver ALP Liver metastasis, seminomas and differentiation of prostatic cancer from benign prostatic
ovarian cancer hyperplasia.
Bone ALP Osteosarcoma, bone metastasis o The greatest clinical use of PSA is in the monitoring of treatment.
LD-1, LD-4, and LD-5 Testicular germ cell tumors o This treatment includes radical prostatectomy, radiation therapy
Cancer at advance stage and antiandrogen therapy.
o The PSA level should fall below the detection limit.
PROTEINS o This may require 2-3 weeks. If it is still at a high level after 2-3
o Carcinoembryonic Protein weeks, it must me assumed that residual tumor is present
- Under normal condition – expression of all protein is
subjected to genetic regulation

HORMONE
o Usually made up of protein
o Increased if the tumor is in the hormone producing organ

[SARANILLO, KA.]
 HORMONES PROTEIN MARKERS
CALCITONIN β2 -MICROGLOBULIN
o Calcitonin is a hormone which decreases blood calcium o β2 -microglobulin is a marker for multiple myeloma, Hodgkin
concentration. lymphoma. It also increases in chronic inflammation and viral
o Its elevated level is usually associated with medullary thyroid hepatitis.
cancer. FERRITIN
o Lung CA, breast, kidney, liver and in nonmalignant conditions o Ferritin is a marker for Hodgkin lymphoma, leukemia, liver, lung
such as pulmonary diseases, pancreatitis, Paget’s disease, and breast cancer.
hyperparathyroidism, myeloproliferative disorders and THYROGLOBULIN
pregnancy. o It is a useful marker for detection of differentiated thyroid
cancer.
HUMAN CHORIONIC GONADOTROPIN (HCG)
o It is a glycoprotein appears in pregnancy. Its high levels is a useful IMMUNOGLOBULIN
marker for tumors of placenta and some tumors of testes. o Monoclonal paraproteins appear as sharp bands in the globulin
o hCG is also at a high level in patients with primary testes insufficiency. area of the serum protein electrophoresis.
o hCG does not cross the blood-brain barrier. Higher levels may o Bence-Jones protein is a free monoclonal immunoglobulin light
indicate metastasis in the brain. chain in the urine and it is a reliable marker for multiple
myeloma.
ONCOFETAL ANTIGENS
o Most reliable markers in this group are α-fetoprotein (AFP) and RECEPTOR MARKERS
carcinoembryonic antigen (CEA) o Estrogen and progesterone receptors are used in breast cancer
as indicators for hormonal therapy.
α-FETOPROTEIN (AFP) o Patients with positive estrogen and progesterone receptors tend
o α-fetoprotein is a marker for hepatocellular and germ cell to respond to hormonal treatment.
carcinoma.
o AFP is useful for screening (AFP levels greater than 1000 µg/L are C-erbB2 (HER-2 Neu)
indicative for cancer except pregnancy), determining prognosis o It is receptor for epidermal growth factor (EGF) but it doesn’t
and monitoring therapy of liver cancers. contain EGF binding domain. It serves as a co-receptor in EGF
o AFP and hCG combined are useful in classifying and staging germ action
cell tumors. One or both markers are increased in those tumors. o In the case of increased expression of C-erbB2 leads the auto-
activation and increased signal transduction
CARCINOEMBRYONIC ANTIGEN (CEA) o An important factor for carcinogenesis and metastasis
o It is a cell-surface protein and a well-defined tumor marker. o Normal cell growth may be over grown in some cancer
o CEA is a marker for colorectal, gastrointestinal, lung and breast
carcinoma. GENETIC CHANGES
o CEA levels are also elevated in smokers and some patients having o Four classes of genes are implicated in development of cancer:
benign conditions such as cirrhosis, rectal polips, ulcerative 1. Protooncogenes which are responsible for normal cell growth
colitis and benign breast disease. and differentiation
o CEA testing should not be used for screening. Some tumors don’t 2. Tumor suppressor genes which are involved in recognition and
produce CEA. It is useful for staging and monitoring therapy. repair of damaged DNA.
3. Apoptosis related genes are responsible for regulation of
CARBOHYDRATE MARKERS apoptosis
o These markers either are 4. DNA repair genes – Alterations on these genes may lead tumor
antigens on the tumor cell development.
surface or are secreted by
tumor cells. SUSCEPTIBLE DNA REPAIR GENES:
o They are high-molecular o BRCA1 and BRCA2 are specific genes in inherited predisposition
weight mucins or blood for developing breast and ovarian cancer, and mutations on
group antigens. these genes are newly measured in some laboratories.
Monoclonal antibodies have been developed against these o Mismatch – repair genes are mutated in some colon cancers
antigens.
o Most reliable markers in this group are CA 15-3, CA 125 and CANCER CLINICAL DNA MARKER RNA PROTEIN
CA19-9. TYPE SAMPLE MARKER
HEAD and Saliva, TP53, Cytokeratins SCC,
NECK serum microsatellite CD44,
CA 15-3 alterations CYFRA,
o CA 15-3 is a marker for breast carcinoma. Elevated CA 15-3 levels presence of telomerase
are also found in patients with pancreatic, lung, ovarian, HPV and EBV
colorectal and liver cancer and in some benign breast and liver DNA
diseases. LUNG Sputum/BA RASand Cytokeratins, CEA,
L. serum TP53 MAGE genes, CA 125,
o not useful for diagnosis. It is most useful for monitoring therapy.
mutations, CEA telomerase,
microsatellite CYFRA
CA 125 alterations
o Although CA 125 is a marker for ovarian and endometrial BREAST Serum Microsatellite Cytokeratins, CA 15-3
carcinomas, it is not specific. CA 125 elevates in pancreatic, lung, alterations hMAM, MAGE, (MS-1), CEA,
genes, CEA CA125
breast, colorectal and gastrointestinal cancer, and in benign
COLON Stool, serum RAS, Cytokeratins, CEA,
conditions such as cirrhosis, hepatitis, endometriosis, APC, and CEA CA19-9,
pericarditis and early pregnancy. TP53 CA15-3,
o In the detection of recurrence, use of CA 125 level as an indicator mutations Telomerase
is about 75 % accurate. PANCREASE Stool, serum RAS and TP53 Cytokeratins, CA 19-9
mutations CEA
BLADDER Urine/wash, TP53 Cytokeratins, CEA, CA125,
CA 19-9
serum mutations, surviving, CA19-9,
o CA 19-9 is a marker for both colorectal and pancreatic microsatellite uroplakin telomerase,
carcinoma. However elevated levels were seen in patients with alterations surviving,
hepatobiliary, gastric, hepatocellular and breast cancer and in CD44
benign conditions such as pancreatitis and benign PROSTATE Urine, PSA, PSA,
gastrointestinal diseases. serum MAGE genes, free PSA,
kallikrein telomerase
o CA 19-9 levels correlate with pancreatic cancer staging.
o It is useful in monitoring pancreatic and colorectal cancer.

[SARANILLO, KA.]
 FORMULA:
CLINICAL CHEMISTRY 322
FLUIDS AND ELECTROLYTES
PRELIM (ADDITIONAL NOTES)

ANION GAP
o Difference between unmeasured cations and anions
- Cations: Na and K
- Anions: Cl and HCO3
o Quality control for analyzer
o Monitor recovery from diabetic ketoacidosis

SAMPLE PROBLEM:
o Na: 140 mmol/L
o Cl: 104 mmol/L
o HCO3: 22 mEq/L
o K: 4.6 mmol/L

DIAGNOSTIC SIGNIFICANCE
INCREASE DECREASE
Uremia/Renal failure (phosphate Hypoalbuminemia (↓ UA)
and sulfate retention)
Ketoacidosis (starvation/diabetes) Hypercalcemia (↓ UC)
Poisoning (methanol, ethanol, Hyperlipidemia
ethylene glycol, salicylate)
Lactic acidosis
Hypernatremia Elevated myeloma proteins
Instrument error

CYSTIC FIBROSIS
o Recognized at infancy (meconium ileus) or early
childhood
o Defective chromosome 7: cystic fibrosis
transmembranous conductance regulator
(CFTR) gene
o CFTR protein: maintain the balance of salt and water on many
surfaces in the body (chloride)

SIGNS AND SYMPTOMS

o Unmeasured anions (UA): 23 mmol/L o Affected organs and glands: pancreas,
o Unmeasured cations (UC): 11 mmol/L Respiratory system
o ↓ Na: no change in AG (Cl shift) Sweat glands
o Other anions (↑ AG): sulfate, lactate, ketone anions o Affecting exocrine glands, thick mucus secretion, ↑ sweat
o (↓ AG): reduced albumin, bromide intoxication electrolytes, ↑ organic and constituents of saliva, overactivity of
o Widened AG: metabolic acidosis due to non-Cl containing acids autonomic nervous system
o Signs: chronic cough, foul smelling stool, persistent URT infection

DIAGNOSIS
o Test requirement: no rash, cuts or skin inflammation
o Neonate: at least 48 hours old
o Specimen: sweat
o Inducer: pilocarpine

1. SWEAT TEST – COULOMETRY

- ↑ Na and Cl

2. PILOCARPINE IONTOPHORESIS (GIBSON AND COOKE)

- Reference method
- >50 mg sweat sample collected within 30 minutes
- Reference value: 5-40 mmol/L
- (+) >65 mmol/L sweat electrolytes

[SARANILLO, KA.]
 ① ASSESMENT OF ACID-BASE BALANCE
CLINICAL CHEMISTRY 322 PARAMETER REFERENCE VALUE
BLOOD GAS AND PH MEASUREMENT
MIDTERM: WEEK 7 LEC (03/08/2023) pH 7.35 – 7.45
ACID-BASE BALANCE pCO2 35 – 45 mmHg
pO2 81 – 100 mmHg
HCO3 21 – 28 mEq/L
o Dissociation of H2CO3 increased HCO3 in RBC causing it to
STEP 1: Evaluate pH
diffuse into the plasma
o pH 7.40: optimum for arterial blood
o HCO3 and H2CO3 are renewable
o chloride-isohydric shift: buffering
o HCO3- + H2CO3 ratio = 20:1
effects of hemoglobin causing venous
blood to be 0.03 unit lower in pH
LUNGS
o each Celsius above 37o: ↑ pH by 0.015
o Respiratory control: CO2 excretion
o TO MAINTAIN pH:
o CO2 diffuses into alveoli and is eliminated through ventilation
1. neutralize acid as they are generated
2. elimination of the acid permanently on a continuous basis

STEP 2: Evaluate lungs

o pCO2: index of efficiency of gas exchange
o not a measure of CO2 concentration
o total CO2 = CO2 + H2CO3 + HCO3-
o ↑ heparin : blood = ↑ pCO2 (12-15%)
o ↑ pCO2: barbiturates, morphine,
KIDNEYS alcoholism
o Excretion of acid: generation of alkali or reabsorption of HCO3- o TO MAINTAIN pH:
from the glomerular filtrate and add it to the blood 1. Retention or elimination of CO2

STEP 3: Evaluate kidneys

o TO MAINTAIN pH:
1. Excreting acid (NH4)
2. Reabsorption of HCO3-

STEP 4: Evaluate oxygenation

o Hypoxia: ↓ pO2
o Reflects availability
o Changes rapidly
o ↓ 60 – 70% venous blood
o O2 dissociation / association = pO2 + affinity of hemoglobin
o ↓ pO2: myocardial infarction, interstitial pneumonia, congestive
heart failure, people living in high altitude area.

o 50-100 mmol/L of acid: must be excreted daily by the kidney STEP 5: Evaluate compensation
(urine pH is 4.6) o the respiratory and metabolic system works together to keep the
body’s acid-base balance within normal limits
PLASMA AND URINE BICARBONATE
RESPIRATORY ACIDOSIS
o ↑ HCO3- : IV infusion of lactate, acetate and HCO3-
o ↓ HCO3- : Diuretics, reduced reabsorption, chronic nephritis
o HCO3- excretion rate:

RESPIRATORY ALKALOSIS
BLOOD BUFFERS
1. HCO3- : HCO3 : major extracellular blood buffer
2. Plasma proteins: weak acid / base
3. Hemoglobin: O2 carrying
1 gram hemoglobin carries 1.39 ml of oxygen
1 mole of hemoglobin binds 1 mole of oxygen METABOLIC ACIDOSIS
4. Inorganic phosphate
HPO4 : H2PO4 = 3:1

HENDERSON – HASSELBALCH EQUATION

o pH depends on the ratio METABOLIC ALKALOSIS
of HCO3- pCO2
o expressed acid-base
relationship and relates
the pH of a solution to the
dissociation properties of
the weak acid

[SARANILLO, KA.]
 ② ACID-BASE DISORDER QUALITY CONTROL
RESPIRATORY ACIDOSIS o 3 levels of control: 1. Acidosis
o ↑ CO2: slow breathing 2. Normal
o Compensation: 3. Alkalosis
- Kidneys: retain HCO3- and o 1 level control sample every 8 hours
excretion of acid o 3 level control every 24 hours
o After compensation: o Single point calibration: used as a “drift
- ↑ pCO2, ↑ HCO3- , pH <7.4 check” to detect changes in response and/or
o Maximal compensation requires deterioration in performance
5 days (90% in 3 days)
o Restriction of NaCl: ↑ HCO3- METHODS
o ↑ 10 mmHg pCO2: ↑ 1 mEq/L HCO3- 1. Gasometer
a. Van Slyke
RESPIRATORY ALKALOSIS b. Natelson
o ↓ CO2: rapid breathing 1. Mercury: vacuum
o Psychogenic stimulation: ↑↑↑ pH 2. Caprylic alcohol: anti-foam
o Compensation 3. Lactic acid
- Kidneys: ↓ reabsorption HCO3- 4. NaOH and NaHSO3
o After compensation:
- ↓ pCO2, ↓ HCO3- , pH >7.4 2. Electrodes
o Compensation completed: 2-3 days a. pH: potentiometry
o ↓10 mmHg pCO2: ↓2mEq/L HCO3- 1. Silver-silver chloride electrode: reference electrode
o (+) hypokalemia 2. Calomel electrode (Hg2Cl2): reference electrode
3. Glass electrode: most common
METABOLIC ACIDOSIS b. pO2
o ↓ HCO3- 1. Clark electrode: polarography-amperometry
o K efflux c. pCO2
o Compensation: 1. Severinghaus electrode: potentiometry
- Lungs: ↓ pCO2 (hyperventilation)
o After compensation: 3. pO2 continuous monitoring
- ↓ HCO3- , ↓ pCO2, pH <7.4 a. Transcutaneous (TC) electrode
o Compensation completed: 12-24 hours ▪ place on directly to the skin
o ↓ 1 mEq/L HCO3- : ↓ 1-1.3 mmHg pCO2 ▪ neonates and infants
o (+) hyperkalemia and hyperchloremia ▪ non invasive

METABOLIC ALKALOSIS
o ↑ HCO3-
o Compensation:
- Lungs: ↑ pCO2 (hypoventilation) ④ FACTORS FOR BLOOD GAS AND PH MEASUREMENTS
o After compensation: 1. TEMPERATURE
- ↑ HCO3- , ↑ pCO2, pH >7.4 - Optimum: 37oC ± 0.1
o Compensation completed: 12-24 hours - Most important factor
o ↑ 10 mEq/L HCO3- : ↑ 6 mmHg pCO2 - ↑ 1oC = ↓ 7% pO2, ↑3% pCO2
o (+) hypokalemia and hypochloridemia - Electrode sample chamber

③ METHODS OF ANALYSIS 2. ↑ PLASMA PROTEINS

ARTERIAL BLOOD GAS (ABG) - ↑ pO2: build up on electrode membrane
o Specimen: arterial blood
o Anticoagulant: lithium heparin 3. BACTERIAL CONTAMINATION
o Ratio: 0.05 ml heparin / ml of blood - ↓ pO2: bacterial consumption
o Syringe: tuberculin
o Winged infusion set not recommended 4. IMPROPER SPECIMEN TRANSPORT
o Collected anaerobically - ↓ pH, ↑pCO2, ↓pO2

5. AIR BUBBLES
- ↑ pO2, 4 mmHg / 2mins
- ↓ pCO2: 4mmHg / 2mins

ALLEN’S TEST
o Test done to determine that collateral circulation is present from
the ulnar artery in case thrombosis occur in the radial artery

PRE-ANALYTICAL CONSIDERATIONS
o Standing specimen: ↓ pH, ↑ pCO2, ↓ pO2
o Chilled: ↓ pH – RBC metabolism consumes O2 and liberates
acidic metabolites
o Glycolysis: ↓ pH
o ↑ anticoagulant = ↓pH: most common error
o ↓ temperature = ↑ oxygen solubility, oxyhemoglobin curve shift
to the left

[SARANILLO, KA.]
 STRUCTURAL CLASS OF HORMONES
CLINICAL CHEMISTRY 322 o PEPTIDES/PROTEINS
INTRO TO ENDOCRINE SYSTEM
MIDTERM: WEEK 8 LEC (03/15/2023)
- Large molecules and cannot cross the cell membrane
- Exists in form of secretory granules (cleavable)
ENDOCRINE SYSTEM
- Water SOLUBLE and not protein bound
o A network of DUCTLESS tissue organs capable of synthesizing,
➢ GLYCOPROTEINS – FSH, hCG, TSH, EPO
storing and secreting a substance (stimulant/inhibitor).
➢ POLYPEPTIDES – ACTH, ADH, GH, Angiotensin
o Considered to be the regulatory system of the body
Calcitonin, Cholecytokinin, Gastrin, Glucagon, Insulin,
(Hemeostasis)
Melanocyte-Stimulating Hormone, Oxytocin, PTH,
Prolactin, Somatostatin
COMPONENTS OF THE ENDOCRINE SYSTEM
o STEROIDS
- Lipid molecules with cholesterol as precursor
- Water INSOLUBLE and protein bound
- Produced by Adrenal glands, Gonads, and Placenta
- Example: Aldosterone, Cortisol, Estrogen, Progesterone,
Testosterone, and Activated Vit D2

o AMINES
- AA derivatives and considered as an intermediary between
steroids and protein hormones
- Example: Adrenaline, Noradrenaline, Thyroxine,
Triiodothyronine

HYPOTHALAMIC CELLS
o Supraoptic nuclei –
FUNCTIONS OF ENDOCRINE SYSTEM Arginine Vasopressin
a. Control chemical and water balance in the body (AVP) aka Anti-Diuretic
b. Control growth and metabolism Hormone (ADH)
c. Control embryonic development and preparation for nurturing a
newborn MAGNOCELLULAR
d. Influence sexual behavior, stimulate growth and maturation of NEURONS
the gonads o Paraventricular nuclei
e. Feedback to the nervous system – Oxytocin

OPERATIONAL MECHANISM PITUITARY GLAND (HYPOPHYSIS)

o Nervous system monitors environment o Is known as the “Master Gland”
- NS sends message to endocrine gland o Located in the Sella turcica of the sphenoid bone
o Endocrine glands produce and secrete hormones into blood o Connected to the by the infundibular stalk to the median
- Hormones hit target organ eminence of the hypothalamus
o Target organ produces substance o General function: Feedback loops, Pulsatile secretion and diurnal
- 1 substance is picked up by receptors and brain stops rhythm
sending message to endocrine glands o Functional by 7th – 9th week of gestation

ENDOCRINOLOGY FEATURE FUNCTION OF PITUITARY GLANDS

o Study of hormones, hormone producing glands, and A. FEEDBACK LOOP
abnormalities of hormones and hormone producing glands. - Short feedback loop
- Long feedback loop
DISORDERS RELATED TO ENDOCRINE SYSTEM - Ultrashort feedback loop
o Tumors - Open loop
o Damage to cells or tissues B. PULSATILE SECRETION
o Hyposecretion - LH median interpulse is 55 mins (peak at 40 mins)
o Hypersecretion - GH median interpulse
C. DIURNAL VARIATION
HORMONES - Regulated through external signals (light-dark changes:
o Are secretions of endocrine glands that act upon a specific tissue Daylight-Darkness)
organs other than its source - ZEITGEBER (TIME GIVER) process of synchronizing external
o Defined as a substance that acts at a site distant from its place of cues with the internal biologic clocks
origin
o Its action may be described as a FEEDBACK MECHANISM 3 MAJOR STRUCTURES
1. ADENOHYPOHYSIS – Anterior Pituitary Gland
FEEDBACK MECHANISM 2. NEUROHYPOPHYSIS – Posterior Pituitary Gland
o Positive Feedback – increase in the product results to elevation 3. INTERMEDIATE LOBE – Pars Intermedialis
of the activity of the system
o Negative Feedback – increase in the product results to
decreased activity of the system

TYPES OF HORMONE ACTION

o ENDOCRINE – classic sample
o PARACRINE – adjacent cell, interstitial space
o AUTOCRINE – self acting
o JUXTACRINE – adjacent cell, direct contact
o EXOCRINE – lumen or gut
o NEUROCINE – neural hormone for nearby cells
o NEUROENDOCRINE – neural hormone for distance cells
o INTRACRINE – action within the cell source

[SARANILLO, KA.]
 NEUROHYPOPHYSIS HORMONES
o Posterior Pituitary Gland o Greek word “hormon” → to set in motion
o Storage region for ADH and Oxytocin o Intercellular chemical signal transported to act on tissues at another site
of the body to influence their activity
o Connected to the supraoptic (ADH) and paraventricular (Oxytocin)
o Transfer information and instructions from one set of cells to another
nuclei of the Magnicellular apparatus of the hypothalamus
o Not a true endocrine gland CHARACTERISTICS OF HORMONES
o Hormone release is stimulated by nipple sucking and serum o Produced by a specific endocrine gland
osmolality o Hormones are released directly from the endocrine gland to the blood
circulation and carried to the site of action as a free hormone or bound
OXYTOCIN to transport protein
o Neurohypophyseal secretions o Acts at a specific site (target site) to induce certain characteristic,
o Stimulates smooth muscle contraction of uterus & mammary glands biochemical changes.
o Labor and parturition
FUNCTIONS OF HORMONES
o Ejection of milk
o Regulate the chemical composition and volume of the ECF
o Help regulate metabolism and energy balance
ANTIDIURETIC HORMONE (ADH) OR VASOPRESSIN (AVP) o Help regulate contraction of smooth and cardiac muscles and secretion
o Neurohypophyseal secretions of glands
o Aka. Arginine Vasopressin (Henry’s), Vasopressin (Bishops) o Help maintain activities of immune system
o ADH or AVP o Plays a role in the smooth sequential integration of growth and
o Central role in water balance development
o Contribute to the basic processes of reproduction, gamete production,
o Acts on V2 receptors lining the epith. of collecting ducts and
nourishment of the fetus and embryo
ascending loop of henle o Help maintain homeostasis

DIABETES INSIPIDUS TYPES OF HORMONES

o Excessive urination with increased urine output o Circulating hormones/Endocrines: acts on distant target cells
o AVP deficiency o Local Hormones
o Result or tumor, trauma, and autoimmunity - Paracrines and Autocrines
o Low levels of AVP and High plasma Osm = DI
o Water deprivation with series of serum and urine Osm
determination may also be used

SYNDROME OF INAPPROPRIATE ADH (SIADH)

o Euvolemic hypoosmolar hyponatremia with hyperosmolar urine
o Inability to dilute urine
FUNCTIONAL TYPES OF HORMONES
ADENOHYPOPHYSIS
o Releasing Hormones:
o Anterior Pituitary Gland - from hypothalamus; promote secretion of Ant. Pituitary hormones
o TRUE ENDOCRINE GLAND o Inhibitory Hormones:
o Largest part of the pituitary gland - from hypothalamus & GIT; suppress the secretion of a particular
o Produces and secretes tropic and/or direct effector peptide/ hormone
glycoprotein hormones o Tropic Hormones:
- stimulate growth & activity of other endocrine glands
ADENOHYPOPHYSEAL CELLS SECRETION o Effector Hormones:
SOMATOtrophs SOMATOtropin (GH) - secreted by all endocrine glands & w/ non endocrine cells as targets
LACTOtrophs ProLACTin
TYPES OF HORMONES ACCORDING TO STRUCTURE
THYROtrophs THYROid Stimulating Hormone
GONADOtrophs GONADOtropins (FSH and LH) STEROIDS
CORTICOtrophs Cleavable proopiomelanocortin (ACTH, b- o Derived from cholesterol
endorphin, b-lipotropin) o transported to blood stream through attachment to transport protein
o E.g. aldosterone, cortisol, estrogen, progesterone, testosterone,
androgens
PHYSIOLOGIC REGULATORY SYSTEMS
o Endocrine system – consists of ductless glands, which secrete BIOGENIC AMINES (AA)
hormone directly into the circulatory system o Tyrosine
Thyroid hormones Adrenal hormones
o Nervous system
- T3-triiodothyronine - Epinephrine
- Neuroendocrine system - T4-thyroxine - Norepinephrine/
o TYPES OF GLANDS Cathecholamines
- Endocrine
PEPTIDES AND PROTEINS
- Exocrine
o synthesized by rough ER
o hypothalamic releasing and inhibiting hormone
TYPES OF ENDOCRINE CONTROL o E.g. oxytocin, ADH, insulin, glucagon, GH, calcitonin, PTH
GLYCOPROTEINS
o AA derivatives with CHO groups
o E.g. TSH, FSH, LH
EICOSANOIDS
o Fatty acids
o with 20 carbon atom fatty acid (arachidonic fatty acid), involved in
cellular activity
o E.g. prostaglandin
MECHANISM OF HORMONE ACTION

MAJOR GLANDS OF ENDOCRINE SYSTEM

o Pituitary Gland o Pancreas
o Thyroid Gland o Reproductive Glands (ovaries & testes)
o Parathyroid Gland o Thymus Gland
o Adrenal Gland o Pineal Gland

[SARANILLO, KA.]
 GROWTH HORMONE (SOMATOTROPIN)
CLINICAL CHEMISTRY 322 o Most abundant
HYPOTHALAMUS AND PITUITARY
MIDTERM: WEEK 9 LEC (03/22/2023)
o Secretion is erratic and short burst
o Ave. interpulse of 2-3hrs (highest during sleep)
TROPIC HORMONES
o Release in promoted by GHRH → pituitary gland
TROPIC HORMONES – acts on specific endocrine organ
o INCREASED: Acromegaly, Gigantism, Chronic malnutrition, renal
o GONADOTROPINS
disease, cirrhosis and sepsis
: FSH – Folliculogenesis (women) and spermatogenesis (men)
o DECREASED: hyperglycemia, obesity, and hypothyroidism
: LH – Leydig’s cells’ testosterone production and ovulation
o It is both metabolic and anabolic: AMPHIBOLIC
➢ TSH – stimulates thyroid activity
- Catabolism promotes gluconeogenesis and lipolysis
➢ ACTH – stimulates adrenal cortex
- Anabolic: increased CHON synthesis
o Hypoglycemic state: hypothalamus to secrete the GHIH
DIRECT EFFECTORS – acts on peripheral tissues
o Hyperglycemic state: stimulates GHRH (↓ sugar ↑ GHRH)
o Growth Hormone (GH) – direct effect on substrate
metabolism in numerous tissues (ie liver)
ACROMEGALY
o PROLACTIN – breast tissue development and (with E1 and
o GH excess in adults.
Progesterone) and initiation and maintenance of lactation
o Mostly due to Pituitary Tumor (>50ng/ml or 220pmol/L)
o Characterized by overgrowth of the bones and soft tissues (face
GONADOTROPINS
and extremeties)
o Follicle Stimulating Hormone (FSH) – folliculogenesis in women
o Usually with diastema (gap between the frontal teeth)
and spermatogenesis in men
o A hypermetabolic condition thus heat intolerance and excessive
o ↑ FSH = Clue for diagnosis of premature menopause
sweating may also be present
o Lutenizing Hormone – testosterone production of Leydig Cells
and ovulation and final follicular growth
Management of hypersecretion of GH
o ↑ FSH and LH; ↓ estrogen = Post menopausal
o Treatment is tumor ablation
o BOTH are important in fertility and menstrual cycle
(transhpenoidal adenomectomy)
o Irradiation
THYROTROPIN
o Somatostatin analogs and dopaminergic
o Thyroid-Stimulating Hormone (TSH)
agonist
o Main stimulus for iodine uptake by thyroid
o Pegvisomant
o Promote development of thyroid follicular cells and thyroid
hormone disease
Screening test
o Somatomedin C or Insulin-like Growth Hormone Test
CORTICOTROPIN
o Acromegaly: increase IGF-1
o Adrenocorticotropic Hormone (ACTH) (from pituitary gland)
o GHD: low IGF-1
o Feedback hormone for/of cortisol (Adrenal gland)
o ↑ cortisol ↓ ACTH and vice versa – to balance
Confirmatory test
o Regulates Adrenal androgen synthesis
o Oral glucose loading test (OGTT)
o Deficiency may lead to atrophy of adrenal gland’s G and R zone
o Overnight fasting
o Pituitary Gland → ACTH → Adrenal Gland
o The patient is given a 100g oral glucose load
o Highest level (peak): 6am-8am
o GH is measured at time zero and 60 and 120 minutes after
o Lowest level: 6pm-11pm
glucose ingestion
o Increased levels:
o Following oral glucose loading, GH is undetectable in normal
- Addison’s disease, ectopic tumors, after protein-rich meals
patients
PROLACTIN (PRL)
GIGANTISM
o Aka. Luteotropic Hormone / Luteotropin
o Juvenile GH excess
o Structurally similar to GH (amino acid structure)
o GH excess before long-bone growth
o Initiates and maintain lactation
o Treatment: Growth hormone replacement therapy
o Promotes breast development in conjunction with Progesterone
and Estrogen (develop in pregnancy)
GH deficiency
o Considered a stress hormone
o In children due to tumors such as craniopharyngiomas (Dwarfism)
o Develop in pregnancy o In adults due to structural and functional abnormalities of the
o Major inhibitor factor: DOPAMINE
pituitary gland
o ↑ Prolactin = associated with hypogonadism (FSH and LH) o Aging
PROLACTINOMA Screening test
o Most common pituitary tumor o Exercise test (Physical Activity Test)
o PREMONEPAUSALS: Irregular menstruation / Amenorrhea,
o Patient prep: Complete Rest for 30 mins (Fasting Serum)
infertility, or galactorrhea
o MEN/POSTMENOPAUSALS: pituitary mass (headache and visual
Confirmatory test
complaints), reduced libido (due to non-functional gonads) o Insulin tolerance test – GOLD STANDARD
o Drugs that can cause increase prolactin:
o Arginine Stimulation Test – 2nd GS
- TCA, phenothiazine, verapamil, Prozac, Opiates
HYPOPITUITARISM
CONDITIONS ASSOCIATED TO HYPERPROLACTINEMIA
o Panhypopituitarism – general loss of pituitary function
o Pituitary adenoma o Renal Failure
o Monotropic hormone deficiency – one type of adenohypotropic cell
o Infertility o Cirrhosis
o Amenorrhea o Primary and Secondary
o Galactorrhea Hypothyroidism Reference:
o Acromegaly o Polycystic Ovary Syndrome o Bishop, M. et al. 2010. Clinical Chemistry Techniques, Principles, and
Correlation. P.455-465. Lipincott Williams and Wilkins. Philadelphia,
- Prolactinoma: >150ng/mm
Pennsylvania, USA.
- Hyperprolactinemia: 25-100 ng/ml o McPherson, R. and Pincus, M. 2011. Henry’s Clinical Diagnosis and
Management by Laboratory Methods. p.367-371. Elsevier Publishing.
IDIOPATHIC GALACTORRHEA Philadelphia, Pennsylvania, USA.
o Lactation occurring in women with normal prolactin levels is o Gardner, G. and Shoback, D. 2011. Greenpan’s Basic and Clinical
defined as idiopathic galactorrhea Endocrinology. P1-26, 65-128. The McGraw-Hill, China.
o This condition is usually seen in women who have been pregnant o Rodriguez, 2018, Clinical Chemistry Handbook
several times and has no pathologic implication o Lecture notes (Picar) and (Baloro)

[SARANILLO, KA.]
 HYPOTHALAMUS ADENOHYPOPHYSIS HORMONES
o Portion of the brain located in the walls TABLE 20-2: ANTERIOR PITUITARY HORMONES
HORMONE TARGET GLAND STRUCTURE FEEDBACK HOMONE
and floor of third ventricle
LH Gonad (tropic) Dimeric glycoprotein Sex steroids (E2/T)
o Collection of specialized cells located at FSH Gonad (tropic) Dimeric glycoprotein Inhibin
the central part of the brain TSH Thyroid (tropic) Dimeric glycoprotein Thyroid hormones: T4/T3
o Control the pituitary gland by production ACTH Adrenal (tropic) Single peptide Cortisol
of chemicals that stimulate or suppress derived from POMC
GH Multiple Single peptide IGF-1
hormone secretion of pituitary (direct effector)
PRL Breast Single peptide Unknown
RESPONSE PATTERN (direct effector)
1. Open-loop negative feedback mechanism
GROWTH HORMONE (SOMATOTROPHIN)
o exerts major effects on cartilage and growth of long bones
o AA transport and nucleic acid & CHON synthesis
o increases hepatic glucose effect w/ anti-insulin effect in muscles
o increases lipolysis elevating plasma free FA (ketogenesis in diabetes)

2. Pulsatility – pulse frequency of secretion FACTORS AFFECTING GH SECRETION

- INCREASING the frequency of GnRH pulses – reduces the TABLE 20-3: OTHER MODIFIERS OF GROWTH HORMONE SECRETION
gonadotroph secretory response decreasing the pulse STIMULATE GH SECRETION INHIBIT GROWTH HORMONE SECRETION
Sleep Glucose loading
frequency, increases the amplitude of the subsequent LH
Exercise β-Agonists (e.g., epinephrine)
impulse. Physiologic stress α-blockers (e.g., phentolamine)
3. Cyclicity Amino acids (e.g., arginine) Emotional/psychogenic stress
- Nervous system – regulates this function Hypoglycemia Nutritional deficiencies
- Hormone secretion is dependent on the time of the day. Sex steroids (e.g., estradiol) Insulin deficiency
α-Agonists (e.g., norepinephrine) Thyroxine deficiency
- Ex. ACTH – peak occurs in the morning
β-blockers (e.g., propranolol)
Hormones that influence secretion and metabolic effects of GH:
HORMONES thyroxine, cortisol, estrogen, somatostatin, somatotropin releasing factor
o TRH: thyrotropin releasing hormones
o GnRH: gonadotropin releasing hormone PROLACTIN (PRL)
o GH-IH: growth hormone inhibiting hormone o acts directly on mammary glands
o GH-RH: growth hormone releasing hormone o controls the initiation and maintenance of lactation
o CRH: corticotropin releasing hormone o induces ductal growth, development of breast lobular alveolar
o PIF: prolactin inhibiting factor system and synthesis of specific milk proteins
o requires priming by estrogens, progestins, corticosteroids,
Table 20-1: HYPOPHYSIOTROPIC HORMONES thyroid hormones, and insulin
HORMONE STRUCTURE ACTION
o Men: 1-20ng/mL
TRH 3 amino acids Releases TSH and prolactin
GnRH 10 amino acids Releases LH and FSH o Women: 1-25 ng/mL
CRH 41 amino acids Releases ACTH
GHRH 44 amino acids Releases GH 3 FORMS OF CIRCULTING PROLACTIN:
Somatostatin 14 and 28 amino acids Inhibits GH and TSH release (additional 1. Non-glycosylated monomer - major form
effects on gut and pancreatic function)
Dopamine (PIF) 1 amino acid Inhibits prolactin release 2. Big prolactin - consists of dimeric and trimeric glycosylated form
3. Macro-prolactin – which is less physiologically active for
PINEAL GLAND
SPECIMEN CONSIDERATION
o Collect 3-4 hours after the patient awakes
o Highest level: 4-8am; 8-10pm

THYROID STIMULATING HORMONE (TSH)

o Increases:
- size of thyroid follicular cells
PITUITARY GLAND (HYPOPHYSIS) - release of thyroxine from thyroid colloid follicles
o small egg-shaped gland located - uptake of iodide by thyroid cells from ECF
at the base of the brain beneath - thyroxine biosynthesis
the hypothalamus o differentiates pituitary (2°) hypothyroidism from primary
o master gland hypothyroidism
o divided into 2 lobes:
- anterior & posterior FOLLICLE STIMULATING HORMONE & LUTEINIZING HORMONE
o FSH: growth and maturity of ovarian follicles, estrogen secretion,
promotes endometrial changes, spermatogenesis
ANTERIOR PITUITARY GLAND o LH: ovulation and secretion of androgens and progesterone,
o Composed of three cell types: initiates secretory phase of mens, formation of corpus luteum
- Chromophobe (50%) and development of testicular cells
- Acidophilic (40%)
- Basophilic (10%) ADRENAL HORMONE ACTH
o GH, PRL, TSH, FSH, LH, ACTH o acts on the adrenal cortex to stimulate growth and secretion of
- regulates the activity of thyroid, adrenals, and reproductive corticosteroids
glands o follows circadian rhythm
o also secretes ENDORPHINS o elevated during times of stress
- acts on the nervous system and reduce feelings of pain
SUMMARY
FIVE TYPES OF CELLS BY IMMUNOLOGICAL TEST o GH: growth of bone and soft tissues
1. Somatotroph – GH o PRL: for lactation
o TSH: release of thyroid hormones
2. Lactotrophs – Prolactin
o FSH: growth of the follicle (female) and
3. Thyrotroph – TSH initial wave of spermatogenesis (male)
4. Gonadotroph – α and β subunits of FSH & LH o LH: ovulation and final follicular growth (female) and
5. Corticotroph – Proopiomelanocortin (POMC) production of testosterone (male)
- ACTH o ACTH: release of cortisol
- β endorphin & β lipotrophin

[SARANILLO, KA.]
 POSTERIOR PITUITARY GLAND GALACTORRHEA
o Oxytocin or pitocin: for contraction of uterus and ejection of milk o inappropriate production of breast milk
primed with estrogen o due to hypersecretion of PRL
o ADH or arginine vasopressin or pitressin: permeability of kidney o symptoms: irregular menstruation, menopausal symptoms, milk
tubules discharges, difficulty in getting erection, breast tenderness and
enlargement
OXYTOCIN
o Major effect: smooth muscle contraction AMENORRHEA
o Stimulates contraction of the gravid uterus o absence of menstrual cycle in females
o Contributes directly to uterine contractions during labor on the o due to hypersecretion of PRL
myometrium and promotes prostaglandin secretion
o Hemostasis at the placental site after delivery IMPOTENCE
o HL: 3-5 minutes o inability to attain penile erection in males
o due to hypersecretion of PRL
SIGNIFICANCE
o Useful test in some pregnant women in predicting pre-term labor INFERTILITY
o Oat cell carcinoma of the lung and adenocarcinoma of the pancreas o lack of FSH and LH in both male and female
o inability to conceive after 1 year of unprotected intercourse
ARGININE VASOPRESSIN
o Maintain osmotic homeostasis CUSHING’S DISEASE
by regulating balance o hypersecretion of ACTH
o Nonapeptide that acts on the o leads to bilateral adrenal hyperplasia and cortisol
DCT and collecting tubules of overproduction
the kidneys o Obesity!!!
o Urine/serum/plasma
osmolality and thirst may ADDISON’S DISEASE
stimulate ADH secretion o secondary (ACTH) or tertiary (CRH) adrenal insufficiency
o 5-10% drop in blood volume and o hyposecretion of glucocorticoids and aldosterone
blood pressure triggers
(baroreceptors) the release ADH POLYURIA
o Responsible for the maintenance of blood volume, pressure and tonicity o deficient ADH production or action
o Basal plasma vasopressin: 2.3-3.1pg/uL - Hypothalamic DI
o Diagnostic test: Overnight water deprivation test - Nephrogenic DI
- Psychogenic or primary polydypsia
DISEASES ASSOCIATED W/ HORMONES OF THE PITUITARY GLAND
DWARFISM SYNDROME OF INAPPROPRIATE ADH SECRETION (SIADH)
o hyposecretion of GH during growth years o autonomous sustained production of AVP in the absence of
o types: known stimuli for its release
- Achrondroplasia o malignancy, CNS diseases, pulmonary disorders drug therapies
- Hypoachondroplasia o decreased urine volume, increased sodium concentration and
- Spondyloepiphyseal Dysplasia urine osmolality
- Diastrophic dysplasia
HYPOPITUITARISM
TEST OF GH INSUFFICIENCY o Panhypopituitarism
o Stimulation tests - tumors - infection
- After exercise or during sleep, GH normally increases - trauma - familial
- Clonidine (potent GH stimulant) - radiation therapy - idiopathic
o Gold standard test: insulin tolerance test - infarction
o 2nd confirmatory test: L-DOPA or Arginine stimulation test o Monotropic hormone deficiency

GH EXCESS TRUE DIABETES INSIPIDUS

o Over production of GH o Hypothalamic/neurogenic/cranial/ central diabetes insipidus
o Gigantism → childhood o Deficiency of ADH with normal ADH receptor, due to
o Acromegaly → Adults hypothalamic or pituitary disease
o Failure of the pituitary gland to secrete ADH
SCREENING TEST o Large volume of urine is excreted (3-20L/day)
o Somatomedin C test
o Fasting serum NEPHROGENIC DIABETES INSIPIDUS
o Complete rest o Normal ADH with normal ADH receptor
- renal resistance to ADH action
GIGANTISM
o Failure of the kidneys to respond to normal or elevated ADH
o hypersecretion of GH during childhood
levels
o Treatment: Desmopressin (dDAVP)
ACROMEGALY
o Urine output: >2.5 L
o hypersecretion of GH during adulthood
o Diagnostic test: Water deprivation test
o Features:
- coarse facial features
SIADH
- soft tissue thickening (lips)
o Occurs when there is uncontrolled secretion of ADH without
- spade like hands
any known stimulus for such release
- protruding jaw (prognathism)
o ADH is release even though the blood volume is normal or
- Sweating
increased and plasma osmolality is low
- impaired glucose tolerance or DM
o Ectopic tumor production of ADH: small cell carcinoma of the
lung
DIAGNOSIS OF ACROMEGALY
o CNS disease
o OGTT and GH measurement
o Pulmonary disease
o Hyperglycemia should suppress GH to <1 ug/L
o Administration of certain drugs
o After treatment, failure to suppress GH below 2 ug/L may cause
o Diagnosis: Water load test
higher prevalence of DM, heart disease, and hypertension

[SARANILLO, KA.]
LABORATORY MEASUREMENT OF SOME HORMONES SECRETED BY GnRH Test
THE PITUITARY GLAND o assesses hypogonadism
o can be done together with anterior pituitary
function test (IST, TRH, GnRH tests)
o Normally,
- Adults: GnRH causes marked rise in LH
(increments of >15 U/L) and smaller rise
in FSH (>2 U/L)
- Children: GnRH causes marked rise in FSH
and smaller rise in FSH

ACTH STIMULATION TEST

o cosyntropin test or tetracosactide test
o small amount of synthetic
ACTH is injected, and amount
of cortisol or aldosterone is
GROWTH HORMONE IMMUNOASSAY
measured
o uses specific GH antibody
o distinguish whether the cause
o require multiple measurements
is adrenal (low cortisol and
- draw specimens every 20-30 minutes over a 12-24 hours
aldosterone production) or
period
pituitary (low ACTH production)
o Insulin tolerance test: to produce hypoglycemia and provoke GH
- cortisol should be increased by twofold to threefold within
release
60 minutes
- Basal: 2-5 ng/mL or ug/L
- fasting (8 hrs)
- Insulin tolerance: >10 ng/mL
- Arginine/L-dopa: >7.5 ng/mL
LH OVULATION DIPSTRIP URINE TEST
o test approximately the same time each day
hGH-EASIA
o reduce liquid intake two hours before testing
o solid phase Enzyme Amplified Sensitivity Immunoassay
o Mab 1-hGH-Mab-HRP
LH IMMUNOASSAY (EIA/IRMA)
o absorbance is measured after colorimetric reaction
o Mab1-LH-Mab2HRP
- Day: <0.2-10 uIU/mL
measured using chromogenic reaction
- Night: 30 uIU/mL
Absorbance proportional to LH concentration
o Mab1-LH-Mab2125I
PROLACTIN IMMUNOASSAY
o homologous competitive binding immunoassay/ sandwich
FERTILITY TEST (MALE)
technique
o Testosterone 300-1100 ng/dl
o uses two or more antibodies directed at different parts of the
o Prolactin 7-18 ng/ml
PRL molecule
o Luteinizing Hormone (LH) 2-18 mIU/ml
o hook effect
o Follicle Stimulating Hormone (FSH): 2-18 mIU/ml
- Adult male: 3-14.7 ng/mL or ug/L
o Estradiol (Day 3): <50 pg/ml
- Adult female: 3.8-23 ng/mL or ug/L
- Pregnancy, 3rd tri: 95-473 ng/mL
FERTILITY TEST (FEMALE)
HORMONE FOLLICULAR DAY OF LH SURGE
ACTH IMMUNOASSAY FSH < 10 mIU/ml > 15 mIU/ml
o chemiluminescence and ELISA LH < 7 mIU/ml >15 mIU/ml
o related test: cortisol PRL < 25 ng/ml
o reacts with intact ACTH and ACTH fragments o FSH: measures your ovarian reserve (ovarian function)
- Adults: 5-80 pg/mL (X 0.22= pmol/L) o low levels of FSH & LH: hypogonadotropic hypogonadism
- Specimen: P, EDTA o high LH with a normal FSH level: PCOD (polycystic ovarian
disease)
DYNAMIC FUNCTION TEST o high prolactin: hyperprolactinemia
o stimulating or suppressing a particular hormonal axis, and
observing the appropriate hormonal response SERUM FSH MEASUREMENT (IRMA)
- If excess is suspected, conduct a suppression test o measures the amount of follicle stimulating hormone (FSH) in
- If deficiency is suspected, conduct a stimulation test blood
- Stimulus: exogenous analogue of a trophic hormone or a o Mab1-serum-Mab2125I
biochemical or physiological stress like hypoglycemia or o used to assess and manage disorders of the endocrine glands,
exercise including suspected infertility
o related tests: LH, PRL, testosterone, estradiol
INSULIN STRESS TEST
o done when hypopituitarism is suspected Normal Values for serum FSH
o also known as Insulin Tolerance Test o Female, menstruating:
o insulin is administered to produce hypoglycemic stress (<2.2 - Follicular phase: 1.4-9.9 mIU/mL (1.4-9.9 IU/L)
mmol/L) - Ovulatory phase: 0.2-17.2 mIU/mL (0.2-17.2 IU/L)
o Tests the ability of Anterior Pituitary Gland to produce ACTH and GH - Luteal phase: 1.1-9.2 mIU/mL (1.1-9.2 IU/L)
- GH >6 ug/L o Postmenopausal: 19.3-100.6 IU/L
- Cortisol > 500 nmol/L o Male: 1-15.4 mIU/mL (1-15.4 IU/L)

TRH Test ADH Measurement

o assesses the adequacy of Anterior Pituitary Reserve, or to o measures the amount of antidiuretic hormone, or vasopressin,
evaluate hypothalamic disease (TSH response to TRH is delayed: in blood
TSH higher at 60’ than 20’) o Related tests: sodium and osmolality
- Hyperthyroidism: pituitary response to TRH is flat - 270-280 mOsm/kg: <1.5 pg/mL (<1.4 pmol/L)
(TSH<2mU/L) - 280-285 mOsm/kg: <2.5 pg/mL (<2.3 pmol/L)
- Hypothyroidism: exaggerated response (>25 mU/L) - 285-290 mOsm/kg: 1-5 pg/mL (0.9-4.6 pmol/L)
o TRH is given as an IV bolus - 290-295 mOsm/kg: 2-7 pg/mL (1.9-6.5 pmol/L)
o Blood sampling done at 0, 20, and 60 minutes - 295-300 mOsm/kg: 4-12 pg/mL (3.7-11.1 pmol/L)

[SARANILLO, KA.]
 THYROID HORMONE BINDING PROTEINS
CLINICAL CHEMISTRY 322 o Thyroxine Binding Protein
THYROID AND PARATHYROID
MIDTERM: WEEK 10 LEC (03/29/2023)
- Transports majority of T3 (affinity for T3 is lower than T4)
- Transports 70-75% of T4
THYROID GLAND
o Thyroxine Binding Pre-Albumin (transthyretin)
o Also known as Butterfly-shaped gland.
- Transports 15-20% of T4
o Consist of two lobes (one on either side of the trachea) located
- No affinity to T3
in the lower part of the neck, just below the voice box (larynx).
o Thyroxine Binding Albumin
o The lobes are connected by a narrow band called the isthmus.
- Transports T3 and 10% of T4
o By 11 weeks of gestation, the gland begins to produce
measurable amounts of hormone.
Note:
o Follicle is the fundamental structural unit of the thyroid gland.
o Protein bound hormones are metabolically inactive.
o 2 types of cells:
o Free hormones (FT3 and FT4) are physiologically active
- follicular cells are secretory (T3 and T4)
o Protein bound hormones do not enter the cell and are
- parafollicular or C cells (Calcitonin)
considered as biologically inert and functions as storage sites for
o Thyroglobulin
circulating thyroid hormones
- acts as a preformed matrix
containing tyrosyl groups;
glycoprotein; THYROID FOLLICLES
- stored in the follicular
colloid of the thyroid gland.
o Controls the biosynthesis and
release of thyroid hormones
from thyroglobulin.
o Regulate carbohydrate, lipid,
and protein metabolism
o Act on the CNS
o Stimulate the heart
o Physical growth and development
THYROID HORMONES
THYROID HORMONES SYNTHESIS o There are two biologically active thyroid hormones:
- Tetraiodothyronine (T4; usually called thyroxine)
- Triiodothyronine (T3)
o Derived from modification of tyrosine

Thyroid hormones derived from two iodinated tyrosine molecules

SYNTHESIS OF THYROID HORMONES

o Active uptake of iodide into follicular cells
o Iodide → iodine – H2O2 (catalysed by TPO)
o Active uptake of iodine at follicular/colloid interface
o Incorporation of iodine onto tyrosine residues of thyroglobulin
o Coupling of iodinated tyrosines
o Storage of T3 and T4

Incorporation of iodine onto tyrosine residues on the

thyroglobulin molecule

[SARANILLO, KA.]
 THYROXINE (T4) HYPOTHYROIDISM
o Principal secretory product o Serum level of thyroid hormone is insufficient to provide for
o Dominant hormone produced from the thyroid gland the metabolic needs of the cells
o All circulating T4 originates in the thyroid gland o Treatment with thyroid hormone replacement therapy:
o It is a good indicator of thyroid secretory rate Levothyroxine
o Represents 80% of thyroid hormone Symptoms include: Causes:
o Serves as the pro-hormone for T3 ▪ Enlarged thyroid gland (goiter) ▪ Tissue damage
o Elevated level causes inhibition of TSH secretion and vice versa ▪ Fatigue ▪ Lack of dietary iodine
▪ Impairment of metal processes
▪ Loss of appetite
TRIIODOTHYRONINE (T3)
▪ Myxedema
o Represents only 20% of the thyroid hormone produced from the gland
o Metabolically active hormone
o Produced by conversion of T4 within the liver and periphery PRIMARY HYPOTHYROIDISM
o Metabolic rate of every cell of the body o Inadequate secretion of thyroid hormones
o Encourages cellular differentiation o There is a problem with the thyroid gland itself
o Tissue growth and development o Laboratory results
o Affect oxygen consumption - Decreased T3, decreased T4
o Calorie and vitamin/mineral metabolism - Decreased free thyroxine index (FT4I)
o Almost 70-80% is produced from the deiodination of T4 - Decreased T3 uptake (T3U), increased TSH
(conversion of T4 to T3 takes place in many tissue, particularly in
the liver and kidneys). HASHIMOTO’S DISEASE (Chronic autoimmune thyroiditis)
o Principal application of this hormone is in diagnosing T3 thyrotoxicosis o Caused by a genetic abnormality in the immune system
o Better indicator of recovery from hyperthyroidism o Involves massive infiltration of the thyroid gland by
o Increased in the plasma level of T3 is the first abnormality seen lymphocytes
in hyperthyroidism o Same symptoms with hypothyroidism
o Most common cause of hypothyroidism
CALCITONIN (CT) Signs and symptoms Causes:
▪ Weigh loss ▪ Pituitary tumors
o Participates in calcium homeostasis by responding to a
▪ Loss of muscle mass ▪ Excessive TSH secretion
hypercalcemia ▪ Hyperactivity yet quick fatigability ▪ Thyroid carcinoma
o Depresses the release of calcium from the bone ▪ Insomnia ▪ Toxic multi-nodular goiter
o Inhibits the bone-dissolving activity of osteoclasts ▪ Increased sweating
▪ Nervousness
HYPERTHYROIDISM ▪ Palpitations
o Excess of circulating thyroid hormones ▪ Goiter
o Signs and symptoms: tachycardia, tremors, weight loss, heat ▪ Bulging eyes
intolerance, and menstrual changes
MYXEDEMA
o Primary hyperthyroidism: elevated T3 and T4 (defects of
o Peculiar non-pitting swelling of the skin
thyroid gland), decreased TSH
o Skin becomes infiltrated by mucopolysaccharides
o Secondary hyperthyroidism: increased FT4 and TSH (defects
o Clinical feature: “puffy face”, weight gain, slow peach,
of pituitary gland)
eyebrows thinned, dry and yellow skin and anemia.
o Myxedema Coma: severe form of primary hypothyroidism
THYROTOXICOSIS
o Caused by excessive thyroid hormone in the circulation
SECONDARY HYPOTHYROIDISM
o Causes the cells to be overactive
o Involves decrease in production of TSH leading to low serum
o T3 thyrotoxicosis or Plummer’s disease: FT3 is increased but
levels of thyroid hormones
FT4 normal with low TSH
o Failure of the pituitary gland
o T4 thyrotoxicosis: T3 is normal or low but T4 increased with
o Laboratory results
low TSH
- All thyroid test values are decreased
o Laboratory results:
- Elevated thyroid hormone serum levels
TERTIARY HYPOTHYROIDISM
- Decreased serum TSH
o Caused by hypothalamic failure leading to a lack of TRH
production
GRAVE’S DISEASE (DIFFUSE TOXIC GOITER)
o Laboratory results:
o Autoimmune disease in which antibodies are produced that
- All thyroid test values are decreased
activate TSH receptor
o Features: exophthalmos (bulging eyes) and pretibial
CONGENITAL HYPOTHYROIDISM/ CRETINISM
myxedema
o Defect in the development or function of the gland
o Laboratory results
o Symptoms: physical and mental development of the child are
- Increased T3, T4, FT4I (Free T4 Index) and T3U (T3
retarded
Uptake)
o Screening test: T4 (Decreased)
- Decreased or Normal TSH
o Confirmatory test: TSH (increased)
RIEDEL’S THYROIDITIS
SUBCLINICAL HYPOTHYROIDISM
o Inflammation of the gland
o T3 and T4 are normal but TSH is slightly increased
o Thyroid turns into woody or stone-hard mass
o Caused by: Viral infection
Bacterial Infection OTHER DISORDERS OF THE THYROID
o Drug-induced thyroid disease
SUBCLINICAL HYPERTHYROIDISM o Amiodarone-induced thyroid disease
o Shows no clinical symptoms but TSH level is low and FT3 and - Cardiovascular drug
FT4 are normal - High iodine content
- Decreased thyroid hormone synthesis
SUB-ACUTE GRANULOMATOUS/ SUB-ACUTE NON-SUPPURATIVE - Hypothyroidal condition
THYROIDITIS/ DE QUERVAIN THYROIDITIS (PAINFUL THYROIDITIS) o Non-thyroidal illness
o Neck pain, low-grade fever and swings in thyroid function test o Thyroid nodules
o Thyroidal peroxidase (TPO) antibodies are absent; ESR and
thyroglobulin test levels are elevated

[SARANILLO, KA.]
 LABORATORY ANALYSES Measurement:
o T3 resin uptake - Double antibody (RIA)
- Analyzes the capacity of thyroid binding globulin to bind - Enzyme-linked immunoassay (ELISA)
thyroid hormones - Immunoradiometric assay (IRMA)
- Indirect measurement of the number of free binding sites - Immunochemiluminescent assay (ICMA)
on the TBG molecule - Measurement depends on:
o Free thyroxine Index (FT4I) ➢ Specificity of antibody used
- Indirectly assesses the concentration of circulating free T4 ➢ Absence of anti-thyroglobulin autoantibodies
- Calculated by multiplying the value of the total T4 by the ➢ Well-differentiated thyroid CA (+)
percentage value of the T3 resin uptake
o Thyroid antibody screens D. THYROID AUTOIMMUNITY/ANTIBODIES
- Assay for the presence of thyroid-stimulating - Thyroid stimulating antibodies (TSAb, TSI)
immunoglobulins - TSH receptor antibodies (TRAb, TSHR-Ab)
o TRH stimulation test - Grave’s disease: antibody directed at TSH receptor
- Measures pituitary TSH stores - Chronic lymphocytic thyroiditis (Hashimoto’s thyroiditis):
- Considered conclusive for hyperthyroidism TPO antibodies
- Thyroid-releasing hormone is injected
- Blood sample are assayed for thyroid-stimulating hormone NUCLEAR MEDICINE
- TSH levels rise rapidly in a normal person RADIOACTIVE IODINE
- TSH levels will not rise in a hyperthyroid patient o Assessing metabolic activity of thyroid tissue
o Iodine Uptake Scan o Assisting in the evaluation & treatment of thyroid CA
- Iodine is taken up by the thyroid gland (RAI uptake) o Percentage of oral dose is taken by the thyroid gland
- Dose of radioactive iodine on an empty stomach o Radioactive iodine uptake
- Concentrated in the thyroid gland or excreted in the urine
over the next few hours High Uptake
- Amount of iodine that goes into the thyroid gland can be - Gland is metabolically active & producing significant
measured by a “Thyroid Uptake” amounts of thyroid hormone
o RIA for Calcitonin - High uptake with undetectable TSH: thyroid is acting
- Marker for medullary thyroid carcinoma autonomously (hypothalamus pituitary-thyroid feedback
o Thyroid Ultrasound system)
- Use of high frequency sound waves to obtain an image of - TSH surrogate (Grave’s disease): Immunoglobulin activates
the thyroid gland and identify nodules TSH receptor = ↑ thyroid hormone; ↑ radioactive iodine
- “solid” or a fluid-filled cyst uptake
- Allows accurate measurement of a nodule’s size Low Uptake
- Determine if a nodule is getting smaller or is growing larger - Gland is metabolically inactive
during treatment
o Thyroid Needle Biopsy “Hot nodules” “Cold nodules”
- Most reliable test to differentiate the “cold” nodule that is - take up significant - Show little or no radioactive
cancer from the “cold” nodule that is benign radioactive iodine iodine
- Unlikely thyroid CA - Indeterminate/may be
- “hot” nodules are rarely cancerous
- Benign cancerous (majority are
- Able to concentrate benign)
TESTS FOR THYROID EVALUATION - Malignant
BLOOD TEST - Unable to concentrate
A. TSH
- Most useful test for assessing thyroid function
- All assays are capable of diagnosing primary
hypothyroidism
- 3 generations of assays

2nd Generation 3rd Generation

▪ 0.1 mU/L ▪ 0.01 mU/L detection limits
detection limits ▪ Routinely used to monitor & adjust thyroid
▪ Routinely used hormone replacement therapy
to monitor & ▪ Screen for hyperthyroidism & hypothyroidism Thyroid in Grave’s disease (diffuse uptake), in toxic adenoma (focal
adjust thyroid ▪ less likely to give false-negative results uptake, and in toxic nodular goiter (focal or patchy uptake), which has
hormone ▪ More accurate distinguish between both autonomous and nonfunctioning nodules
replacement euthyroidism & hyperthyroidism
therapy ▪ ability to detect subclinical disease (mild
THYROID ULTRASOUND
▪ screen for degree of thyroid dysfunction due to large
hyperthyroidism reciprocal change in TSH levels even for small
o Assessment of thyroid anatomy
& hypothyroidism changes in Free T4 o Characterization of palpable abnormalities
o Small size (<1cm) thyroid nodules seen
B. SERUM T4 AND T3
- RIA: radioimmunoassay FINE NEEDLE ASPIRATION BIOPSY
- Chemiluminometric assay o Most accurate tool in
- Immunometric technique evaluation of thyroid
- Total T4 and T3 nodules
- Free T4 and T3 o Identification and
treatment of thyroid
C. THYROGLOBULIN malignancy
- Protein synthesized & secreted by thyroid follicular cells o Palpation with
- Pro-hormone in circulation Ultrasound
- Proof of presence of thyroid tissue (benign/malignant) o Indeterminate with
- Ideal tumor marker for thyroid cancer patients FNAB: surgical removal
for definitive diagnosis
- Patients treated for thyroid CA: undetectable thyroglobulin
levels

[SARANILLO, KA.]
 PARATHYROID GLAND 1,25-dihydoxy Vitamin D (Active metabolite)
o Located on or near the thyroid capsule; sometimes within the o In small intestinal:
thyroid gland - Upregulates expression of a number of genes that stimulate
o May also be found between the hyoid transepithelial calcium transport from the intestinal lumen
bone in the neck and mediastinum into the blood
o Smallest endocrine gland in the body - Site of greatest absorption is the duodenum
o Secreted parathyroid hormone (PTH): - Also stimulates absorption of phosphate
HYPERCALCEMIC hormone o In bone:
- Differentiation of osteoclast precursors to osteoclasts
ROLE OF PARATHYROID HORMONE - Stimulates osteoblasts to influence osteoclasts to mobilize
o Prime role: to prevent hypocalcemia (regulates blood calcium) bone calcium
o It preserves calcium and phosphate within the normal range - Does not directly affect mature osteoclast physiology
o Major physiologic action is mineral homeostasis - Mineralization of bone
o Involved in the metabolism of both calcium and phosphorus by o Abnormal bone results when vitamin D is deficient or its
the kidney and bone metabolism is defective
o In bone o Increases blood calcium by augmenting intestinal absorption of
- Increases bone resorption (breaking down) of calcium into calcium. Blood calcium feeds back to parathyroid tissue and
plasma affects synthesis and secretion of PTH.
o In the Kidney o Direct transcriptional control over the PTH gene in the
- Increases renal reabsorption of calcium parathyroids
- To drive 1α-hydroxylation of 25-hydroxy vitamin D to - The 1,25(OH)2D –
produce the active metabolite of vitamin D, 1,25(OH)2D vitamin D receptor
(1,25-dihydroxy vitamin D) complex binds to the
o Stimulates conversion of inactive Vitamin D to activated vitamin D3 vitamin D response
o Indirectly stimulates intestinal absorption of calcium element upstream of
o As calcium levels increase, PTH secretion is suppressed allowing the PTH gene and
urinary loss of calcium and calcium remain in bone downregulates PTH
o If calcium levels decrease, PTH is released gene transcription.
- PTH stimulates
CALCITRIOL is production of
a form of 1,25(OH)2D, and
vitamin D that 1,25(OH)2D, in turn,
is used to treat feeds back to
and prevent decrease PTH
low levels of secretion, all to maintain blood calcium in the normal range.
calcium in the
blood of LABORATORY ANALYSES
patients whose o PTH C-terminal analysis:
kidneys or - Examines the intact PTH molecule
parathyroid - Specific for detecting hyperparathyroidism
glands (glands o PTH N-terminal analysis:
in the neck - Measures both the whole PTH molecule
that release - Amino-terminal fragments in the serum
natural o Nephrogenous cAMP:
substances to - Measures urinary portion of cAMP produced by the kidneys
control the - Directly influence by PTH and its excretion by the renal
amount of tubules
calcium in the blood) are not working normally.
PRIMARY ANALYTES OF INTEREST:
o TOTAL AND IONIZED CALCIUM
HORMONAL CONTROL OF CALCIUM METABOLISM - Ionized (free) calcium is the biologically active component
o Parathyroid hormone (PTH) and vitamin D are the two hormones of circulating calcium (50% of circulating)
that play the dominant role in the regulation of calcium - Total calcium is being measured when calcium is measured
homeostasis in a serum chemistry panel.
o INORGANIC PHOSPHORUS
VITAMIN D - Retention causes a decrease in ionized calcium, leading to
o it should first be noted that vitamin D is in reality a hormone and, increased parathyroid hormone
as is true of hormones in general, it is made at (a) site(s) different o PARATHYROID HORMONE
than the organs whose function it affects. - ↑ calcium, ↓ phosphorus
o Vitamin D shares striking similarities in origin with steroid
hormones ANALYTES OF SECONDARY INTEREST
o A metabolic product of the cholesterol synthetic pathway o CALCITONIN
o Tissues involved in the synthesis: skin, liver and kidneys - ↓ calcium, ↑ phosphorus, counteracts parathyroid
o Tissues it affects: gut, bone, and parathyroid hormone (PTH)
o De novo synthesis of vitamin D begins in the skin o VITAMIN D
o 7-dehydrocholesterol is transformed to vitamin D3 by the action - Stimulates intestinal absorption of calcium
of ultraviolet light. o MAGNESIUM
o Vitamin D3 is biologically inert and must be further metabolized - Magnesium deficiency can inhibit the secretion of PTH and
to the biologically active metabolite also blunt its action on target tissues
o An enzyme in the liver, 25-hydroxylase, metabolizes vitamin D3 o BICARBONATE
to 25-hydroxy vitamin D. - PTH decreases bicarbonate reabsorption
o Serum 25-hydroxy vitamin D indicates the adequacy of vitamin D
stores in the body.
o An enzyme in the kidneys, renal 1-hydroxylase, under regulation
from PTH, completes the metabolism of vitamin D to the active
metabolite, 1,25-dihydroxy vitamin D.

[SARANILLO, KA.]
 CLINICAL DISORDERS HYPERPARATHYROIDISM
HYPOPARATHYROIDISM HYPERCALCEMIA
PRIMARY HYPOPARATHYROIDISM o Diagnosed when serum calcium levels rise
o Hyposecretion of PTH o Higher than 102 ng/L
o Caused by: o Sustained at level >100 mg/L
- Surgical removal of the gland (thyroidectomy, lymph node
dissection) PRIMARY HYPERPARATHYROIDISM
- Trauma following surgery o Most common cause of hypercalcemia in the outpatient setting
- Radiotherapy directed at thyroid gland o Physiologic defect lies with the parathyroid glands themselves
o Laboratory results: o Autonomous nature of PTH production
- Low serum calcium o Single adenoma, multiple adenomas, or hyperplasia of the
- High phosphorus parathyroid glands.
o Laboratory results:
IDIOPATHIC HYPOPARATHYROIDISM - Serum calcium is increased
o Rare and hereditary - PTH is increased
o Laboratory results: - Phosphorus is normal to decreased
- Very low serum calcium level
- Very high phosphorus level GENETIC SYNDROMES ASSOCIATED WITH PRIMARY
HYPERPARATHYROIDISM
PSEUDOHYPOPARATHYROIDISM o Multiple endocrine neoplasia, type 1 (MEN-1): This may result
o Characterized by lack of responsiveness of renal or other organ in tumors of the parathyroids,
systems to PTH pituitary, and pancreas. It
o This results from uncoupling of the PTH receptor from adenylate results from loss of a tumor
cyclase, due to a mutant stimulatory G protein (Gs). suppressor gene that maps to
o PTH binds its receptor but cannon activate the second human chromosome 11.
messenger, cAMP, and thus there is no response. o Multiple endocrine neoplasia,
o Laboratory results: type 2a (MEN-2a): medullary
- Serum PTH levels are typically normal to increased in this thyroid hyperplasia or cancer,
condition and pheochromocytoma. This
results from an activating
OTHER CAUSES OF HYPOPARATHYROIDISM mutation in the ret photo-
o Autoimmune destruction of parathyroid tissue associated with oncogene, which resides on
other autoimmune diseases such as type 1 diabetes, human chromosome 10.
Hashimoto’s thyroiditis, and Addison’s disease. o Familial hyperparathyroidism: without other associated tumors.
o Mutations in the PTH gene The gene is not known but has been mapped to human
o Abnormal deposition of copper or aluminum in the parathyroid chromosome 1.
glands o Familial hypocalciuric hypercalcemia (FHH): result of a mutation
in the calcium sensing receptor thus an increased “set point” for
calcium homeostasis. It is associated with mild hypercalcemia
and hyperparathyroidism, yet decreased (or low normal) urinary
calcium excretion. It is the decreased urinary calcium excretion
that distinguishes FHH from primary HPT, making this test very
valuable in distinguishing between the two.

SECONDARY HYPERPARATHYROIDISM
o Condition associated with the body’s attempt to compensate for
hypocalcemic state
o Seen in renal failure due to inexcretion of phosphorus
o Decreased calcium stimulates secretion of PTH
o Laboratory results:
- Serum calcium is low
- PTH is increased
- Phosphorus is increased

TERTIARY HYPERPARATHYROIDISM
o Autonomous PTH secretion and hypercalcemia in the setting of
renal failure or after renal transplantation
o Prolonged stimulation of the parathyroid glands, stimulates the
development of parathyroid hyperplasia or adenoma similar to
that seen in primary hyperparathyroidism
o PTH is often extremely elevated
o Occurs with secondary hyperparathyroidism
o Treated with parathyroidectomy

[SARANILLO, KA.]
 PHOSPHATE BUFFER SYSTEM
CLINICAL CHEMISTRY 322 o higher concentrations inside the cell
ACID-BASE BALANCE AND BLOOD GASES
MIDTERM: WEEK 12 LEC (ADDITIONAL NOTES)
o Regulates intracellular pH
o pKa = 6.8 (close to intracellular pH)
ACID-BASE PHYSIOLOGY
o Takes several hours to correct acid-base imbalances
o NORMAL [H+] = 40 nmol/L or 40 nEq/L (range 38–42 nmol/L)
o Most of this intracellular buffering occurs in the bone.
o Normal arterial pH: 7.40 (7.35-7.45)
o important source of buffering acid load acutely by an uptake of
- Measured as Ph
H+ in exchange for Na+, K+, and bone minerals.
- pH = -log [H+]
o These bone minerals rescue the HCO3−/CO2 system in severe
acidosis.
PRODUCTION OF ENDOGENOUS ACIDS AND BASES
o acid = proton donor
LUNGS
o base is a proton acceptor
o the second line of defense against pH disturbance.
o DIET is a major contributor to endogenous acid and base
o pCO2 is maintained around 40 mmHg.
production.
o achieved by expelling the CO2 that is produced by cellular
metabolism through the lungs ALVEOLAR VENTILATION
ENDOGENOUS ACIDS
o Oxidation of dietary carbohydrates, fats, and amino acids yields CO2.
o takes several hours to complete.
o ↑H+ = ↑ Ventilatory Rate = ↓pCO2 →→

ALVEOLAR VENTILATION
o H2CO3 - volatile acid o controlled by chemoreceptors (medulla, carotid body and aortic arch.
o Nonvolatile (fixed) acids from cellular metabolism o maintains normal pCO2 to prevent an acute change in pH.
o Sulfuric Acid (50%) - Sulfur-containing Amino Acids (I.E., Cysteine o Blood [H+] and pCO2 are important regulators
And Methionine) And o ↓ Ventilatory Rate = ↑ pH (near normal)
o Phosphoric Acid – Phosphoproteins
o Lactic And Pyruvic Acids – Glucose KIDNEYS
o Acetoacetic/ Β-hydroxybutyric Acids - Triglycerides o The maintenance of [HCO3−] is achieved by three renal
o Hydrochloric acid - cationic amino acids (i.e., lysine, arginine, and mechanisms:
histidine) - Reabsorption of filtered HCO3−
o OTHER SOURCES: > starvation produces ketoacids > strenuous - Generation of new HCO3− by titratable acid (TA) excretion
exercise - lactic acid > corticosteroids (catabolism of muscle - Formation of HCO3− from generation of NH4+
CHONs)
REABSORPTION OF FILTERED HCO3
ENDOGENOUS BASES o HCO3− is freely filtered at the glomerulus.
o anionic amino acids (glutamate and aspartate) o The daily filtered load of HCO3− is 4,320 mEq.
o citrate or lactate generated during metabolism of carbohydrate (24 mEq/L × 180 L/day = 4,320 mEq/day).
o Vegetarian diets contain high amounts of anionic amino acids o HCO3− reabsorption by various segments of the nephron can be
and small amounts of sulfur- and phosphate-containing proteins. summarized as follows:
o Therefore, these diets generate more bases than acids. In - Proximal tubule, 80%
general, the production of acid exceeds that of base in a person - Loop of Henle, 10%
ingesting a typical North American diet. - Distal tubule, 6%
- Collecting duct, 4%
MAINTENANCE OF NORMAL PH
PROXIMAL TUBULE
BUFFERS o Bicarbonate
o All acids that are produced must be removed from the body in reabsorption occurs due
order to maintain normal blood pH. to H+ secretion into the
o Buffers - first line of defense against wide fluctuations in pH. tubular lumen via Na/H+
o BICARBONATE/CARBON DIOXIDE - most important buffer in exchanger.
blood o H-ATPase Transporter:
o Other buffer systems are disodium phosphate/monosodium responsible for proton
phosphate (Na2HPO42−/ NaH2PO4−) and plasma proteins. transport into the lumen.
o HEMOGLOBIN (HB) SYSTEM - reduced Hb (HHb−), and o Na/H Exchanger – major
oxyhemoglobin (HbO22−). exit point of H+ from the
o Bones also participate in buffering (intracellular) cell to the lumen
o Kidneys takes hours to days. o Na/K-ATPase pump – provides energy and electrochemical
gradient for H+ secretion and HCO3− exit
HCO3 - / H2CO3 BUFFER SYSTEM o Na/HCO3 symporter – HCO3− exit point located in the
o first line of defense in protecting pH basolateral membrane
o HCO3 - / H2CO3 Ratio = 20:1 o CARBONIC ANHYDRASE II – catalyze hydration of CO2 to form
o Henderson– Hasselbalch equation H2CO3 - Cytoplasmic
o H2CO3 – Measured as pCO2 o CARBONIC ANHYDRASE IV – splits H2CO3 into H2O and CO2. -
o At normal body temperature: Apical membrane
- pH= 7.4
- pCO2 = 40mmHg LOOP OF HENLE (10% )
- pKa = 6.1 o Most HCO3− reabsorption occurs in the
- solubility coefficient (α) of CO2 = 0.03 thick ascending Henleʼs loop.
- Normal plasma [HCO3−] is 24 mEq/L o The mechanisms for H+ secretion into
the lumen and HCO3− exit across the
PLASMA PROTEIN basolateral membrane is similar to the
o Plasma proteins contain several ionizable groups in their amino proximal tubule.
acids with pKa close to the extracellular pH that buffer either
acids or bases
o HEMOGLOBIN – Gas exchange
o NOTE: Extracellular buffering to an acid load is complete within
30 min.

[SARANILLO, KA.]
 DISTAL TUBULE Factors affecting HCO3- reabsorption by the distal tubule
o THREE DISTINCT SEGMENTS:
- distal convoluted tubule – contains only H-ATPase in its
apical membrane.
- connecting tubule – consists of principal cells and
intercalated cells
- cortical collecting duct - consists of principal cells and
intercalated cells
o INTERCALATED CELLS – 3 TYPES OF INTERCALATED CELLS
- Type A, Type B, Type C

GENERATION OF NEW HCO3− BY TITRATABLE ACID EXCRETION

URINARY BUFFERS
o PHOSPHATES (HPO4-) – responsible for excretion of 40% of H+ in
the proximal tubule
o HPO4 :H2PO4 (3:1)
- TITRATABLE ACIDITY - number
of equivalents of hydroxyl
ions required to titrate a unit
volume of acidic urine to the
INTERCALATED CELLS pH of the blood
o Type A – H+ secreting cells - pKa = 6.8 an ideal urinary
- contain H-ATPase and K/H exchanger in buffer. (Intracellular Buffer)
the apical membrane. - 1 H+ secreted = 1 HCO3-
- Cl/HCO3 exchanger – AE1 gene formed
expression
o Type B - secrete HCO3− into the lumen. AMMONIUM (NH3+)
- Pendrin - a Cl/HCO3 exchanger located o responsible for excretion of
in the apical membrane 60% of H+
- H-ATPase - basolateral membrane. H+ 1. PCT: L-Glutamine + H2O ➜ L-
is secreted into the peritubular capillary glutamate + NH4+
o Type C (formerly non-A, non-B) cells o NH4+ secreted in
express HATPase and pendrin (Cl/HCO3 the lumen via
exchanger) in the apical membrane. Na/H antiporter
- also participate in HCO3− handling. (instead of H)
2. Reabsorption in the TALH (Na/K/CL cotransporter) (replacing K)
PRINCIPAL CELLS 3. Renal Medulla: NH4+ -> NH3 + H+ - NH3 accumulation
o major role in transepithelial sodium, potassium and water 4. Diffusion into Collecting Tubules: NH3 + H+ ➜ NH4+
transport, but they may also play a role in acid secretion. o New HCO3− is formed from the metabolism of glutamine and its
- apical H+ secretion organic anions (α-ketoglutarate). Glutamine⟷ NH4 ++
- basolateral Cl−/HCO − exchange activity o Glutamate ⟷ NH3 + a -Ketoglutarate ⟷ 2HCO –
- cytosolic carbonic anhydrase
- H+-K +/ATPase alpha-subunits ACID BASE DISORDERS
- ammonia transporters
- Rhbg
- Rhcg

COLLECTING DUCT
o HCO3- regulation involves:
o Type A Intercalated cells (outer and inner medullary)
- Pendrin (apical)
- H+/ATPase (basolateral)
- H+-K +-ATPase (apical)
o reabsorb HCO3− and secrete protons
o does not secrete HCO3− into the lumen.

ALDOSTERONE
o HCO3− reabsorption and H+ secretion by the intercalated (Type A) cell.
o stimulates Na+ reabsorption by the principal cell.
o As a result of this Na+ reabsorption, the lumen becomes
electrically negative, which promotes H+ secretion.

Factors affecting hco3- reabsorption (or h+ secretion) by the proximal tubule

[SARANILLO, KA.]
 EVALUTATION OF ACID-BASE DISORDERS
o pH: 7.40
o HCO3 - : 24 mEq/L
o pCO2 : 40 mmHg
o pO2: 90mmHg (81-100)
Respiratory Opposite: Metabolic Equal (ROME)

METABOLIC VS RESPIRATORY

ANALYSIS OF MIXED ACID–BASE DISORDERS

o Mixed acid–base disorders should be suspected whenever:
1. There is no compensatory response or overcompensation for a
primary simple acid–base disorder.
2. The pH and [HCO3−] are normal, but the AG is high (mixed
metabolic acidosis and metabolic alkalosis).
3. The pH is near normal, but [HCO3−] is low (mixed metabolic
acidosis and respiratory alkalosis).
4. The pH is low (7.40), but [HCO3−] is normal (mixed metabolic
alkalosis and respiratory alkalosis).

EVALUATION OF MIXED ACID-BASE DISORDER

1. Identify the PRIMARY DISORDER.
2. Calculate ANION GAP.
3. Determine the △AG/ △HCO3 -.
4. Calculate the EXPECTED COMPENSATION VALUE (WINTERS
FORMULA)
5. Identify the Secondary Disorder.

[SARANILLO, KA.]
 CORTEX STEROIDOGENESIS
CLINICAL CHEMISTRY 322 o Control of steroid hormone biosynthesis is complex, including
ADRENAL GLANDS
MIDTERM: WEEK 12 LEC (04/12/2023)
adrenocorticotropic hormone (ACTH) and angiotensin (Ang) II
o All adrenal steroids are derived by sequential enzymatic
ADRENAL GLANDS
conversion of a common substrate: cholesterol
o Multifunctional organ that produces
o Adrenal parenchymal cells accumulate and store circulating low-
the steroid hormones and
density lipoproteins (LDL)
neuropeptides essential for life
o The adrenal gland can also synthesize additional cholesterol
o Most pathologic conditions of the
using acetyl-CoA, ensuring normal adrenal steroidogenesis in
adrenal gland are linked by their
patients with variable lipid disorders and in patients on lipid-
impact on blood pressure and
lowering agents
electrolyte balance
o Corticotropin-releasing hormone (CRH)
o (circadian signals, serum cortisol, and stress)
CORTEX MEDULLA
o release of stored ACTH
Corticosteroid: lipid derivatives Catecholamine: Tyrosine derivative
3 Zones of Cortex:
o transport of free cholesterol into adrenal mitochondria
1) G Zone (Zona Glomerulosa) Norepinephrine/Noradrenaline o accumulate and store circulating low-density lipoproteins (LDL)
- Mineral corticoids: Aldosterone o synthesize additional cholesterol using acetyl-CoA
2) F Zone (Zona Fasciculata) Epinephrine/Adrenaline o Mitochondrial membrane cytochrome P450 (CYP450) converts
- Glucocorticoids: Cortisol Cholesterol to Pregnenolone
3) R Zone (Zona Reticularis) o After conversion,
- Sex steroids precursor: Pregnenolone is returned
Dehydroepiandrosterone
to the cytosol for
subsequent zonal
o Adult adrenal glands are shaped like pyramids, located just conversion by microsomal
above and medial to the kidneys in the retroperitoneal space enzymes in each layer by
(suprarenal glands). F-layer enzymes and/or
o Cortex appears yellow, the medulla is dark mahogany androgens by enzymes in
o Glucocorticoids from the cortex are carried directly to the the R-layer
adrenal medulla via the portal system, where they stimulate
production of epinephrine (EPI) G ZONE
o Sympathetic and parasympathetic axons reach the medulla o Pregnenolone (3β-hydroxysteroid dehydrogenase) →
through the cortex. o Progesterone (21β- hydroxylase) →
o En route, these axons release neurotransmitters (e.g., o 11-Deoxycorticosterone(11β-hydroxylase) →
catecholamines, neuropeptide Y) that modulate cortex blood o Corticosterone(18-hydroxylase) → Aldosterone
flow, cell growth, and function.
o Medullary projections into the cortex have been found to F ZONE
contain cells that also synthesize and release neuropeptides o Pregnenolone (17α-hydroxylase) → 17α-OH Pregnenolone
- vasoactive inhibitory peptide (VIP) (3β-Hydroxysteroid dehydrogenase) → 17α-OH Progesterone
- Adrenomedullin o Progesterone (17α- hydroxylase) → 17α-OH Progesterone
- atrial natriuretic peptide (ANP) (21β-hydroxylase) → 11-Deoxycortisol
o 11-Deoxycortisol (11β- hydroxylase) → Cortisol
Cortisol → Cortisone
o <Urinary 17-hydroxycorticosteroids>

R ZONE
o 17α-OH Pregnenolone & 17a-OH Progesterone
(17α-hydroxylase) → DHEA
o DHEA (3β-Hydroxysteroid dehydrogenase) → Androstenedione
→ Testosterone → Estradiol
o DHEA (S) → Androstenediol (3β- Hydroxysteroid dehydrogenase)
→ Testosterone → Estradiol
o <Urinary 17- ketosteroids>

G ZONE SUMMARY:
o Zona glomerulosa cells (outer 10%)
o synthesize mineralocorticoids (aldosterone) sodium retention
volume
o ↓ Potassium
o Acid-base homeostasis
o Low cytoplasmic-to-nuclear ratios and small nuclei with dense
chromatin with intermediate lipid inclusions.

F ZONE
o Zona fasciculata cells (middle 75%)
o synthesize glucocorticoids, (cortisol, and corticocortisone)
o critical to blood glucose homeostasis and blood pressure.
o Fasciculata cells are cords of clear cells, with a high cytoplasmic-
to-nuclear ratio and lipids laden with "foamy" cytoplasm.

R ZONE
o Zone reticularis cells (inner 10%)
o Sulfate DHEA (dehydroepiandrosterone) to DHEAS, which is the
main adrenal androgen.
o The zone is sharply demarcated with lipid-deficient cords of
irregular, dense cells with lipofuscin deposits.

[SARANILLO, KA.]
o Cortisol is the primary feedback regulator of ACTH- stimulated CORTISOL
hormone production in the adrenal cortex. o Synthesis: 15-20 mg/day
o ACTH generally does not significantly impact G-layer aldosterone o Maintain blood glucose by inducing lipolysis and amino acid
synthesis release from muscle breakdown for conversion into glucose
o Decreased activity of any enzymes required for biosynthesis can (gluconeogenesis) → storage as liver glycogen
occur as an acquired or inherited (autosomal recessive) trait.
o Defects that decrease production of cortisol cause increases in URINARY CORTISOL METABOLITES
ACTH and CRH secretion in an attempt to stimulate cortisol levels 1. 17-hydroxycorticosteroid
and lead to adrenal hyperplasia or overproduction of androgens - Utilizes phenyl hydrazine in H2SO4 + alcohol (Porter-Silber method)
2. 17-ketogenic steroids
- Sodium bismuthate for Norymberski reaction
- Meta-dinitrobenzene for Zimmerman reaction

G ZONE
o Conversion of Pregnenolone to Aldosterone.
o 15-20 mg/day
o Synthesis occurs due to: PATHOLOGIC CONDITIONS
- Low Aldosterone synthase activity in other zones CONGENITAL ADRENAL HYPERPLASIA (CAH)
o Final oxidation of Corticosterone to Aldosterone o Inherited family of enzyme disorders
o Decreased cortisol and aldosterone production
- Low G-cell 17α-hydroxylase activity
o Clinical presentation depends on enzyme affected
o Prevents substrate diversion into other pathways
o Laboratory findings:
o Aldosterone secretion is regulated by: - Increased upstream substrates
- Renin-Angiotensin System (RAS) which functions to - Overflow across open pathways
maintain mainly sodium balance. - Decreased products downstream
o Renin: proteolytic enzyme by renal cells in the juxtaglomerular o Partial defects: puberty
apparatus o 95%: 21-hydroxylase deficiency
o Production stimulated by: - 17-OH progesterone & androgen buildup
- Volume depletion - Cortisol decreased
- Low filtered salt - Treatment: Oral glucocorticoids
- Sympathetic nerve stimulation
ISOLATED HYPOALDOSTERONISM
o Insufficient aldosterone production
o Adrenal gland destruction
o Chronic heparin therapy
o Unilateral adrenalectomy (transient)
o Patients with mild renal insufficiency
o DM with mild metabolic acidosis
o High serum K+
o Low urinary K+ excretion (urine K+ <urine Na+)
o Low reninemia
o Treatment: Florinef (synth. Mineralocorticoid)
- Enhances salt retention, secretion of Potassium and Acid

HYPERALDOSTERONISM
o Excess aldosterone production
o Metabolic alkalosis
o HTN
o Hypokalemia
- Easy fatigability
- Muscle weakness (paralysis)
- Polyuria (loss of renal concentrating ability)
ALDOSTERONE - Palpitations
o Increase blood pressure through volume expansion by increasing - Impaired insulin secretion
sodium reabsorption → water retention
o Stimulates Hydrogen + and Potassium + excretion → metabolic PRIMARY ALDOSTERONISM
alkalosis with volume expansion, hypertension and hypokalemia o Low Renin
o Enhanced with high sodium diets o Autonomous over secretion of aldosterone
o Diagnosis:
Stimulated by: Inhibited by: - PA/PRA (Plasma Renin Activity) greater than 25
Angiotensin II Atrial Natriuretic Peptide (ANP) - Low plasma renin that fails to increase with volume depletion
ACTH Intracellular calcium - High aldosterone that fails to decrease with saline or
Elevated Serum K+ Certain drugs: angiotensin inhibition
Progesterone - Ketoconazole
Dopamine - ACE inhibitors SECONDARY ALDOSTERONISM
- NSAIDS
o Elevated Renin
- Heparin o RAS-activated aldosterone secretion

PSEUDOALDOSTERONISM
o LIDDLE’S SYNDROME
- Increase epithelial sodium channel
o BARTTER’S SYNDROME
- Bumetanide-sensitive Chloride channel mutation
o GITELMAN’S SYNDROME
- Thiazide-sensitive transporter mutation

[SARANILLO, KA.]
 HYPERCORTISOLISM
Overproduction of CRH & ACTH Adrenal glucocorticoid secretion Effects
o Immune (suppression, poor healing)
o Dermatologic (thin, friable tissue, wide purple striae)
o Vascular (vessel fragility, ecchymoses)
o Metabolic (hyperglycemia & insulin resistance)
o Bone (loss)
o Adipose (increased fat with redistribution to
DIAGNOSIS ALGORITHM upper back and central locations buffalo hump.
o Urinary potassium excretion o Muscle (wasting, weakness, heart failure)
- >30 mEq/day: suggestive of hyperaldosteronism o Neurologic(neuropathy)
- <30 mEq/day: renal K+ retention (diuretic use & GIT loss) o Renal (edema, HTN, calciuria)
o Upright PA/PRA ratio
- Overnight fluid deprivation (PRA) CUSHING’S SYNDROME
- *Volume expansion (2L normal saline in 4h: normally o Excess glucocorticoid production
supresses aldosterone) o Hypercortisolism
- PA/PRA ration: >25 (1'Aldosteronism) o Prolonged exogenous steroid use
o ACTH o Most common causes:
- ACTH & Cortisol: highest early in the morning (8am) - ACTH-secreting pituitary adenoma
- ACTH & Cortisol: lowest at night (10pm-12am) - Autonomous Cortisol production from adrenal tumor (ACTH
- ACTH pulse amplitude: rises between 2-4am suppressed)
- ACTH suppressed: Elevated glucocorticoids - Excess ACTH or CRH production (usually malignant)
- ACTH peaks: Protein-rich meals
ADH & CRH stimulation CUSHING’S SYNDROME DIAGNOSIS
Hypoglycemia: ADH & CRH stimulation 1. Document Cortisol excess
Acute stress - Random plasma cortisol: of little value for the diagnosis of
Cushing’s syndrome
ADRENAL INSUFFICIENCY: - Baseline AM cortisol: have no diagnostic value
ADDISON’S DISEASE - Urine free cortisol
o Low cortisol: Hypocortisolism o Increased once corticosteroid binding globulin is
o Primary adrenal problem: destruction of 90% of the adrenal saturated
gland cortex o 24-hour urine free cortisol (Tandem Mass
- Autoimmune - Bilateral adrenal hemorrhage Spectroscopy): most sensitive & specific
adrenalitis (70%) - Adrenoleukodystrophy 2. Determine if Diurnal Rhythm is Lost
- Fungal diseases - Infiltrative processes - Late night values remain high
- HIV infection - Metastasis - Plasma cortisol (highest 6-8 am) (lower 10pm-12am)
- Tuberculosis - Single midnight cortisol (>7.5 ug/dL) = 100% specific
o Secondary to ACTH deficiency - Single salivary cortisol level (11pm) with 8am salivary
o Glucocorticoid therapy (most common) cortisol after overnight dexamethasone suppression (1mg)
o Tumors = 100% specific & 92% sensitive
o Hemorrhage o midnight (12am) values <1.3ng/ml (RIA)
o Infiltrative processes o midnight (12am) values >1.5ng/ml (Competitive-
o Developmental abnormalities protein binding)
o Malignancies 3. Determine Loss of Normal Cortisol
o Signs and Symptoms: - Suppression by dexamethasone
- Weakness - Hyponatremia o Dexamethasone acts as exogenous cortisol
- Fatigues - Hyperkalemia substitute
- Anorexia - Hypercalcemia o Suppress ACTH production of pituitary gland (N)
- Nausea - Prerenal azotemia o Suppress Cortisol production of adrenal gland (N)
- Diarrhea - Mild metabolic acidosis o Screen patients for overproduction of cortisol
- Abdominal pain
- Weigh loss ANDROGEN EXCESS
o Diagnosis o Ambiguous genitalia in infant girls
- Low baseline cortisol levels (8am, supine) o Precocious puberty in children
- Elevated ACTH >200 pg/ml o Short stature
- Random cortisol >20 ug/dl o Virilization in boys
- Cosyntropin (synthetic stimulator of cortisol/aldosterone) - Penile enlargement
stimulation tests - Androgen-dependent hair growth
o Metyropone Test – Block 11B-hydroxylase - 2' sexual char.
o Insulin tolerance test o Virilization in girls
- Gold standard for secondary and tertiary hypocorticolism - Hirsutism
- Acne
- Clitorimegaly

[SARANILLO, KA.]
ANDROGEN INSUFFICIENCY URINE & PLASMA CATECHOLAMINE MEASUREMENTS
o Virilization in women. o Catecholamine are:
- Infertility - Hydrophilic
- Masculinizing effects - Circulate in low levels (50% albumin-bound)
- Hirsutism - Short half lives (seconds to 2 minutes)
- Acne - Produce wide, rapidly fluctuating levels
- Male-pattern baldness
- Menstrual irregularities CAUSES OF SYMPATHETIC HYPERACTIVITY
o Autonomous dysfunctions
o In Men: o Panic attacks (emotions)
- Inhibits gonadotropins release by pituitary o Pheochromocytoma (catecholamine-producing tumor)
- Lowers testicular testosterone production o Stress response: Hypoglycemia Injury
- Virilization in men Infraction Infection
o Infertility Psychosis Seizures
o Feminizing effects o Drugs:
o Hypogonadal symptoms - Decongestants
▪ Loss of muscle mass - Appetite suppressors
▪ Decreased hair growth - Stimulants
▪ Decreased testes size - Bronchodilators
- MAO inhibitors
ADRENAL MEDULLA - Thyroid hormone
o Composed primarily by chromaffin cells that secrete - Cortisol
catecholamines - Nicotine withdrawal
- Short-acting sympathetic antagonists
MEDULLARY HORMONES - Clonidine & propranolol
o Norepinephrine (Primary amine) - Tyramine intake
- produced by sympathetic ganglia
- Highest concentration id found in brain
- neurotransmitter in both CNS and SNS
o Epinephrine (Adrenaline, Secondary amine)
- Most abundant medullary hormone
- Produced from norepinephrine, comes only from adrenal
- "Fight or flight hormone", released in response to
physiologic injuries or psychological threats (stress and
anxiety)
o Dopamine
- Cathecolamine produced in the body by the
decarboxylation of 3,4-dihydroxyphenylalanine (DOPA)
- Major intact cathecolamine present in urine

MEDULLARY CATECHOLAMINE RELEASE

o STIMULATION → Catecholamine release
- (STIMULATION-Transmitting messages via efferent axons)
o Medulla functions as an atypical sympathetic ganglion
o Catecholamines serve as 1st responders to stress in seconds
(cortisol takes 20 mins)
- Promotes fight-or-flight response
- Increases cardiac output & blood pressure
- Diverts blood toward muscle & brain
- Mobilizes fuel from storage

BIOSYNTHESIS & STORAGE OF CATECHOLAMINES

o Medulla chromaffin cells
o Norepinephrine (NE) passively diffuse into the cytosol
o NE is converted into Epinephrine (EPI) by Phenylethanolamine N-
methyltransferase (PNMT) {Cortisol dependent: ↑Cortisol=↑EPI}
o EPI is transported into secretory vesicles by vesicle monoamine
transporters (VMAT) in pheochromocytes

[SARANILLO, KA.]
 CLINICAL CHEMISTRY 322 MYOTONIC DYSTROPHY
GONADAL HORMONES
MIDTERM: WEEK 12 LEC
o Inherited in an autosomal dominant fashion
- Hypogonadism
HORMONOGENESIS
- Muscle weakness
o Testosterone: predominant hormone secreted by the testes
- Frontal balding
o Controlled by pituitary hormones:
- Diabetes
- Follicle-stimulating hormone (FSH):
➢ acts primarily on germinal stem cells - Muscle dystonia
- Luteinizing hormone (LH): o Testicular failure typically presents in the fourth decade of life
➢ Acts primarily on the Leydig cells
➢ Located in the testicular interstitium TESTICULAR INJURY AND INFECTION
➢ Synthesize testosterone o Postpubertal mumps infection o Testicular damage
- Mumps orchitis - Viral orchitis
HORMONAL CONTROL OF TESTICULAR FUNCTION - Permanent testicular injury - HIV infection
o GnRH: determines the production of LH and FSH o Radiation and chemotherapy for cancer
o ↓ Pulse generation of GnRH = ↓ LH and FSH = HYPOGONADISM
SERTOLI CELL-ONLY SYNDROME
TESTICULAR STEROIDOGENESIS o Lack of germ cells
o Conversion of cholesterol to pregnenolone o Men present with:
- Cholesterol: trapped by endocytosis from the blood or - Small testes
synthesized within the Leydig cells - High FSH levels
o LH binds to the glycoprotein receptor in the cell wall - Azoospermia
- Induces intracellular cyclic AMP production - Normal testosterone levels
- Activates protein kinase A o Testicular biopsy is the only procedure to confirm this diagnosis
- Catalyzes protein phosphorylation
DISORDERS OF SEXUAL DEVELOPMENT & TESTICULAR HYPOFUNCTION
HORMONAL CONTROL OF TESTICULAR FUNCTION o Low testosterone levels together with low or inappropriately
o Testosterone: principal androgen hormone in the blood normal FSH or LH levels
o 2% - 3% free o Hypogonadotropic Hypogonadism
o About 50% of testosterone is bound to albumin - Kallmann’s Syndrome - Age
o About 45% is bound to sex hormone-binding globulin (SHBG) - Hyperprolactinemia - Pituitary Disease
o Testosterone & inhibin: provide feedback control to the
hypothalamus and pituitary KALLMANN’S SYNDROME
o Testosterone concentration fluctuates in a circadian fashion o Inherited, X-linked recessive trait that manifests as
- Reflecting the parallel rhythms of LH and FSH levels hypogonadism during puberty
o Highest levels: found at about 8am o Anosmia (inability to smell)
o Lowest levels: found at about 8pm o Midline defects (cleft palate and lip)
o Certain men also have red-green color blindness, congenital
DISORDERS OF SEXUAL DEVELOPMENT & TESTICULAR HYPOFUNCTION deafness, or cerebellar dysfunction
o Hypergonadotropic Hypogonadism
o ↓ testosterone, ↑ FSH or LH, and impaired sperm production HYPERPROLACTINEMIA
- Klinefelter’s Syndrome o Prolactin elevation
- Testicular Feminization Syndrome o Drug-induced
- 5-Reductase Deficiency o Prolactin-producing tumors of the pituitary
- Myotonic Dystrophy
- Testicular injury and infection AGE
- Sertoli Cell-only Syndrome
o Gradual reduction in testosterone after age 30
o Average decline of about 110 ng/dL every decade
TESTICULAR FEMINIZATION SYNDROME
o Elevation of SHBG by about 1% per year
o Most severe form of androgen resistance syndrome
o Total testosterone levels may be normal in aging men but the
o Lack of testosterone action in the target tissue
free (unbound) levels of testosterone are more reliable
o Physical development pursues the female phenotype
indicators of biochemical reduction
- Fully developed breast
o Reduced secondary sex hair growth
- Female distribution of fat and hair
o Loss of muscle bulk and strength
o Men present with primary amenorrhea
o Loss of bone density
- Lack of female internal genitalia becomes apparent
o Low serum testosterone levels
o Testicles are often undescended
o Failure to promptly remove testicles results in malignant
PITUITARY DISEASE
transformation
o Injury to the pituitary o Autoimmune hypophysitis
o Normal levels of testosterone o Granulomatous metastatic disease
o Tumors
o Elevated FSH and LH levels o Hemochromatosis (rare)
o Surgical trauma
o No response to administration of exogenous testosterone
o Vascular injury
5’ REDUCTASE DEFICIENCY
DIAGNOSIS OF HYPOGONADISM
o Genotype – XY
o Circadian rhythm and the time of sampling must be considered
o Reduction in levels of the enzyme 5-reductase → decreased
o Multiple estimation of free and bound testosterone levels should
testosterone levels
be done on different days before a diagnosis
o Physical development is similar to the female phenotype
o Clinical signs and symptoms of hypogonadism
o Puberty: residual enzyme activity sufficiently converts testosterone to
dihydrotestosterone, resulting in development of a male phenotype - Loss of secondary sexual characteristics, osteoporosis
PRIMARY DISEASE SECONDARY DISEASE
KLINEFELTER’S SYNDROME o Destruction of the testes o Destruction of the pituitary
o Most common karyotype is 47, XY o FSH and/or LH levels are o FSH and/or LH levels
o Men with this disorder have small (2.5 cm), firm testicles elevated inappropriately normal or low
o Gynecomastia o Pituitary MRI should be one in
young individuals
o Reduced production of testosterone
o Older individuals
o FSH and LH levels are elevated
o Secondary or tertiary (hypothalamic) dysfunction
o Azoospermia → sterility
- Reduced hypothalamic pulse generator frequency
o Reduced bone density and breast cancer
- Resulting in low or inappropriately normal FSH and/or LH levels

[SARANILLO, KA.]
 TESTOSTERONE REPLACEMENT THERAPY THE LUTEAL PHASE
o Parenteral testosterone o Estrogen levels peak 1 day before ovulation, at which point a
o Transdermal testosterone therapy positive feedback system results in an LH surge
o Testosterone gel o Start of the luteal phase is marked by the extrusion of the ovum
o Buccal testosterone approximately 36 hours after this LH surge → luteinization of the
o Complications of testosterone replacement: Graafian follicle to form the corpus luteum
- Polycythemia o Corpus luteum secretes progesterone to aid in the implantation
- Prostate enlargement of the embryo
- Possible growth-promoting effect on undiagnosed prostate o In the absence of fertilization
cancer o Gradual decline in the production of progesterone & estrogen by
- Worsening of obstructive sleep apnea the corpus luteum
- Peripheral edema o Loss of endometrial blood supply
- Gynecomastia o Shedding of the endometrium approximately 14 days after
ovulation occurred
HORMONAL PRODUCTION BY THE OVARIES o Typical duration of menstrual bleeding: 3-5 days
o Steroids pathway and synthetic enzymes are present in the o Blood loss averaging 50 mL
ovaries o Onset of menses marks the end of the luteal phase
o Cholesterol is either synthesized from acetate or actively
transported from the low-density lipoprotein (LDL) particle in HORMONAL CONTROL OF OVULATION
blood o GnRH pulse generator of the arcuate nuclei and medical preoptic
- Substrate for hormonal production nuclei of the hypothalamus
o Positive and negative feedback responses
ESTROGEN o Estrogen & Progesterone
o Carbon-18 compounds o LH & FSH
o Principal estrogen: estradiol o AFTER MENOPAUSE: ↓ Estrogen, ↑ FSH, ↑ LH
o Estrone and estriol are primarily metabolites of intraovarian and o REPRODUCTIVE YEARS:
extraglandular conversion - ↑ FSH levels: Early in the follicular phase
o Promote breast, uterine, and vaginal development. Thelarch and - ↑ LH: Midcycle surge
menarch Stimulates a series of events
o Affect the skin, vascular smooth muscles, bone cells, & central Culminates in ovulation
nervous system FSH ↓
o Lack or estrogen that naturally occurs with the onset of o Anovulation and amenorrhea
menopause o Injury to the hypothalamus
- Atrophic changes o Presence of either psychosocial or physical stressors
o During the reproductive period - Changes in hormonal cues
- Estrogen is responsible for follicular phase changes in the
uterus ESTRIOL
- Deficiency: irregular and incomplete development of the o Major estrogen produced by the placenta during pregnancy
endometrium o Estrone and estradiol are also produced from DHEAS
- Produced by the maternal & fetal adrenal glands
PROGESTERONE
o Carbon-21 compounds NOTE:
o Produced by the corpus luteum ESTRONE: for menopause (estrone mag-isa na lang)
o Induces the secretory activity of those endometrial glands that ESTRADIOLE: principal estrogen, reproductive years (mag-asawa/dalawa)
have been primed by estrogen ESTRIOL: during pregnancy (mag-aanak/tatlo na)
o Readying the endometrium for embryo implantation
o Dominant hormone responsible for the luteal phase
o Deficiency: failure of implantation of the embryo
o Thickening of the cervical mucus
o Reduction of uterine contractions
o Thermogenic effect: basal body temperature rises after
ovulation

THE MENSTRUAL CYCLE

o 2 phases of parallel events occurring at the ovaries and
endometrium
o Within the ovaries
o Follicular and luteal phases
o Endometrial events
o Proliferative and secretory phases

THE FOLLICULAR PHASE

o Begins with the onset of menses and ends on the day of LH surge
o Early in the follicular phase, the ovary secretes very little
estrogen or progesterone
o Rise in FSH, however, stimulates estrogen production (FSH = estrogen)
o Estrogen secreted by the developing follicle within the ovary
stimulates:
- Uterine epithelial cells
- Blood vessel growth
- Endometrial gland development to increase the thickness of
the endometrium
o Intense secretory capacity of the uterine glands provides a
secretion that aids the implantation of the embryo

[SARANILLO, KA.]
 ROUTES OF EXPOSURE
CLINICAL CHEMISTRY 322 o Toxins can enter the body via several routes; ingestion,
INTRO TO TOXICOLOGY
FINALS: WEEK 13 LEC (03/26/2023)
inhalation and transdermal absorption are the most common.
o Ingestion is the most often seen in a clinical setting.
TOXICOLOGY
o Hydrophobic substances have the ability to diffuse across cell
o Toxicology is the study of poisons (“toxikos” means poisonous)
membranes and, therefore, can be absorbed anywhere along the
o A branch of science that deals with the study of poison
gastrointestinal tract.
o Poison – causes harmful effects when administered
o Toxins must be absorbed into the circulation to exhibit its
systemic effect
FOUR MAJOR DISCIPLINES WITHIN TOXICOLOGY
o Absorption is taken up by processes intended for dietary
MECHANISTIC TOXICOLOGY
nutrients
o Mechanistic toxicology elucidates the cellular and biochemical
o Most are absorbed by passive diffusion
effects of toxins
(weak acids → stomach; weak bases → intestines)
o For basis, for rational therapy design and develops tests to assess
o Toxins are not absorbed in the GIT and produces local effects
the degree of exposure of the poisoned individuals
(bleeding, diarrhea, malabsorption of nutrients thus causing
systemic effects)
DESCRIPTIVE TOXICOLOGY
o Descriptive toxicology uses the results from animal experiments
OTHER FACTORS INFLUENCE ABSORBANCE OF TOXINS FROM THE
to predict what level of exposure will cause harm in humans
GASTROINTESTICAL TRACT
o Ex: biorats, guinea pigs
o Rate of dissolution
o Gastrointestinal motility
FORENSIC TOXICOLOGY
o Resistance to degradation in the gastrointestinal tract,
o Forensic toxicology is primary concerned with the medicolegal
o Interaction with other substances
consequences of toxin exposure
o For establishing and validating the analytic performance of the
MODES OF TOXIC ACTION
methods used to generate evidence in the legal situation
o This includes the consideration, at the fundamental level of
o Includes the cause of death of the person
organ, cell and molecular function of all events leading to toxicity
in vivo: uptake, distribution, metabolism, mode of action and
CLINICAL TOXICOLOGY
excretion.
o Clinical toxicology is the study of interrelationships between
o Important aspects include the following:
toxin exposure and disease states.
o Focuses not only the diagnostic testing but also the therapeutic
1. BIOCHEMICAL AND MOLECULAR TOXICOLOGY consider events
intervention of what the poison or drug can do.
at the biochemical and molecular levels, including:
- Enzymes that metabolize xenobiotics, (foreign to the body
REGULATORY TOXICOLOGISTS
or eco system)
o Regulatory toxicologists are responsible for interpreting the
- Generation of reactive intermediates
data from mechanistic and descriptive studies to establish
- Interaction of xenobiotics or their metabolites with
standards that define the level of exposure that will not pose a
macromolecules
risk to public health or safety.
- Gene expression in metabolism and modes of action,
o Formulation of laws and regulation to minimize the effects of
- Signaling pathways in toxic action
toxic chemicals on human health and the environment
2. BEHAVIORAL TOXICOLOGY deals with the effects of toxicants on
OTHER APPLICATIONS OF TOXICOLOGY
animal and human behavior, which is the final integrated
o Veterinary toxicology is the diagnosis and treatment of
expression of nervous function in the intact animal.
poisoning in animals other than humans, particularly livestock
- This involves both the peripheral and central nervous
and companion animals
systems, as well as effects mediated by other organ
o To prevent the transmission of toxins to the meat, dairy, etc.
systems, such as the endocrine glands. (affect CNS)
o Takes care of the animals that undergo the experiment
3. NUTRITIONAL TOXICOLOGY deals with the effects of diet on the
o Environmental toxicology is concerned with the movement of
expression of toxicity and with the mechanisms of these effects.
toxicants and their metabolites and degradation products in the
- Ex: drugs, vitamins, natural substances
environment and in food chains and with the effect of such
contaminants on individuals and, especially, populations.
4. CARCINOGENESIS includes the chemical biochemical, and
o Concerned of the poisonous substances that may affect our
molecular events that lead to the large number of effects on cell
environment
growth collectively known as cancer
o Ex: factory wastes going into the waters
5. TERATOGENESIS includes the chemical, biochemical, and
o Industrial toxicology is a specific area of environmental
molecular events that lead to deleterious effects on
toxicology that deals with the work environment and constitutes
development
a significant part of industrial hygiene.
- Teratoma – deletion of development of the cells
o Checks if workers become exposed to the poisonous toxins
6. MUTAGENESIS is concerned with toxic effects on the genetic
EXPOSURE TO TOXINS
material and the inheritance of these effects. (mutation)
o 50% of poisoning cases are intentional suicide attempts.
o Accidental exposure accounts for about 30% cases.
7. ORGAN TOXICITY considers effects at the level of organ function
o remaining cases are a result of homicide or occupational
(neurotoxicity, hepatotoxicity, nephrotoxicity, etc.)
exposure.
o Suicide has the highest mortality rate.
o Accidental exposure occurs most frequently in children.
o An accidental drug overdose of either therapeutic or illicit drugs
is relatively common in adults.
o Occupational exposure primarily occurs in industrial and
agricultural settings.

[SARANILLO, KA.]
 CHEMICAL USE CLASSES o The TD50 in the dose that would be
o This includes the toxicology aspects of the development of new predicted to produce a toxic
chemicals for commercial use. In some of these use classes, response in the 50% of the
toxicity, at least to some organisms, is a desirable trait; in others, population
it is an undesirable side effect. o The LD50 is the dose that would
predict death in 50% of the
1. AGRICULTURE CHEMICALS include many compounds, such as population
insecticides, herbicides, fungicides, and rodenticides, in which o Similar experiments can be used to
toxicity to the target organism is a desired quality whereas evaluate the doses of therapeutic
toxicity to “nontarget species” is to be avoided. drugs. The ED50 is the dose that
2. CLINICAL DRUGS are properly the province of pharmaceutical would be predicted to be effective
chemistry and pharmacology. However, toxic side effects and or have a therapeutic benefit in 50%
testing for them clearly fall within the science of toxicology of the population.
3. DRUGS OF ABUSE are chemicals taken for psychological or other o The therapeutic index is the ratio of the TD50 to the ED50
effects and may cause dependence and toxicity. Many of these
are illegal, but some are of clinical significance when used SOURCES OF TOXIC COMPOUNDS
correctly o Exposure Classes include toxicants in food, air, water, and soil as
4. FOOD ADDITIVES are of concern to toxicologists only when they well as toxicants characteristic of domestic and occupational
are toxic or being tested for possible toxicity settings.
5. INDUSTRIAL CHEMICALS are so numerous that testing them for o Use Classes include drugs of abuse, therapeutic drugs,
toxicity or controlling exposure to those known to be toxic is a agricultural chemicals, food additives and contaminants, metal,
large area of toxicological activity solvents, combustion products, cosmetics, and toxins.
6. NATURALLY OCCURING SUBSTANCES include many phytotoxins,
mycotoxins, and minerals, all occurring in the environment. The ACUTE AND CHRONIC TOXICITY
recently expanded and now extensive use of herbal remedies o Acute toxicity refers to a single, short-term exposure to a
and dietary supplements has become a cause of concern for substance, the dose of which is sufficient to cause immediate
toxicologists and regulators. Not only is their efficacy frequently toxic effects.
dubious, but their potential toxicity is largely unknown. o Chronic toxicity refers to repeated frequent exposure for
7. COMBUSTION PROCUTS are now properly a use class but are a extended periods, at doses that are insufficient to cause an
large and important class of toxicants, generated primarily from immediate acute response
fuels and other industrial chemicals. o Chronic toxicity may affect different systems then those
associated with acute toxicity.
DOSE-RESPONSE RELATIONSHIP
o Toxicity is a relative event that depends not only on the toxic ANALYSIS OF TOXIC AGENTS
properties of the chemical and the dose administered but also o Analysis of toxic agents in a clinical setting is a two-step
on individual and interspecific variation in the metabolic procedure.
processing of the chemical. o The first step is a screening test, which is a rapid, simple,
o A poison can be defined as any substance that causes a harmful qualitative procedure
effect on exposure. o Intended to detect specific substances or classes of toxicants.
o Dose is a key issue. o A negative result can rule out a drug or toxicant
o The concept that any o A positive result should be considered a presumptive positive
substance has the potential to until confirmed by a second, more specific method
cause harm if given at the o Immunoassays are commonly used to screen for drugs. In some
correct dosage (even water) is instances, these assays are specific for a single drug (e.g.,
a central theme in toxicology. tetrahydrocannabinol [THC]).
o Most correlate the dose of a o Thin-layer chromatography is a relatively simple, inexpensive
toxin that will result in a method of detecting various drugs and other organic
harmful response. compounds.
o One such system correlates a o Gas chromatography (GC) is a widely used, well-established
single acute oral dose range with the probability of a lethal technique for the qualitative and quantitative determination of
outcome in an average 70 kg man (Table 30-1) many volatile substances.
o The predicted response in this system is death, which is valid. o The reference method for the qualitative identification of most
o However, most toxins can express pathologic effects other than organic compounds is GC, using a mass spectrometer as the
death at lower degrees of exposure. detector.

o A more in depth-characterization is by evaluating data from a

cumulative frequency histogram of toxic responses over a range
of doses.
o This experimental approach is
typically used to evaluate
several responses over a wide
range of concentrations.
o One response monitored is the
toxic response: the response
that has been associated with
an early pathologic effect at
lower than lethal doses.
o For a substance that exerts
early toxic effects by damaging
liver cells, the response
monitored may be increases in
serum alanine aminotransferase (ALT) or -glutamyltransferase
(GGT) activity.
o The dose response relationship implies that there will be an
increase in the toxic response as the dose is increased.

[SARANILLO, KA.]
 THE FIELD OF TOXICOLOGY
CLINICAL CHEMISTRY 322 o Toxicology addresses a variety of questions. For example, in
TOXICOLOGY
FINALS: WEEK 14 LEC
agriculture, toxicology determines the possible health effects
from exposure to pesticides or herbicides, or the effect of animal
DEFINITION OF TERMS
o TOXICOLOGY feed additives, such as growth factors, on people.
- is a broad multidisciplinary science whose goal is to determine the effects of o Toxicology is also used in laboratory experiments on animals to
chemical agents on living organisms establish dose-response relationships.
- Branch of science that deals with poison
- Poison can be defined as any substance that causes harmful effect when
o Toxicology also deals with the way chemicals and waste products
administered either by accident or intentional to a living organism affect the health of an individual
o HARMFUL OR ADVERSE EFFECTS
- Are those that are damaging to either the survival or normal function of the SUB-DISCIPLINES OR SUB-SPECIALTIES OF TOXICOLOGY
individual
o Environmental toxicology o Clinal Toxicology
- Side effects – not all is affected to the organism
o Occupational (industrial) toxicology o Descriptive toxicology
- Adverse effect – damaging to either the survival or normal function of the
o Regulatory Toxicology o Forensic toxicology
individual (harmful and damaging)
o Food toxicology o Analytical toxicology
o TOXICITY
o Mechanistic toxicology
- describes the degree to which a substance is poisonous or can cause injury
- The toxicity depends on a variety of factors: dose, duration and route of
exposure, shape and structure of the chemical itself, and individual human ENVIRONMENTAL TOXICOLOGY
factors. o Is concerned with the study of chemicals that contaminate food
o TOXIC
- Relates to poisonous or deadly effects on the body by inhalation (breathing), water, soil or the atmosphere
ingestion (eating), or absorption (skin or passive diffusion), or by direct o Is also deals with toxic substances that enter bodies of waters
contact with a chemical such as lakes, streams, rivers and oceans.
o TOXICANT
- Any chemical that can injure or kill humans, animals, or plants; a poison.
o This sub-discipline addresses the question of how various plants,
- The term “toxicant” is used when talking about toxic substances that are animals, and humans are affected by exposure to toxic
produced by or are a by-product of human-made activities substances
- For example, dioxin (2,3-7,8-tetrachlorodibenzop-dioxin (TCDD)), produced
as a by-product of certain chlorinated chemicals, is a toxicant.
o TOXIN OCCUPATIONAL (INDUSTRIAL) TOXICOLOGY
- Usually is used when talking about toxic substances produced naturally. o Is concerned with health effects from exposure to chemicals in
- A toxin is any poisonous substance of microbial (bacteria or other tiny plants the workplace
or animals), vegetable, or synthetic chemical origin that reacts with specific
cellular components to kill cells, alter growth or development, or kill the
o This field grew out of a need to protect workers from toxic
organism substances and to make their work environment safe.
o TOXIC SYMPTOM
- This term includes any feeling or sign indicating the presence of a poison in REGULATORY TOXICOLOGY
the system
o TOXIC EFFECTS o Gathers and evaluates existing toxicological information to
- Refers to the health effects that occur due to exposure to a toxic substance; establish concentration-based standards of “safe” exposure.
also known as a poisonous effect on the body o The standard is the level of a chemical that a person can be
- Side effects: not usually harmful to the body
- Adverse effects: harmful to the organism
exposed to without any harmful health effects
- Toxic effects: highest levels of toxicity (leads to death)
o SELECTIVE TOXICITY FOOD TOXICOLOGY
- Means that a chemical will produce injury to one kind of living matter o Is involved in delivering a safe and edible supply of food to the
without harming another form of life, even though the two may exist close
together (Ex: out of two organisms, only one is affected) consumer
o DOSE o During processing, a number of substances may be added to
- Is the actual amount of a chemical that enter the body. food to make it look, taste, or smell better. Fats, oils, sugars,
- The dose received may be due to either acute (short) or chronic (long-term)
exposure.
starches and other substances may be added to change the
- An acute exposure occurs over a very short period of time, usually 24 hours texture and taste of food. All of these additives are studied to
- Chronic exposures occur over long periods of time such as weeks, months, or determine if what amount, they may produce adverse effects.
years.
- The amount of exposure and the type of toxin will determine the toxic effect
o DOSE RESPONSE CLINICAL TOXICOLOGY
- A relationship between exposure and health effect, that can be established o Concerned with diseases and illnesses associated with short
by measuring the response relative to an increasing dose (Therapeutic drug term or long-term exposure to toxic chemicals
management (TDM))
- This relationship is important in determining the toxicity of a particular
o Clinical toxicologists include emergency room physicians who
substance. must be familiar with the symptoms associated with exposure to
- Relies on the concept that a dose or time of exposure will cause an effect or a wide variety of toxic substances in order to administer the
response on the exposed organism appropriate treatment
- The larger or more intense the dose, the greater the response or effect
- “The dose makes the poison” (too much is bad)
o THRESHOLD DOSE DESCRIPTIVE TOXICOLOGY
- Also referred to as the no observed adverse effect level (NOAEL), or the no o Is concerned with gathering toxicological information from
effect level (NEL)
- These terms are often used by toxicologists when discussing the relationship
animal experimentation
between exposure and dose o These types of experiments are used to establish how much of a
o INDIVIDUAL SUSCEPTIBILITY chemical would cause illness or death
- This term describes the differences in types of responses to hazardous
substances, between people.
- Each person is unique = great differences in the response to exposure FORENSIC TOXICOLOGY
o SENSITIVE SUB-POPULATION o Is used to help establish cause and effect relationships between
- Describes those persons who are more at risk from illness due to exposure exposure to a drug or chemical and the toxic or lethal effects that
to hazardous substances than the average, healthy person
- These includes very young people, chronically ill, very old, pregnant women
result from that exposure
and women of child bearing age. Also, it depends on the type of
contaminants or other factors (age, weight, lifestyle). ANALYTICAL TOXICOLOGY
- Used to describe some population o Identifies the toxicant through analysis of body fluids, stomach
o ACUTE TOXICITY
- Single, short-term exposure to a substance content, excrement, or skin.
- Dose: cause immediate toxic effects
o CHRONIC TOXICITY MECHANISTIC TOXICOLOGY
- Repeated exposure for extended period of time
- Dose: insufficient to cause an immediate acute response
o Makes observations on how toxic substances cause their effects
- May affect different systems and associated with acute toxicity o The effects of exposure can depend on a number of factors,
o TD 50 (toxic dose) including the size of the molecule, the specific tissue type or
- Is the dose that would be predicted to produce a toxic response in 50% of cellular components affected, whether the substance is easily
the population
o LD50 (lethal dose) dissolved in water or fatty tissues, all of which are important
- Is the dose that would predict the death in 50% of the population when trying to determine the way a toxic substance causes
o ED 50 (effective dose) harm, and whether effects seen in animals can be expected in
- Is the dose that would be predicted to be effective or have a therapeutic
benefit in 50% of the population
humans.

[SARANILLO, KA.]
 EXPOSURE TO TOXINS CHEMICAL USE CLASSES
o 50% of poisoning cases are intentional suicide attempts o This includes the toxicology aspects of the development of new
o Accidental exposure accounts for about 30% of cases. chemicals for commercial use. In some of these use classes,
o The remaining cases are a result of homicide or occupational toxicity, at least to some organisms, is a desirable trait; in others,
exposure it is an undesirable side effect.
o Suicide has the highest mortality rate
o An accidental drug overdose of either therapeutic or illicit drugs 1. AGRICULTURE CHEMICALS include many compounds, such as
is relatively common in adults insecticides, herbicides, fungicides, and rodenticides, in which
o Occupational exposure primarily occurs in industrial and toxicity to the target organism is a desired quality whereas
agricultural settings toxicity to “nontarget species” is to be avoided.
2. CLINICAL DRUGS are properly the province of pharmaceutical
ROUTES OF EXPOSURE chemistry and pharmacology. However, toxic side effects and
o Toxins can enter the body via several routes: ingestion, testing for them clearly fall within the science of toxicology
inhalation and transdermal absorption are the most common. 3. DRUGS OF ABUSE are chemicals taken for psychological or other
o Ingestion is the most often seen in a clinical setting. effects and may cause dependence and toxicity. Many of these
o Hydrophobic substances have the ability to diffuse across cell are illegal, but some are of clinical significance when used
membranes and, therefore, can be absorbed anywhere along the correctly
gastrointestinal tract. 4. FOOD ADDITIVES are of concern to toxicologists only when they
o Toxins: absorbed in the circulation to exhibit systematic effects are toxic or being tested for possible toxicity
o Absorption: taken up by processes intended for dietary nutrients 5. INDUSTRIAL CHEMICALS are so numerous that testing them for
o Most toxicants are absorbed by PASSIVE DIFFUSION toxicity or controlling exposure to those known to be toxic is a
o Weak acids: stomach; Weak bases: Intestines large area of toxicological activity
6. NATURALLY OCCURING SUBSTANCES include many phytotoxins,
OTHER FACTORS INFLUENCE ABSORBANCE OF TOXINS FROM THE GI TRACT mycotoxins, and minerals, all occurring in the environment. The
o Rate of dissolution recently expanded and now extensive use of herbal remedies
o Gastrointestinal motility and dietary supplements has become a cause of concern for
o Resistance to degradation in the gastrointestinal tract toxicologists and regulators. Not only is their efficacy frequently
o Interaction with other substances dubious, but their potential toxicity is largely unknown.
7. COMBUSTION PROCUTS are now properly a use class but are a
MODES OF TOXIC ACTION large and important class of toxicants, generated primarily from
o Important aspects include the following: fuels and other industrial chemicals.

1. BIOCHEMICAL AND MOLECULAR TOXICOLOGY consider events DOSE-RESPONSE RELATIONSHIP

at the biochemical and molecular levels, including: o Toxicity is a relative event that depends not only on the toxic
- Enzymes that metabolize xenobiotics, (foreign to the body properties of the chemical and the dose administered but also
or eco system) on individual and interspecific variation in the metabolic
- Generation of reactive intermediates processing of the chemical.
- Interaction of xenobiotics or their metabolites with o A poison can be defined as any substance that causes a harmful
macromolecules effect on exposure.
- Gene expression in metabolism and modes of action, o Dose is a key issue.
- Signaling pathways in toxic action o The concept that any
substance has the potential to
2. BEHAVIORAL TOXICOLOGY deals with the effects of toxicants on cause harm if given at the
animal and human behavior, which is the final integrated correct dosage (even water) is
expression of nervous function in the intact animal. a central theme in toxicology.
- This involves both the peripheral and central nervous o Most correlate the dose of a
systems, as well as effects mediated by other organ toxin that will result in a
systems, such as the endocrine glands. (affect CNS) harmful response.
o One such system correlates a
3. NUTRITIONAL TOXICOLOGY deals with the effects of diet on the single acute oral dose range with the probability of a lethal
expression of toxicity and with the mechanisms of these effects. outcome in an average 70 kg man (Table 30-1)
- Ex: drugs, vitamins, natural substances o The predicted response in this system is death, which is valid.
o However, most toxins can express pathologic effects other than
4. CARCINOGENESIS includes the chemical biochemical, and death at lower degrees of exposure.
molecular events that lead to the large number of effects on cell
growth collectively known as cancer o A more in depth-characterization is by evaluating data from a
cumulative frequency histogram of toxic responses over a range
5. TERATOGENESIS includes the chemical, biochemical, and of doses.
molecular events that lead to deleterious effects on o This experimental approach is
development typically used to evaluate
- Teratoma – deletion of development of the cells several responses over a wide
range of concentrations.
6. MUTAGENESIS is concerned with toxic effects on the genetic o One response monitored is the
material and the inheritance of these effects. (mutation) toxic response: the response
that has been associated with
7. ORGAN TOXICITY considers effects at the level of organ function an early pathologic effect at
(neurotoxicity, hepatotoxicity, nephrotoxicity, etc.) lower than lethal doses.
o For a substance that exerts
early toxic effects by damaging
liver cells, the response
monitored may be increases in
serum alanine aminotransferase (ALT) or -glutamyltransferase
(GGT) activity.
o The dose response relationship implies that there will be an
increase in the toxic response as the dose is increased.

[SARANILLO, KA.]
ANALYTICAL TECHNIQUE FOR BROAD-SPECTRUM DRUG DETECTION CYANIDE
o Simple, inexpensive and rapid spot tests o Hydrocyanic acid (HCN); also referred to as prussic acid, is a
o Immunoassays (screening for drugs) colorless gas with the odor of almond detectable by only about
o Chromatographic and/or mass spectrophotometric techniques 50% of the population (common suicide agents)
a. Thin-layer Chromatography (TLC) o The ionized state is cyanide (CN) (Binding to Heme Fe)
b. High-performance Liquid Chromatography (HPLC)
c. Gas Chromatography (GC) o Patients exposed to toxic concentrations of cyanide exhibit rapid
d. Gas chromatography-mass spectrophotometry (GC-MS) onset of symptoms typical of cellular hypoxia:
e. Liquid chromatography-mass spectrophotometry (LC-MS) - Flushing
- Headache
AGENTS THAT CAUSE CELLULAR HYPOXIA - Tachypnea (abnormality rapid breathing)
1. Carbon Monoxide - Dizziness
2. Cyanide - Respiratory depression – which progress rapidly to: Coma,
3. Methemoglobin-Forming Agents Seizure, Complete heart block and Death

CARBON MONOXIDE o Treatment requires rapid identification of CN as the intoxicant

o Carbon monoxide (CO) is a colorless, odorless, tasteless gas that followed by administration of sodium nitrite to cause formation
is the product of incomplete combustion of carbonaceous of methemoglobin, which avidly binds and clears CN, and
material thiosulfate to enhance clearance via metabolism

o Common exogenous sources of carbon monoxide: o Analytical method

a. Cigarette smoke - Spectrophotometric method
b. Gasoline engines - Iron-selective electrode
c. Improperly ventilated heating units - Isotope dilution GM-MS analysis

o Not only decreases the oxygen content of blood, but also o Reference values
decreases oxygen availability to tissue thereby producing a - Normal CN conc. is less than 0.2 ug/ml of whole blood
greater degree of tissue hypoxia - The patients likely becomes comatose when blood CN
concentration is greater than 2 ug/ml and concentration
o Factors other than carboxy-hemoglobin concentration that greater than 5 ug/ml are lethal
contribute to the toxicity:
a. Length of exposure METHEMOGLOBIN-FORMING AGENTS
b. Metabolic activity o Heme iron in hemoglobin is normally in the ferrous state (Fe2+)
c. Underlying disease, especially cardiac or cerebrovascular o When oxidized to the ferric state (Fe3+), methemoglobin is
disease formed, and this form of hemoglobin does not bind oxygen
o The principal physiological system to maintain hemoglobin iron
o Effects of carbon monoxide poisoning. Delayed development of in the reduced state is nicotinamide-adenine dinucleotide
neuropsychiatric sequelae which may include: (NADH) – methemoglobin reductase (diaphorase I)
- Personality changes o Congenital methemoglobinemia may result from a deficiency of
- Motor disturbances NADH – methemoglobin reductase
- Memory impairment
o Toxic effects of methemoglobinemia are a consequence of
o Treatment for carbon monoxide poisoning: hypoxia associated with:
- Removal of the individual from the contaminated area and - With the diminished O2, content of the blood
the administration of oxygen (oxygen therapy) - With the decreased O2, dissociation from hemoglobin
species in which some, but all, subunits contain heme iron
o The half-life of carboxyhemoglobin is 5 to 6 hours when the in the ferric state
patient breathes room air; it is reduced to about 1.5 hours when o The PO2 is normal in these patients and therefore so is the
the patient breathes 100% oxygen hemoglobin oxygen saturation
o Thus, a normal PO2 in a cyanotic patient is a significant indication
o Analytical method: for possible presence of methemoglobinemia
- Gas chromatography methods, considered as reference o Specific therapy for toxic methemoglobinemia involves the
procedures, are accurate and precise even for every low administration of methylene blue to hasten the conversion of
concentration of carbon monoxide methemoglobin to hemoglobin
- Spectrophotometric methods are rapid, convenient,
accurate and precise except at very low concentrations of o Analytical method
carboxyhemoglobin (less than 2% to 3%) - Measured by automated multiwavelength spectrometers
- Specimens should be kept on ice or refrigerated but not frozen
o Reference Values: Carboxyhemoglobin in: - Freezing results in an increase in methemoglobin conc.
- Rural smokers are about 0.5% - Methylene blue and sulfhemoglobin causes spectral
- For urban non-smokers 1% to 2% interference in the measurement of methemoglobin with
- For smokers 5% to 6% some CO-oximeters

Values may be increased by about 3% in hemolytic anemia o Reference values:

- Normal concentration of methemoglobin is less than 1.5%
of total hemoglobin
- Methemoglobin concentration up to 20% cause cyanosis
- Concentrations between 20% to 50% may cause:
a. Dyspnea d. Weakness
b. Exercise intolerance e. Syncope
c. Fatigue
- Methemoglobin concentration of 50% to 70%
a. Dysrhythmias
b. Seizures
c. Metabolic acidosis
d. Coma
- Methemoglobin concentration greater than 70% may be
lethal

[SARANILLO, KA.]
 ALCOHOLS OF TOXICOLOGICAL INTEREST METHANOL
o Ethanol (CH3Ch2-OH) o Wood alcohol
o Methanol (CH3OH) o Is used as:
o ISOPROPANOL (CH3-CHOH-CH3) - Solvent in a number of commercial products (is a major
component of a number of paints and varnishes, as well as
ETHANOL being found in paint removers)
o Ethyl alcohol; grain alcohol - Constituents of antifreeze and window cleaning fluids
o The principal pharmacological action of ethanol is depression of - Components of canned fuel
the Central Nervous System (CNS)
o The CNS effects of methanol are
o The CNS effects vary, depending on the blood ethanol substantially less severe than those of
concentrations, from: ethanol
1. Euphoria and decreased inhibitions – less than or equal to o Methanol is oxidized by the liver ADH (at
50 mg/dl about one tenth the rate of ethanol) to
2. Increased disorientation and loss of voluntary muscle formaldehyde.
control resulting in irregular movements – 100 to 300 o Formaldehyde in turn is rapidly oxidized
mg/dl by aldehyde dehydrogenase to formic
3. Coma and death – greater than 400 mg/dl acid, which may cause serious acidosis
and optic neuropathy, resulting in
o Ethanol is a teratogen, and blindness or death
alcohol consumption during o Serum formate concentrations correlate better with the degree
pregnancy has been known to of acidosis and the severity of CNS and ocular toxicity than do
result in a baby being born with serum methanol concentration.
fetal alcohol spectrum disorders
(FASD) o Treatments for methanol intoxication:
These effects may include: - The administration of ethanol or preferably fomepizole to
- Physical, mental, inhibit the metabolism of ethanol
behavioral, and/or learning disabilities with possible - Sodium bicarbonate therapy to help alleviate the metabolic
lifelong implications acidosis
- Folate administration to enhance folate-mediated
o Other alcohol-related conditions include Alcohol-Related metabolism of formate, and the use of hemodialysis to
Neurodevelopmental Disorders (ARND) and Alcohol-Related enhance clearance of methanol and formate
Birth Defects (ARBD)
o FASD, ARND, and ARBD affects newborns every year than Down o Headspace gas chromatographic analysis – is the method of
syndrome, cystic fibrosis, spina bifida and sudden infant death choice for the measurement of methanol
syndrome combined o Fatal dose: 60 – 250 ml
o FASD, ARND, and ARBD are 100% preventable when a woman
completely abstains from alcohol during her pregnancy ISOPROPANOL
o Ethanol is metabolized principally by liver alcohol o Isopropanol is readily available to the general population as a
dehydrogenase (ADH) to acetaldehyde, which is subsequently 70% aqueous solution for use as rubbing alcohol
oxidized to acetic acid by aldehyde dehydrogenase o It has about twice the CNSS depressant action of ethanol, but is
not as toxic as methanol
o Metabolism of Ethanol o Isopropanol has a short half-life of 1 – 6 hours, as it is rapidly
- After entering the circulation, ethanol is metabolized by the metabolized by ADH to acetone, which is eliminated much more
liver. The enzyme, alcohol dehydrogenase, oxidizes the slowly (17 to 27 hours), primarily in alveolar air and urine
ethanol to form acetaldehyde o Acetone has CNS depressant activity
similar to that of ethanol, and because of
its half-life, it prolongs the apparent CNS
effects of isopropanol
- o Severe isopropanol intoxication, like that
- The acetaldehyde formed is then excreted or converted to
of ethanol, has been known to result in
acetate and in turn acetate is converted to acetyl CoA which
coma or death
leads the two-carbon molecule into the TCA cycle
o Isopropanol and its metabolite, acetone,
- The maximum rate of ethanol conversion in the normal
may be determined by headspace gas
adult is approximately 7g of ethanol per hour. This load is
chromatography or by nuclear magnetic
equivalent to approximately 6-10 ml of ethanol (roughly
resonance (NMR) spectroscopy.
one beer, one glass of wine, or one shot of whiskey)
- The major route of ethanol disposal is by way of metabolism
ETHYLENE GLYCOL (1,2-ethanediol)
- Approximately 90-98% of the ethanol consumed is
o Is a common component of hydraulic fluid and antifreeze
converted by the liver
o The immediate effects of ethylene glycol ingestion are similar to
- The remainder is excreted primarily by the kidney, with the
those of ethanol
rate of elimination in urine being approximately one half
o However, metabolism by hepatic ADH and ALDH results in the
the rate of absorption by the stomach
formation of several toxic species, including oxalic acid and
- A small fraction is lost through perspiration and
glycolic acid, which results in severe metabolic acidosis
respirations, with the lung disposing of approximately 0.6 –
o This is complicated by the rapid formation and deposition of
1.5% of the total ethanol load
calcium oxalate crystals in renal tubules
o Calcium oxalate crystals formation may result in renal tubular
o Detection limit: 12 hours
damage
o Fatal dose: 300-400 ml pure alcohol consumed in less than 1 hour
o Toxic level: 250-400 mg/dl

[SARANILLO, KA.]
 ANALYSIS OF ETHANOL URINE ETHANOL
BLOOD ETHANOL o Urine has been used as an alternate, less invasive specimen for
o Specimens for the determination of ethanol are serum, plasma, the determination of ethanol, compared with blood.
or whole blood o During the post-absorptive phase following alcohol ingestion,
o The venipuncture site should be cleanses with alcohol-free the concentration of ethanol in urine is roughly 1.3 times that in
disinfectant, such as aqueous benzalkonium chloride (Zephiran) blood.
o Ethanol distributes into the aqueous compartments of blood o The use of urine alcohol measurement is discouraged by some
because the water content of serum (~98% [approximately 98%]) laboratories because the ratio of 1.3 is highly variable, and the
is greater than that of whole blood (~86), results indicating urine alcohol concentration represents an average of the blood
higher alcohol concentrations are obtained with serum alcohol concentration during the time period in which urine
o Because of the volatile nature of alcohols, specimens must be collected in the bladder
kept capped to prevent evaporative loss to the atmosphere o A better correlation of urine with blood alcohol concentration is
o Blood may be stored when properly sealed for 14 days at room obtained by first emptying the bladder and then collecting urine
temperature or at 4C, with or without preservative after 20 to 30 minutes
o For longer storage or for nonsterile postmortem specimens, o Urine ethanol test is the detection of alcohol in urine represents
sodium fluoride should be used as a preservative to prevent a ingestion of alcohol within the previous 8 hours
decrease or occasionally an increase (via fermentation) in
ethanol concentrations. DETERMINATION OF ALCOHOLS
o To measure ethanol in blood, o Several analytic methods can used for the determination of
enzymatic analysis is the method of ethanol in serum
choice for many laboratories o The enzymatic, GC and osmometry methods are the most
o In this method ethanol is measured by commonly used.
oxidation to acetaldehyde with NAD*, o Serum osmolality increases by about 10 mOms/kg for each 60
a reaction catalyzed by ADH. With this mg/dL increase in serum ethanol. It is a useful screening test
reaction, formation of NAD, o GC is the reference method for ethanol determination
measured by 340 nm, is proportional o This method can simultaneously
to the amount of ethanol in the quantitate other alcohols, such as
specimen methanol and isopropanol
o ADH method is specific for ethanol o Quantitation of peaks can be done by
o Serum or plasma is the most common specimen for ethanol constructing a standard curve or by ratio
determination by ADH method; the method also performs well to an internal standard (n-propanol)
with urine or saliva shown in Figure 30-2.
o W.H. Porter personal observation, a false positive result occurs
in urine ethanol ANALGESIC (NONPRESCRIPTION)
Definition of terms:
BREATH ETHANOL o Pharmacokinetics is derived from two Greek words:
o The fundamental principle for use of breath analysis is the - “pharmakon” means drug or poison
ethanol in capillary alveolar blood rapidly equilibrates with - “kinesis” means motion
alveolar air in a ratio of approximately 2100:1 (blood:breath). - Pharmacokinetics is the study of drug movement
o Breath alcohol expressed as g/210L is approximately equivalent throughout the body. It involves the four processes of
to gram per deciliter of alcohol in whole blood absorption, distribution, metabolism, and excretion
o Available devices for commercial evidential breath alcohol o Side Effects – physiologic effects not related to the desired drug
measurement effects; even with a correct drug dosage, side effects occur and
- Infrared absorption spectrometry – most common are predicted
- Dichromate-sulfuric acid oxidation-reduction (photometric) o Adverse Effects – more severe than side effects; undesired
- GC (flame ionization or thermal conductivity detection) effects that may be unpleasant or even dangerous, and can occur
- Electrochemical oxidation (fuel cell) for a number of reasons such as:
- Metal-oxide semiconductor sensors - the drug may have other effects on the body besides the
therapeutic effect
SALIVA ETHANOL - The client is sensitive to the drug being given
o Ethanol distributes between blood and saliva by passive diffusion - The drug’s action in the body causes other responses that
largely according to the water content of these fluid (85% w/v are undesirable or unpleasant
for whole blood; 99% for saliva) - The client is taking too much or too little of the drug, leading
o A small test device has been developed to measure ethanol in to adverse effects
saliva o Absorption – refers to what happens to a drug from the time it
- Saliva is absorbed onto a swab, which is then inserted into is introduced to the body until it reaches the circulating fluids
the test cartridge and tissues. Areas in the body where drug can be absorbed:
- Ethanol measurement is based on an ADH reaction coupled - GI tract either orally or rectally
with a diaphorase-mediated color indicator reaction, which - Mucous membranes
provide for visual end-point detection on a thermometer- - Skin
like scale after 2 minute incubation - Lungs
o A test card device for the qualitative measurement of ethanol in - Muscle
saliva or urine also based on ADH-diaphorase-coupled detection - Subcutaneous tissues
scheme
o The test card is designed to produce a positive response for ANALGESICS
ethanol concentration greater than 0.02 g/dl o substances that relive pain without causing loss of consciousness.
o Plastic test strip suitable for insertion under the subject’s tongue o When used in excess, analgesics such as acetaminophen and
or into collected saliva salicylate have been known to result in a toxic response.
o After saturation of the reaction pad with saliva and a 2-minute
incubation period, an ADH-diaphorase-coupled indicator color
bar becomes visible if the ethanol concentration is 0.02 g/dl or
greater

[SARANILLO, KA.]
 ACETAMINOPHEN o Distribution
o Has analgesic and antipyretic actions: - Can cross the placenta and enter breast milk
o Therapeutics actions - Extensively bound to plasma albumin
- Acts directly on the thermoregulatory cells in the o Elimination
hypothalamus to cause sweating and vasodilation - Excreted by the kidneys
- The release of heat causes reduction of fever o Contraindications and cautions
o Indications: - Presence of known allergy to salicylates or tartrazine dye
- Treatment of fever associated with influenza - Clients with peptic ulcer disease and bleeding abnormalities
- Prophylaxis of children receiving diphtheria-pertussis-tetanus - Clients with impaired renal function because drug is excreted
immunizations. in the urine
- Relief of musculoskeletal pain associated with arthritis. - Clients with influenza or chickenpox, because of the risk of
Reye’s syndrome
PHARMACOKINETICS - Those who will undergo surgery or invasive procedure because
➢ ABSORPTION of the risk of bleeding
- Readily absorbed from the GI tract, with peak levels in ½ to 2
hours ADVERSE EFFECTS:
- Absorption through rectal administration is slow due to the ➢ GASTROINTESTINAL EFFECTS
presence of fecal matter and decreased blood flow in the colon - Most common side effects are gastric distress, heartburn and
➢ METABOLISM nausea
- Because of its short half-life, it can be administered every 4 - Long term use may cause gastric ulceration, perforation and
hours as needed with a maximum dose of 4grams/day bleeding
- It is extensively metabolized in the liver - Ulcers result from increased secretion of pepsin, decreased
- It can be metabolized two pathways: Major and Minor production of cytoprotective mucus, decreased submucosal
a. Major pathway – acetaminophen undergoes conjugation blood flow and direct irritant action of salicylate to gastric
with glucuronic acid and other compounds to form mucosa
nontoxic metabolites ➢ BLEEDING
b. Minor pathway – it is oxidized by a 450-containing - can promote bleeding by inhibiting platelet aggregation
enzyme into a highly reactive and toxic compound - caution is needed when aspirin is used in conjunction with
anticoagulant
o Distribution – crosses the placenta and enters the breastmilk ➢ SALICYLISM
o Elimination – excreted in the urine - A syndrome that begins to develop when aspirin levels go high
o Contraindication above therapeutic levels
- allergy to acetaminophen - Signs include tinnitus, sweating, headache and dizziness
- Should be used cautiously in pregnancy or lactation and in - If salicylism develops, salicylates should be discontinued until
hepatic dysfunction and chronic alcoholism due to the symptoms subside
associated toxic effects on the liver ➢ REYE’S SYNDROME
o Adverse Effects - Characteristic symptoms are encephalopathy and fatty liver
- It does not cause gastric ulceration or renal impairment degeneration
- Includes headache, hemolytic anemia, renal dysfunction, skin - Relationship between salicylate use and Reye’s syndrome is still
rash and fever unknown
- Signs and symptoms of acute toxicity include signs of poisoning ➢ HYPERSENSITIVITY REACTIONS
such as nausea, vomiting, diarrhea, sweating and abdominal - Reactions are most likely in adults who have predispositions
discomfort. Acetylcysteine (Mucomyst) is given as an antidote such as asthma, hay fever or nasal polyps
to minimize liver damage - Starts with profuse, watery rhinorrhea and may progress to
o Analytical Methodology generalized urticaria, bronchospasm, laryngeal edema and
- Most commonly measured by immunoassay methods shock
- A different approach uses arylacylamide aminohydrolase with ➢ ADVERSE EFFECTS ASSOCIATED WITH USE DURING PREGNANCY
a coupled color generating reaction - the principal risks for pregnant women are anemia and
- Chromatographic methods are highly accurate and therefore postpartum hemorrhage
may be considered reference procedures; however, they are - It may suppress spontaneous uterine contractions, thereby
more difficult to perform especially on an emergency basis prolong gestation and labor
- Commercially available Qualitative one step lateral flow - It can induce premature closure of ductus arteriosus
immunoassay – cutoff 25ug/mL - Has been associated with low birthweight, stillbirth, renal
toxicity, intracranial hemorrhage in preterm infants and
SALICYLATES neonatal death
o One of the oldest anti-inflammatory drugs used by ancient people
to reduce fever and relive pain. These drugs were extracted from o Analytical method
barks and plants during the old times - The classic methods for the measurement of salicylate in serum
are based on the method of TRINDER:
o Therapeutic actions o These procedures rely on the reaction between
- Inhibit action of prostaglandins (chemical released by active salicylate and Fe3+ to form a colored complex that is
white blood cells that can increase body temperature measure at 540nm
- Inhibit platelet aggregation by inhibiting the synthesis of - Photometric method – used to assess salicylate overdose
prostacyclin - Fluorescent polarization immunoassay
- Salicylate hydroxylase – mediated photometric procedure –
o Indications: considered more specific
- Suppression of inflammation - A qualitative, one step lateral flow immunoassay (cutoff
- Relieving mild to moderate pain 100mg/L) – are commercially available
- Reducing temperature
- Treatment of inflammatory conditions such as rheumatoid BARBITURATES
arthritis and osteoarthritis o Can be considered as substances of abuse of as analgesics
o Barbiturates intoxication results in cardiac arrest and respiratory
PHARMACOKINETICS: depression
➢ ABSORPTION: o Short acting Sedative – (Secobarbital)
a. Principal site of absorption is the small intestine o Intermediate-acting Sedative – (Amo-, Butabarbital)
b. Rapidly absorbed by the stomach following oral administration o Long-Active Sedative (Phenobarbital)
c. Absorbed slowly when administered by rectal suppository
➢ METABOLISM
a. Has a half life of 15 to 20 minutes, due to the rapid conversion
of salicylic acid
b. At low levels, has a half-life of 2 hours
c. Primarily metabolized by the liver

[SARANILLO, KA.]
 ANTICHOLINERGIC DRUGS o Analytical Methodology
Tricyclic Antidepressants (TCA) - Chromatographic methods – most commonly HPLC
o Therapeutic Actions - Immunoassay
- TCAs inhibit uptake at presynaptic junction of the o may be used for qualitative or semi-quantitative
norepinephrine and serotonin, which results in the detection of tricyclic antidepressants which are
accumulation of these neurotransmitters in the synaptic cleft useful for screening purposes
and increased stimulation of postsynaptic receptors. - Tricyclic antidepressants are adequately detected in urine,
- This mechanism is consistent with the amine theory of using a commercial TLC kit and by colloidal gold immunoassay
depression, which asserts that depression stems from a
deficiency in monoamine-mediated transmission. Phenothiazines (Dopamine Receptor Antagonists)
- They elevate mood, increase alertness, improve appetite and o Therapeutic actions
normalize sleep patterns. - They block dopamine receptors in the chemoreceptor trigger
zone (CTZ), thereby suppressing nausea and vomiting.
o Indications - The chemoreceptor trigger zone is an area in the brain,
- TCAs are preferred drugs for treatment of major depression. specifically in the medulla that communicates with the
However, they do not relieve symptoms immediately. vomiting center to induce vomiting.
Therapeutic effects may be seen within 1 to 3 weeks of therapy
and full relief from symptoms can occur up to 1 to 2 months. o Indications
- Treatment of enuresis in children older than 6 because of their - Reduce vomiting from antineoplastic and radiation therapy
anticholinergic effects related to cancer.
- Control vomiting after surgery.
o Pharmacokinetics
- TCAs are well-absorbed from the gastrointestinal tract, o Pharmacokinetics
reaching peak levels in 2 to 4 hours. - Aside from oral administration, these drugs can also be given
- They are highly bound to protein and are lipid-soluble; this through intravenous or intramuscular injection.
allows distribution in the tissues; primarily excreted in the - Onset of actions begins within 30 minutes - 1 hour after oral
urine. administration.
- They cross the placenta and enter breast milk. - These drugs are biotransformed by the liver and excreted in the
urine.
o Contraindications and Cautions
- Contraindicated in patients with recent myocardial infarction o Adverse effects
because of the possible occurrence of reinfarction or extension - Phenothiazines cause a variety of adverse reactions including
of the infarct with the cardiac effects of the drug. extrapyramidal symptoms such as pseudoparkinsonism,
- Contraindicated in patients who have undergone myelography dystonia, akathisia and tardive dyskinesia.
within the previous 24 hours or in the next 24 hours. - Chlorpromazine may cause sedation and orthostatic
- Patients should not take TCAs while taking monoamine oxidase hypotension.
inhibitors due to potential adverse effects or toxic reactions
when these drugs are combined. Symptoms include severe o Analytical Methodology
hyperpyretic crisis with severe convulsions, elevation in blood - Qualitative detection of phenothiazines or their metabolites in
pressure and death. urine is sufficient to document ingestion for symptomatic
- Caution should be used with TCAs in patients with pre-existing patients. Suitable methods of detection include TLC
cardiovascular disorders because of the cardiac stimulatory
effects of the drug and in any condition that would be Antihistamines
exacerbated by the anticholinergic effects such as angle- o Therapeutic Action
closure glaucoma, urinary retention, prostate hypertrophy, or - Compete with histamines for H1 receptor sites in the client’s
GI surgery. arterioles, capillaries and secretory glands in the mucous
- Caution is also necessary in patients with history of seizures membranes; they do not inhibit histamine release.
because the seizure threshold may be decreased secondary to - Possess anticholinergic and antipruritic effects.
stimulation of receptor sites.
- Use with caution for patients with renal and liver disease. o Indications
- Drugs of choice for systemic treatment of allergic rhinitis and
o Side effects – The anticholinergic actions of tricyclic antidepressants conjunctivitis.
are responsible for side effects frequently experienced even at - Reduce rhinorrhea, lacrimation, nasal and conjunctival pruritus
therapeutic doses and are therefore commonly present in overdose. and sneezing.
These effects include:
- Tachycardia o Pharmacokinetics
- hyperpyrexia - Oral antihistamines are well absorbed orally; onset of action is
- dilated pupils usually 1-3 hours after administration.
- dry skin and mouth - They are metabolized in the liver and excreted in feces and
- flushing urine.
- decreased GI motility - They cross the placenta and enter breast milk
- urinary retention
o Contraindications and Cautions
o Adverse effects - Contraindicated in pregnant and lactating patients.
- Orthostatic hypotension is the most serious of the common - Used with caution in patients with renal or hepatic impairment
adverse responses to TCAs. It is due to the blockage of alpha1 - and patients with history of arrhythmias or prolonged QT
adrenergic receptors on blood vessels. intervals because cardiac arrhythmias may occur with the use
- TCAs block muscarinic receptors, and can cause anticholinergic of antihistamines.
effects such as dry mouth, blurred vision, photophobia,
constipation, urinary hesitancy and tachycardia. o Adverse Effects
- Sedation is also a common response which is caused by the - Most common side effect is sedation.
blockage of histamine receptors in the central nervous system. - Anticholinergic side effect such as dry mouth, stuffy nose,
- TCAs can affect cardiac function by decreasing vagal influence blurred vision, constipation and urinary retention.
on the heart and acting directly on the bundle of His to slow the
conduction. Both effects increase the risk of dysrhythmias. o Analytical Methodology
- TCAs lower seizure threshold. - Urine drug screen
- Hypomania may develop as a result of increased effect of TCAs - Quantitation of antihistamines in serum is not useful because
there is a poor correlation between dose, drug concentration,
and degree of toxicity.

[SARANILLO, KA.]
 TRACE ELEMENTS
CLINICAL CHEMISTRY 322 o Clinically important trace elements
METALS AND METALLOIDS / TRACE ELEMENTS
FINALS: WEEK 14 LEC
o Health effects, toxicity and deficiency
o Laboratory methods and instrumentation
METALS AND METALLOIDS
ARSENIC CADMIUM
o Homicide agent o Mining industrial METHODS AND INSTRUMENTATION
o Suicide agent o Inhalation, ingestion o Atomic Absorption Spectrometer (AAS)
o Manmade, occupational o Tubular dysfunction o Atomic Emission Spectrometer (AES)
o Inhalation, ingestion o Itai-itae disease o Flame Atomic Absorption Spectroscopy (FAAS)
o Nonspecific symptoms (contaminated rice) o Graphite Furnace Atomic Absorption Spectroscopy [GFAAS])
o Blood, urine, hair & o Whole blood o Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)
fingernail o Urine o Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

LEAD MERCURY ALUMINUM

o Household paints o Industrial o Crystalline silver white ductile metal
o Inhalation, Ingestion o Contaminated Food o Most abundant metal
o Oxygen, silicon & fluorine
o Tubular dysfunction, o Blood diarrhea,
o Aluminum containing minerals
basophilic stippling hypotension, bradycardia, o Good conductivity of heat & Electricity
o Decreases IQ renal dysfunction
o Whole blood: o Whole blood HEALTH EFFECTS & TOXICITY
zinc protoporphyrin o 24hr urine o Toxicity is not well o Increased left ventricular
o Urine: Aminolevulinic Acid understood mass & decreased
o Encephalopathy myocardial function
PESTICIDES o Osteomalacia or aplastic o Renal insufficiencies
bone disease o Anemia
o Occupational, insecticide, herbicide
o Proximal myopathy o Bone disease
o Ingestion, Inhalation, transdermal absorption o Increased risk of infection o Progressive dementia
o Bradycardia, muscular twitching, cramps, respiratory culture o Microcytic anemia o Impaired neurologic
Development
THERAPEUTIC DRUGS LABORATORY
USAGE ADVERSE EFFECTS LAB o ICP MS
DIAGNOSIS o GFAAS
Salicylates Anti-pyretic, Reye’s syndrome, Chromogenic o Urine and serum levels are useful in determining toxic exposures,
Anti-inflammatory, Metabolic acidosis assay (Trinders
monitoring exposure overtime, and monitoring chelation therapy
Analgesic Interferences with rxn.) Spectro-
Platelet Aggregation photometer
Acetaminophen Analgesis Hepatoxic HPLC, ARSENIC
(Liver necrosis) Immunoassay o Metallic & non-metallic properties
o Earth’s crust (1.5 2.0 mg/kg)
AMPHETAMINE METHAMPHETAMINE o Food (25 50 ug/d)
o Ingestion o Anthropogenic sources
o Desired and Adverse Effects: irritability, psychosis, hypertension,
HEALTH EFFECTS & TOXICITY
arrythmias, increased mental and physical capacity
o Nausea, emesis, abdominal pain, rice water diarrhea
o Pancytopenia, anemia, & basophilic stippling
METHYLENEDIOXY-METHYLAMPHETAMINE o ECG changes
o Amphetamine derivative o Encephalopathy & neuropathy
o Orally in tablets o Renal insufficiency & renal failure
o Inhalation, Injection or smoking o Hepatic systems
o Hallucination, euphoria, violent behavior, tachycardia, o Mees Lines”, hyperkeratosis, hyperpigmentati on, & alopecia
hypertension, cardiac & Liver toxicity, renal failure o Cirrhosis & hepatomegaly
o Hypertension & PVD
o CNS neuropathy & tremor
CANNABINOIDS o Squamous cell, hepatocellular, skin, bladder, lung, & renal
o Found in “Marijuana” carcinomas
o Ingestion, Smoking o “Black foot disease”
o Euphoria; impairment of short-term memory & intellectual
function LABORATORY
o Metabolite in urine: THC-COOH o ICP MS
o GFAAS
o HG AAS
COCAINE “CRACK”
o Arsenic speciation is desired, a separation method is employed either
o Insufflations, IV injection, smoking, inhalation (local anesthetic) online or off line prior to metal analysis
o Euphoria arrhythmia, seizure & myocardial infarction
o Benzoylecgonine CADMIUM
o Soft, bluish white metal which is easily cut with a knife
OPIATES o Burning of fossil fuels such as coal and oil & incineration of municipal
1. Naturally Occurring: opium, morphine, codeine waste materials
2. Chemically modified: heroin
3. Synthetic: fentanyl HEALTH EFFECTS & TOXICITY
o Toxicity is believed to be a result of “Protein Cd” adducts
o Ingestion or injection (pain reliever, anesthetic) o Severe nausea, vomiting, and abdominal pain
o Renal dysfunction
o Sedation, cardiopulmonary failure, myoglobinuria, analgesia
o Nasal epithelial damage and lung damage similar to emphysema
o “EDTA can be used as a chelating agent in cadmium poisoning
PHENCYCLIDINE (PCP)
o Ingestion, Inhalation of PCP LABORATORY
o Hostility, paranoia, coma, stimulant, depressant, hallucinogenic, o GFAAS & ICP MS
agitation o ICP AES
o Urinary excretion is about 70% in the RBC
SEDATIVES o 0.001% & 0.01% of the body burden per 24 hours.
o BARBITURATES (Phenobarbital)
o BENZODIAZEPINES (Diazepam Valium Lorazepam – Ativan)
-Mouth, IM, IV, Rectal
-Lethargy (fatigue), slurred speech, coma, respiratory depression

[SARANILLO, KA.]
 CHROMIUM IRON
o Greek word “CHROMA” o 4th most abundant element
o Rubies red & emeralds green o Most abundant transition metal
o 21st most abundant element in the earth’s crust o Classified as a trace element
o Stainless steel o Oxygen transport
o Participates in redox chemistry
HEALTH EFFECTS & TOXICITY o Agent in redox & electron transfer reactions
o Enhances insulin action
o Metabolism of glucose, fat, & cholesterol HEALTH EFFECTS
o Adequate daily intake of chromium (50 200ug/d) Increased in serum Iron:
a. Condition of increased erythrocyte destruction ( haemolytic anemia)
DEFICIENCY b. Decreased blood formation (lead poisoning, pyridoxine deficiency)
o Insulin resistance c. Increased release of iron from the body of stores (release of ferritin
o Impaired glucose tolerance in acute hepatic cell necrosis)
o Hyperlipidemia d. Defective iron storage (pernicious anemia)
o Glucose intolerance, glycosuria, hypercholesterolemia, decreased e. Increased rate of absorption (hemochromatosis and transfusion
longevity, decrease in sperm counts, and impaired fertility siderosis)

TOXICITY Decreased in serum Iron:

o Cr (6+) Powerful oxidizing agent a. Generalized iron deficiency (lack of sufficient dietary iron)
o CrO4 2+ At physiological Ph b. Inadequate absorption of iron
o It similarity to essential phosphate & sulfate anions c. Chronic loss of iron as a result of bleeding or nephrosis
o Skin ulcer, renal & hepatic necrosis d. Impared releases of iron from the reticuoendothelial system
o Severe dermatitis & skin ulcers Allergic dermatitis with eczema (infection)
o Cr (3+) - protein complex e. Malignant
o Airway irritant, airway obstruction, & possibly lung cancer f. Rheumatoid arthritis
o Lung, kidneys, liver, skin, & immune system
o Transient renal effects Increase in TIBC: Decreased in TIBC:
o Elevated urinary B 2 Microglobulin (an indicator of renal tubular damage) a. Iron defificency a. Chronic infections
b. Late pregnancy b. Malignancy
LABORATORY: c. Oral contraceptives c. Iron poisoning
o GFAAS, NAA, ICP MS d. Viral Hepatitis d. Nephrosis
o Plasma, serum, and urine do not indicate the total body status of the e. Kwashiorkor
individual, whereas urine levels may be useful for metabolic studies f. Thalassemia

COPPER LABORATORY
o Relatively soft yet tough metal o Cofactor of several “TOTAL IRON CONTENT”
o Electrical & heat conducting metalloenzymes o Fe(3+) bound to transferrin
o Forms alloys w/ zinc, tin, & nickel o Heme synthesis o Serum or heparinized plasma
o Cu (0), Cu (1+), Cu (2+), Cu (3+) o Cellular respiration o Oxalate citrate and EDTA
o Collagen synthesis o Early morning sampling
HEALTH EFFECTS o Specimen with visible hemolysis should be avoided
o Ceruloplasmin, cytochrome C oxidase, superoxide dismutase,
tyrosinase, metallothionein, dopamine hydroxylase, lysyl oxidase, TIBC
clotting factor V and unknown enzyme that cross links keratin in hair o Amount of iron that could be bound if transferrin amount of iron that
could be bound if transferrin and other minor iron binding proteins
DEFICIENCY present in the serum or plasma sample were saturated
o Premature infants
o Severe diffuse diseases of small bowel, lymphosarcoma, & scleroderma “PERCENT SATURATION”
o Malnutrition, malabsorption, chronic diarrhea, hyperalimentation, & o The percent saturation, also called the transferrin saturation, is the
prolonged feeding with low copper, total milk diet ratio of serum iron to TIBC.
o Neutropenia & hypochromic anemia in early stages o Approximately 20% to 50%, but it varies with age and sex.
o Osteoporosis & various bone and joint abnormalities
o Decreased pigmentation of the skin and general pallor “TRANSFERIN”
o Hypotonia, apnea, and psychomotor o Measured by immunochemical methods such as nephelometry.
o Retardation o Iron deficiency
o Coronary heart disease o Chronic infections & malignancy
o “Menkes Disease” o Iron overload & hemochromatosis
-  This invariably fatal, progressive brain, disease characterized by
peculiar hair, called kinky or steely, and retardation of growth.  “FERRITIN”
- Progressive mental deterioration, coarse feces, disturbance of o Immunochemical methods
muscle tone, seizures, and episodes of severe hypothermia. o Iron deficiency anemia
o Iron overload & hemochromatosis
HEALTH EFFECTS & TOXICITY o Chronic infections, malignancy, and viral hepatitis
o Interferes with absorption of iron and zinc.
o Copper is an irritant to epithelia & mucus membranes. LABORATORY
o Hepatic & renal damage with hemolysis. o AAS
o Copper induced emesis has characteristic “blue green color” o ICP-ms
o “Wilson’s Disease” o Iron quantification in liver is not used for the evaluation of acute iron
-  Genetically determined copper accumulation disease that toxicity. Hepcidin testing has not yet been shown to be clinically
usually presents between ages of 6 and 40 years useful.
-  Neurological disorders, liver dysfunction, and Kayser Fleischer
18-3 LABOTAORY MARKERS OF IRON STATUS IN SEVERAL DISEASE STATES
rings in the cornea caused by copper deposition
SI TRANSFERRIN FERR. % SAT. TIBC
Iron deficiency ↓ ↑ ↓ ↓ ↑
TABLE 18 -2 INTERPRETATION OF COPPER TESTING RESULTS
Iron overdose ↑ ↓ ↑ ↑ ↓
SERUM COPPER URINE COPPER
Hemochromatosis ↑ Slightly ↓ ↑ ↑ ↓
Nutritional deficiency ↓ ↓
Malnutrition ↓ ↓ ↓ Variable ↓
Menkes syndrome ↓ ↑
Chronic infection ↓ ↓ ↑ ↓ ↓
Acute copper toxicity ↑ or ↑↑ ↑
Acute liver disease ↑ Variable ↑ ↑ Variable
Chronic copper toxicity ↑ ↑
Chronic anemia ↓ N or ↓ N or ↓ N or ↓
Wilson’s disease N or ↓ ↑ or ↑↑ ↑
Smoking, inflammatory conditions ↑ or ↑↑ N
Estrogen, pregnancy ↑ or ↑↑ N

LABORATORY
o Flame aas, icp ms, icp aes, and asv
o Serum copper & urine copper
o Direct measurement of free copper & Serum ceruloplasmin

[SARANILLO, KA.]
 LEAD SELENIUM
o Soft, bluish white, highly malleable and ductile o Naturally occurring metalloid
o Poor conductor of electricity & heat and resistant to corrosion o Similar to sulfur (chemical & physical)
o Essential trace elements
TOXICITY o Major constituent of 40 minerals & minor constituent of 37 others
o 60 μg/Dl
o 45 μg/g HEALTH EFFECTS
o IQ declines are seen in children w/ blood lead levels of ≥10μg/dL o Glutathione peroxidase (in the form of selenocysteine)
o Clumsiness, gait abnormalities, headache, behavioral changes, o Deiodinaseenzymes and thioredoxin reductase
seizures, &severe cognitive and behavioral problems.
o Abdominal pain, constipation, and colic. DEFICIENCY
o Cardiomyopathy, skeletal muscle weakness, and osteoarthritis
LABORATORY o Selenium intakes and the rate of cancer of the large intestine,
o ICP-ms rectum, prostate, breast, ovary, and lungs and leukemia.
o Icp-aes& gfaas o “keshandisease”
o The most common specimen type is whole venous blood, the result - an endemic cardiomyopathy that affects mostly children and
of which is commonly referred to as the BLL women in childbearing age dizziness, malaise, loss of appetite,
o Elevated lead levels in capillary blood specimens should be nausea, chills, abnormal electrocardiograms, cardiogenic
confirmed with a venous specimen shock, cardiac enlargements, and congestive heart failure
o “kashin-beck disease”
MERCURY - an endemic osteoarthritis that occurs during adolescent and
o “Quicksilver” preadolescent years
o Heavy, silvery metal
o Liquid at room temp. & pressure TOXICITY
o Hg(0), Hg(1+), and Hg(2+). o Nausea, vomiting, and diarrhea Tachycardia Nails and skin and hair
o Organic mercury loss, as well as neurologic problems such as unsteady gait or
paralysis
HEALTH EFFECTS % TOXICITY
o Cause or trigger autism in children LABORATORY
o Headache, tremor, impaired coordination, abdominalcramps, o ICP-Ms
diarrhea, dermatitis, polyneuropathy, proteinuria, and hepatic o GfaaS
dysfunction o Determination of urinary and blood selenium is an useful measure
of selenium status
LABORATORY
o ICP-ms ZINC
o Cold vapor aas o Bluish-white lustrous metal.
o Mercury is usually determined as total mercury levels in blood and o Stable in dry air and becomes cover with a white coating when
urine without regard to chemical form. exposed to moisture.
o Alloys, especially brass (with copper), in galvanizing steel, in die
MANGANESE casting, in paints, in skin lotion.
o 12thmost abundant element
o Treatment for “Wilson’s disease”
o Found in over 250 minerals
HEALTH EFFECTS
o Constituent of metalloenzymes and as an enzyme activator
o Oxidoreductases, transferases, hydrolases, leases, isomerases, and
HEALTH EFFECTS lipases.
o Arginase, pyruvate carboxylase, and manganese superoxide o Synthesis and metabolism of DNA & RNA
dismutase.
o Synthesis and metabolism of proteins
o Hydrolases, kinases, decarboxylases, and transferases o Metabolism of glucose and cholesterol, membrane structure
maintenance, insulin function, and growth factor affects
o Many of these activations are not specific to manganese and other
metal ions (magnesium, iron, or copper)
DEFICIENCY
DEFICIENCY
o Growth retardation, slows skeletal maturation, causes testicular
o Blood clotting defects, hypocholesterolemia, dermatitis, and atrophy, and reduces taste perception.
elevated serum calcium, phosphorus, and alkaline phosphatase
o Old age, pregnancy, lactation, and alcoholism
activity
o “acrodermatitis enteropathica”
- First develop a characteristic facial and diaper rash Growth
o Epilepsy, hip abnormalities, joint disease, congenital malformation,
retardation, diarrhea, impaired T-cell immunity, insufficient
heart and bone problems, and stunted growth in children
wound healing, infections, delayed testicular development in
adolescence, and early death
TOXICITY
o Nausea, vomiting, headache, disorientation, memory loss, anxiety, o “adolescent”
- Slow growth or weight loss, altered taste, delayed puberty,
and compulsive laughing or crying.
dwarfism, impaired dark adaptation, alopecia, emotional
o Resembles Parkinson’s diseasewith akinesia, rigidity, tremors,mask-
like faces. instability and tremors.
- Lymphopenia and death
o locuramanganica(manganese madness)

LABORATORY TOXICITY
o ICP-Ms o Gastrointestinal symptoms
o gfaaS o Decrease in heme synthesis
o Urine manganese is used in conjunction with serum manganese to o Hyperglycemia
evaluate possible toxicity or deficiency
o “zinc fume fever” – Chemically induced pneumonia, severe
pulmonary inflammation, fever, hyperpnea, coughing, pains in legs
and chest, and vomiting
MOLYBDENUM
o Hard, silvery white metal LABORATORY
o Molybdenite, wulfenite, & powellite o faaS, ICP-AES, ICP-Ms
o Low urine zinc levels in the presence of low serum zinc levels usually
HEALTH EFFECTS confirm zinc deficiency.
o Xanthine oxidase, aldehyde oxidase, and sulfite oxidase o Serum zinc cannot be interpreted as evidence of normal zinc stores
o “Molybdopterin” o Zinc concentration in RBCs is approximately 10 times that in serum.
o Dietary molybdenum deficiency is rare o Copper status should be monitored in patients undergoing long-
o Molybdenum cofactor deficiency is a recessively inherited error of term zinc therapy.
metabolism
o Elevated uric acid in blood and an increased incidence of gout
LABORATORY
o ICP-Ms
o gfaaS
o Blood levels are less than 60 μg/LElevated lead levels in capillary
blood specimens should be confirmed with a venous specimen

[SARANILLO, KA.]
 LIDOCAINE (XYLOCAINE)
CLINICAL CHEMISTRY 322 o used to correct ventricular arrhythmia and to prevent ventricular
THERAPEUTIC DRUGS MANAGEMENT fibrillation
FINALS: WEEK 17 LEC

CARDIOACTIVE DRUGS Route, Absorption and Elimination

DIGOXIN - administered by continuous IV infusion after a loading dose
o a cardiac glycoside used in the treatment of congestive heart failure - cannot be administered orally
o inhibits Na, K, ATPase which causes a decrease in intracellular potassium, - elimination by hepatic metabolism
resulting in increased intracellular calcium in cardiac myocytes - primary product of hepatic metabolism = monoethylglycinexylidide
o ↓K ↑Ca
o therapeutic range = 1.5 – 4.0 ug/ml
Route, Absorption and Elimination o toxicity range = >4.0 ug/mL
- orally administered o CNS depression = > 4-8 ug/mL
- absorption varies because of the influence of dietary factors, o seizure and decrease BP and cardiac output: > 8ug/mL
gastrointestinal motility, and formulation of drug
PROPRANOLOL
- eliminated by renal filtration
- half-life of plasma digoxin – 38 hours in an average adult o a beta-receptor blocking drug; used in the treatment of angina pectoris,
hypertension, coronary artery disease
o hyperthyroid patients display a resistance to digoxin actions o toxic effects = bradycardia, arterial insufficiency (Raynaud’s type),
o hypothyroidism patients are more sensitive to digoxin pharyngitis
o immunoassay – used to measure total digoxin concentration in serum
o peak serum level = 8 hours after an oral dose
AMIODARONE (CORDARONE)
o half-life = 38 hours (average adult) o blocks potassium channels in the cardiac muscle
o toxic level = > 2ng/mL o use for treatment of ventricular arrhythmias
o toxic effects = nausea, vomiting, visual disturbances, premature o an iodine-containing drug which can cause hyperthyroidism or
ventricular contractions and atrioventricular node blockage hypothyroidism
o therapeutic range = 1.0 – 2.5 ug/mL
QUINIDINE o toxic effect = bradycardia, hepatitis, photodermatitis
o earliest known antiarrhythmic agents
o a naturally occurring drug that can be used to treat various cardiac
VERAPAMIL
arrhythmic situation o for treatment of angina, hypertension and supraventricular arrhythmias
o 2 most common formulation: quinidine sulfate and gluconate o therapeutic range = 80-400ng/mL
o toxic effects = hypotension, peripheral edema, ventricular fibrillation
Route, Absorption and Elimination
- orally administered ANTIBIOTICS
- gastrointestinal absorption is complete and rapid for the sulfate AMINOGLYCOSIDES (GENTAMICIN, TOBRAMYCIN, AMIKACIN,
- Sulfate: peak serum concentration are reached about 2 hours after an
KANAMYCIN, NEOMYCIN, STREPTOMYCIN)
oral dose
o used for treatment of gram (-) bacterial infections
- Gluconate: peak serum concentration is reached 4 – 5 hours after an
o IV or IM administered
oral dose
o causes nephrotoxicity and ototoxicity
- absorbed quinidine = 70 – 80% bound to serum proteins
o not well absorbed from the gastrointestinal tract
- eliminated by hepatic metabolism
o eliminated by renal filtration
o chromatography or immunoassay = used to determine aminoglycosides
o chromatography or immunoassay = used to determine plasma quinidine
concentration o it requires trough and peak measurements
o peak serum level = 2 hours after an oral dose (sulfate); 4-5 hours o toxic range:
(gluconate) ➢ 30 ug/mL (amikacin and kanamycin) – peak levels
o therapeutic range = 2.3 – 5 ug/mL ➢ 12-15 ug/mL (gentamicin and tobramycin) – peak levels
o toxic range = > 5 ug/mL
o toxic effects = nausea, vomiting, abdominal discomfort, cardiovascular
VANCOMYCIN
toxicity o a glycopeptides
o effective against gram positive cocci and bacilli
PROCAINAMIDE (PRONESTYL) o administered by IV infusion
o used to treat cardiac arrhythmia o toxic side effects occurs in the therapeutic range (5-10 ug/mL)
o toxic effects = red man syndrome; nephrotoxicity and ototoxicity
Route, Absorption and Elimination o eliminated by renal filtration and excretion
- orally administered o chromatography and immunoassay = used to determine vancomycin
- gastrointestinal absorption is rapid and complete o therapeutic range = 5-10 ug/mL
- peak plasma concentration = 1 hour o toxic levels
- absorbed procainamide = 20% bound to plasma proteins ➢ > 10 ug/mL – nephrotoxicity
- eliminated by a combination of renal filtration and hepatic metabolism ➢ > 40 ug/mL – ototoxicity

o immunoassay = used to measure both procainamide and its active CHLORAMPHENICOL

metabolite o it distributes to all tissues, and it concentrates in the CSF
o peak serum level = 1 hour after the dose (pinakamabilis) o 50% protein bound; rapidly absorbed in the GIT
o toxic effects = reversible lupus-like syndrome, nephritic syndrome, o toxic effects = blood dyscrasia, cytoplasmic vacuolation (erythroid &
urticaria myeloid cells)
o toxic level = > 25 ug/mL
DYSOPYRAMIDE
o used to treat cardiac arrhythmias
o used a quinidine substitute
ANTIEPILEPTIC DRUGS (1ST GENERATION)
PHENOBARBITAL
Route, Absorption and Elimination o Slow acting barbiturate that controls every type of seizures
- orally administered o peak is about 10 hours after oral dose
- peak serum concentration = 1-2 hours o 50% protein bound in the circulation
- it binds to several plasma proteins o eliminated by hepatic metabolism
- eliminated by renal filtration (majorly/mostly) and to a lesser extent by o half life = 70 – 100 hours
hepatic metabolism o peak serum level = 10 hrs after oral dose
o therapeutic range = 20-40 ug/mL (Phenobarbital); 5-12 ug/mL
o chromatography or immunoassay can be used to determine the plasma (primidone)
concentration of disopyramide o toxic effects = drowsiness, depression and reduced mental capacity
o therapeutic range = 3-5 ug/mL
o toxic range = 10 ug/mL PHENYTOIN (DILANTIN)
o toxic effects = bradycardia and atrioventricular node blockage o treatment for seizure disorders
o short term prophylactic agent in brain injury preventing loss of functional tissue
o IV and oral administration
o 87-97% protein bound
o toxicity is observed when total serum concentration is within therapeutic range
o eliminated by hepatic metabolism
o major toxicity = initiation of seizures
o therapeutic range = 10-20 ug/mL (bound); 1-2 ug/mL (free form)
o toxic range = > 20 ug/mL
o injectable form: fosphenytoin

[TAURO, K.]
 VALPORIC ACID (DEPAKENE) TIAGABINE
o used for treatment of petit mal (absence seizure) and grand mal; o for treating partial seizures
o orally administered o 96% protein bound
o 93% protein bound in the circulation o half life = 4-13 hrs
o eliminated by hepatic metabolism o metabolized by hepatic mixed-function oxidase pathway
o used for treatment of petit mal (absence seizure) and grand mal; o hepatic dysfunction prolongs half-life
o orally administered o therapeutic concentration = 20 -100 ug/mL
o eliminated by hepatic metabolism o side effects = symptom of confusion, difficulty in speaking clearly, mild
o therapeutic range = 50-120 ug/mL sedation and a tingling sensation in the body’s extremities especially in
o toxic levels the hands and fingers
➢ > 120 ug/mL (nausea, lethargy and weight loss)
➢ > 200 ug/mL (pancreatitis, hallucinations, heyperammonemia) TOPIRAMATE
o for partial and generalized seizures
CARBAMAZEPINE (TEGRETOL) o orally administered
o effective for grand mal seizures and treating seizures accompanied by o peak concentration = within 1-4 hrs
pain o 15% protein bound
o has serious toxic effects o eliminated by renal filtration
o orally administered
o 70-80% protein bound in the circulation ZONISAMIDE
o eliminated by hepatic metabolism o therapy for partial and generalized seizures
o idiosyncratic effects = rashes, leucopenia, nausea, vertigo, febrile o administered orally
reactions o absorbed from the GI tract
o therapeutic levels = 8-12 ug/mL o 60% protein bound and accumulates in the RBC’s
o toxic levels = > 15 ug/mL (hematologic dyscrasias, aplastic anemia) o metabolized by the liver
o for monotherapy its half life is 50 – 70 hrs
ETHOSUXIMIDE (ZARONTIN) o children requires higher dosage
o drug of choice for controlling petit mal seizure o serum concentration = 10-38 ug/mL
o orally administered
o is free in serum and not protein bound
PSYCHOACTIVE DRUGS
o therapeutic level = 40-=100 ug/mL
o toxic levels = > 100 ug/mL
LITHIUM
o toxic effects = GI disturbances, ataxia, SLE, aplastic anemia, pancytopenia o orally administered
o used to treat manic depression (bipolar disorder)
o distribution is uniform throughout total body water
ANTIEPILEPTIC DRUGS (2ND GENERATION) o eliminated by renal filtration (urine) and subject to reabsorption
FELBAMATE o it inhibits thyroid hormone synthesis and release – inhibit iodine uptake
o for severe epilepsies such as Lennox-Gastaut syndrome and refractory o flame emission photometry and atomic absorption – viable methods
epilepsy o therapeutic range = 0.8 – 1.2 mmol/L
o administered orally o toxic levels
o peak serum concentration = reached within 1-4 hrs ➢ 2 mmol/L – apathy, lethargy, speech difficulties
o 30% bound to serum proteins ➢ > 2mmol/L – seizures, muscle rigidity and coma
o eliminated by renal and hepatic metabolism
o half life = 14-22 hrs. in adults TRICYCLIC ANTIDEPRESSANTS (TCA)
o clearance is higher in children o used for the treatment of depression, insomnia, extreme apathy and loss
o contraindicated by hepatic dysfunction of libido (loss of sexual desire)
o metabolism is enhanced by enzyme inducers like phenobarbital, o orally administered
primidone, phenytoin and carbamazepine o are highly protein bound (85-95%)
o adverse effects = fatal aplastic anemia and hepatic failure o eliminated by hepatic metabolism
o toxicity of TCAs is dose dependent
GABAPENTIN (NEURONTIN) o chromatographic method
o used for partial seizures o examples = imipramine, amitriptyline, doxepin, nortriptyline, trazadone
o maximum bioavailability = 60% (nacoconsume ng body) o toxic effects = drowsiness, blurred vision, memory loss, seizure, cardiac
o administered orally arrhythmia, parkinsonian syndrome, unconsciousness
o unbound to plasma proteins o peak serum concentration = 2-12 hrs
o excreted unchanged in the urine (not metabolized by liver) o therapeutic levels = 100-300 ng/mL
o pagkainom: neurodine ; pagkalabas = neurontin o major metabolites = desipramine
o half-life = 5-9 hrs
o therapeutic concentration = 12-20 ug/mL FLUOXETINE (PROZAC)
o adverse effects = dizziness, ataxia, fatigue and nystagmus o blocks the reuptake of serotonin in central serotonergic pathways
o used for treatment of obsessive compulsive disorders
LAMOTRIGINE o toxic effects = attempted suicide, decreased libido and sexual function
o for partial generalized seizures o therapeutic levels = 90-300 ng/mL
o administered orally
o rapidly and completely absorbed in the GI tract CLOZAPINE
o approximately 50% protein bound o atypical antipsychotic used to treat otherwise treatmentrefractory
o eliminated by hepatic metabolism schizophrenia, including its negative symptoms, suicidal tendencies and
o half-life = 15-30 hrs various types of cognitive deficiencies associated with the disease
o rate of elimination is dependent on age and physiologic condition o research has found that there is not a well-established clinical serum
o valproic acid is an inhibitor of lamotrigine concentration beneficial effects of the drug have been demonstrated at
o therapeutic range = 2.5 – 15 ug/mL 35-420 ng/mL
o TDM indicated to check for compliance and in patients with altered
LEVETIRACETAM pharmacokinetics
o indicated in partial and generalized seizures o TDM may also be used in avoiding symptoms of toxicity and overdosing,
o administered orally which may present in seizure
o doesn’t bind to serum proteins
o half-life = 6-8 hrs OLANZAPINE
o rate of elimination = increased in children and pregnant women; o a thienobenzodiazepine derivative that treats schizophrenia, acute manic
decreased in the elderly episodes and the recurrence of bipolar disorders
o its clearance correlates with GFR that may be used in monitoring patients o administered as fast-acting intramuscular injection at dose of 2.5-10mg
with renal impairment per injection
o therapeutic range = 8-26 ug/mL o orally administered
o 85% absorbed, although approximately 40% inactivated by first-pass
OXCARBAZEPINE metabolism
o for monotherapy of partial seizures and in secondary generalized o women and non-smoker tend to have a lower clearance and thus a
tonic-clonic seizures higher serum concentration compared with men and smokers
o 40% protein bound o TDM helps to optimize clinical response while balancing it with adverse
o peak centration is 8 hrs effects with a therapeutic range of 20-50 ng/mL
o metabolized by the liver
o half-life = 8-10 hrs
o higher clearance rate in children
o serum concentration = 12-35 ug/mL

[TAURO, K.]
 BRONCHODILATORS ANTINEOPLASTIC DRUGS
THEOPHYLLINE METHOTREXATE
o belongs to the methylated xanthine class o effective therapy for a variety of neoplastic conditions; also an
o its action is specific to the relaxation of bronchial smooth muscle immunosuppressive agent
o it is used in the treatment of asthma and other chronic obstructive o it inhibits DNA synthesis in all cells, by blocking dihydrofolate reductase
pulmonary disease o leucovorin (antidote) used to reverse the action of methotrexate –
o drug for primary apnea of prematurity – absence of respiratory effort in leucovorin rescue
newborn infants o toxic levels = 0.01 umol/L
o intravenous and oral administration o toxic effects = leucopenia, GI ulceration, thrombocytopenia, cirrhosis
o 50% protein-bound
o eliminated by renal filtration and hepatic metabolism BUSULFAN
o the best predictor of toxicity is the blood level of the drug and not early o an alkalizing agent used to treat leukemias and lymphomas prior to bone
signs of symptoms of toxicity marrow transplantation
o therapeutic range = 10-20 ug/mL o for controlling and managing
o toxic range = > 20 ug/mL o overdosage may cause hepatic occlusive disease
o toxic effects = GI bleeding, seizures, tachycardia, syncope
ANTI-INFLAMMATORY AND ANALGESIC DRUGS
IMMUNOSUPPRESSIVE DRUGS SALICYLATES/ASPIRIN (ACETYLSALICYLIC ACID)
CYCLOSPORINE o commonly used analgesic, antipyretic and anti-inflammatory drug
o inhibits the cellular immune response by blocking production of o it is a direct stimulator of the respiratory system and an inhibitor of the
interleukin-2 Kreb’s cycle
o used to prevent rejection of allogenic organ transplants o has anticoagulant property – antiplatelet activity
o for suppression of graft-versus host disease (GVHD) o function = it decreases thromboxane and prostaglandin formation
o orally administered with 5-50% absorption through inhibition of cyclooxygenase
o organs such as the heart, liver and pancreas require high dosage (300 o side effects = gastrointestinal disturbance; interference with platelet
ng/mL) aggregation
o it has marked affinity with RBC, RBC cyclosporine is temperature o toxic effects = mixed acid-base disturbance (metabolic acidosis and
dependent respiratory alkalosis); Reye’s syndrome
o eliminated by hepatic metabolism o methods for testing = Trinder assay; enzymatic assay (salicylate
o specimen of choice = whole blood (with lysis of RBC to yield the total hydroxylase); HPLC; EMIT
amount) o therapeutic levels = 5 mg/dl (treatment of headache)
o toxic effects = renal tubular and glomerular dysfunction
o toxic range = 350 – 300 ng/mL ACETAMINOPHEN (TYLENOL)
o chromatographic methods are available o an inhibitor of prostaglandin metabolism
o commonly used as analgesic and antipyretic drug
TACROLIMUS (PROGRAF/FK-506) o overdosage leads to hepatotoxicity
o 100 times more potent than cyclosporine o reference method = HPLC
o orally administered o therapeutic levels = 25 ug/mL
o early use suggested a low degree of toxicity compared with cyclosporine o toxic levels
therapeutic concentrations ➢ > 50 ug/mL
o associated with thrombus formation ➢ 100-300 ug/mL (hepatic necrosis)
o GI uptake is highly variable
o eliminated by hepatic metabolism IBUPROFEN
o metabolic products are primarily secreted into the bile o has analgesic and anti-inflammatory actions
o increases in immunoreactive tacrolimus may be seen in cholestasis as a o has a lower risk to toxicities than salicylates and acetaminophen
result of cross reactivity o toxic effects = nausea, vomiting, blurred vision, abdominal pain, edema
o high performance liquid chromatography mass Spectrophotometry o therapeutic levels = 10-50 ug/mL
(HPLC/MS) = most common method o toxic levels = > 100 ug/mL
o several immunoassays are available

SIROLIMUS (RAPAMYCIN) NEUROLEPTICS (ANTIPSYCHOTIC MAJOR TRANQUILIZER)

o antifungal agent with immunosuppressive activity
o for patients receiving kidney transplants
o block the action of dopamine and serotonin in the limbic system
o extremely potent and requires TDM due to its inherent toxicity
o used in the treatment of acute schizophrenia
o adverse events = thrombocytopenia, anemia, leucopenia, infections and
o monitoring of these drugs in serum is difficult due to abundant
hyperlipidemia
metabolites for each drug resulting to extensive metabolism in the liver
o commonly use in conjunction with cyclosporine or tacrolimus
o 2 classes = phenothiazines (chlorpromazine) and butyrophenones
o it is rapidly absorbed after once-daily oral administration with peak blood
(haloperidol)
levels at about 1 hour
o examples = risperdal, olanzapine (zyprexa), quetiapine (seroquel),
o oral bioavailability = 15% when taken in conjunction with cyclosporine
aripiprazole (abilify)
o serum concentration is affected extensively by intestinal and hepatic
o toxic effects = cholestasis, orthostatic hypotension, aplastic anemia,
first-pass metabolism
muscle rigidity
o half-life of 62 hrs
o binds more to lipoproteins than to serum protein
o whole blood – ideal specimen for analysis
o TDM commences using a trough level specimen obtained 5-7 days after
initiation of therapy
o therapeutic range = 4-12 ug/L when it used in conjunction with
cyclosporine and 12-20 ug/L if cyclosporine is discontinued
o assayed using chromatography

MYCOPHENOLIC ACID
o mycophenolate mefetil (MMF) is the prodrug, which is rapidly converted
in the liver to its active form of mycophenolic acid (MPA)
o lymphocyte proliferation inhibitor
o used most commonly as supplemental therapy with cyclosporine and
tacrolimus in renal transplant patients
o low trough levels increase the risk of acute rejection
o orally administered
o once in circulation, it is 95% protein bound
o therapeutic serum concentrations = 1.0 – 3.5 ug/mL
o MPA and its metabolites can be assayed using plasma specimen as the
most likely sample of choice when using chromatography
o immunoassay is less specific, yet common method in assaying plasma
MPA

LEFLUNOMIDE (LFM)
o inhibits lymphocyte proliferation
o for treatment of rheumatoid arthritis

[TAURO, K.]
 CLINICAL CHEMISTRY 322 FIRST-PASS METABOLISM
THERAPEUTIC DRUG MONITORING
FINALS: WEEK 17
o All substances, including drugs, absorbed from the intestine
(except the rectum) enter the hepatic portal system
THERAPEUTIC DRUG MONITORING
o Blood from the gastrointestinal tract is routed through the liver
o Analysis, assessment, and evaluation
before it enters into general circulation
o Circulating Concentration of drugs
o Certain drugs are subject to significant hepatic uptake and
o Serum, plasma, whole blood
metabolism during this passage through the liver.
o Absorptive characteristics of a drug may change with age,
PURPOSE
pregnancy, or pathologic conditions
o Ensure that a given drug dosage produces
o Predicting the final circulating concentration from a standard
- Maximal therapeutic benefit
oral dose can be difficult
- Minimal toxic adverse effects
o With the use of TDM, however, effective oral dosage regimens
can be determined
TDM IS UNNEEDED WHEN…
o Most drug therapies
III. FREE VERSUS BOUND DRUGS
o Dosage regimens have been established that are safe and
o Binding with serum constituents
effective in most of the population
o Drug-protein complexes
o However, weak correlation between dosage and therapeutic
o Drug dynamics: Only free fraction can interact with its site off
effects or toxic outcomes
action and result in a biologic response
o Difficult to predict what dose to be used
o Trial and error: if a standard drug dose does not display therapeutic
o Therapeutic Range – total plasma content
benefit and increasing the dose does provide benefit without toxic o High free fraction – patient experience toxic adverse effects
effects, an appropriate dosage adjustment has been made. o Low free fraction – therapeutic benefit unrealized
o If overdosing or underdosing results in severe consequences to
the patient, trial and error is not justified CHANGES IN SERUM-BINDING PROTEINS ARE DUE TO…
- Overdosage: above the normal dose o ↑ Serum Proteins, ↓ free fraction and ↑ Bound fraction
- Underdosage: below the normal dose 1. Inflammation
2. Malignancies
o STANDARD DOSAGE 3. Pregnancy
- Statistically derived from observations in a healthy o ↓ Serum Proteins, ↑ free fraction and ↓ Bound fraction
population 4. Hepatic disease
o DISEASED STATES 5. Nephrotic syndrome
- Altered physiologic conditions 6. Malnutrition
- Standard dose do not produce predicted concentration in
circulation CHANGES IN % FREE DRUG
Due to competition for binding sites Measurement:
COMMON INDICATIONS FOR TDM: o ↑ Serum Proteins, ↓ free o Drugs that are highly
o The consequences of overdosing and underdosing are serious fraction and ↑ Bound fraction protein-bound
o There is a small difference between a therapeutic and a toxic dose 1. UREA o Clinical signs are
o There is a poor relationship between the dose of drug and 2. BILIRUBIN inconsistent
circulating concentrations but a good correlation between 3. HORMONES
circulating concentrations and therapeutic or toxic effects
o There is a change in the patient’s physiologic state that may IV. DRUG DISTRIBUTION
unpredictably affect circulating drug concentrations o Lipid solubility
o A drug interaction is or may be occurring o Volume of distribution (Vd=D/Ct)
o TDM helps in monitoring patient compliance o High Vd = hydrophobic drugs
o Low Vd = ionized or bound in plasma
FACTORS AFFECTING CIRCULATING CONCENTRATION OF A DRUG o Where: Vd = volume of distribution (liters)
1. Routes of administration (IV, IM, parental, etc.,) : D = an injected dose (mg) or grams (g)
2. Absorption (Small intestine, large intestine, Stomach) : C = concentration in plasma (mg/L or g/L)
3. Free versus Bound drugs o The free fraction of circulating drugs is subject to diffusion out of
4. Drug distribution the vasculature into interstitial and intracellular spaces
5. Drug elimination o Largely dependent on the lipid solubility of the drug
o Highly hydrophobic: easily traverse cellular membranes and
partition into lipid compartments, such as adipose and nerve cells
I. ROUTES OF ADMINISTRATION
o Polar but not ionized: also cross cell membrane but do not
o Therapeutic benefit: appropriate concentration at its site of action
sequester into lipid compartments
o Bioavailability: unchanged fraction of the administered dose as
o Ionized species diffuse out of the vasculature but at a slow rate
it enters systemic circulation
o The volume of distribution (Vd) index is used to describe the
o The goal of most therapeutic regimens is to acquire blood,
distribution characteristics of a drug
plasma, or serum concentration that has been correlated with an
effective concentration at the site of action
1. Directly into the circulation: IV Intravenous V. DRUG ELIMINATION
2. Into muscles: IM Intramuscular o ∆C/∆T = -kC : Equation that defines how the change in
3. Just under the skin: SC Subcutaneous concentration per unit time is directly related to the
4. Inhaled or absorbed through the skin: Transcutaneous concentration of drug (C) and the constant (k)
5. Rectal delivery: Suppository o The k value is a simple proportionality factor that describes the
6. Oral delivery: most common route of delivery percentage change (negative because it is decreasing) per unit
time; it is commonly referred to as the elimination constant or
II. ABSORPTION the rate of elimination
o Formulation of the Drug o Hepatic metabolism or renal filtration, or a combination of the
- Tablets and Capsules: dissolution before absorption two, eliminates most drugs. For certain drugs, elimination by
- Liquid solutions: more rapidly absorbed these routes is highly variable
o Passive diffusion: requires that the drug be in a hydrophobic
(non-ionized state) HALF-LIFE
o Stomach: weak acids ; Small intestines: weak bases o Time needed for the serum concentration to decrease by one half
o Determine graphically
DISEASED STATES o Conversion of elimination constant (k)
1. Changes in intestinal motility 3. inflammation - T 1/2 = 0.693 / k
2. pH 4. food & other drugs

[SARANILLO, KA.]
 DRUG CATEGORY DRUGS IN THAT TREATMENT USE LIDOCAINE
CATEGORY o Correct ventricular arrhythmia
Cardiac drugs Digoxin, digitoxin, Congestive heart o Prevent ventricular fibrillation
quinidine, procainamide, failure, o Acute myocardial infarction
N-acetyl-procainamide (a angina,
metabolite of arrhythmias
procainamide) SERUM CONCENTRATIONS
Antibiotics Aminoglycosides Infections with o Therapeutic range
(gentamicin, tobramycin, bacteria that are - 1.5-4 ug/ml
amikacin) Vancomycin, resistant to less o Toxic range
Chloramphenicol toxic antibiotics - 4-8 ug/ml: Central nervous system depression
Antiepileptics Phenobarbital, phenytoin, Epilepsy, - More than 8 ug/ml: seizures, severe decrease in blood
valproic acid, prevention of pressure and cardiac output
carbamazepine, seizures, - The toxic effects are due to the parent drug and the additive
ethosuximide, sometimes sometimes to
Monoethlglycinexylidide (MEGX)
gabapentin, lamotrigine stabilize moods
Bronchodilators Theophylline, caffeine Asthma, chronic - Measurement is through chromatography and
obstructive immunoassay
pulmonary
disorder (COPD), QUINIDINE
neonatal apnea o Quinidine sulfate
Immunosuppressants Cyclosporine, tacrolimus, Prevent rejection o Quinidine gluconate
sirolimus, mycophenolate of transplanted o Naturally occurring drug
mofetil, azathioprine organs,
o Treat cardiac arrhythmia
autoimmune
disorders
o PEAK ASSESSMENT
Anti-cancer drugs Methotrexate Psoriasis, - Quinidine sulfate: after 2 hours
rheumatoid - Quinidine gluconate: after 4-5 hours
arthritis, various o Toxic effects: nausea, vomiting, abdominal discomfort, and
cancers, non- cardiovascular toxicity
hodgekin’s
lymphomas, PROCAINAMIDE
osteosarcoma o Treat cardiac arrythmia
Psychiatric drugs Lithium, valproic acid, Bipolar disorder
o Hepatic metabolite of its parent drug
some antidepressants (manic
(imipramine, amitriptyline, depression),
- N-acetyl procainamide
nortriptyline depression</TR o Measurement: immunoassay
Protease inhibitors Indinavir, ritonavir, HIV / AIDS o Peak serum concentration: 1 hours
lopinavir, saquinavir, o Toxic effects: Myocardial depression and arrythmia
atazanavir, nelfinavir
DISOPYRAMIDE
o Treat cardiac arrythmia
o Substitute for Quinidine
o Measure of Disopyramide: Chromatography and Immunoassay
o Therapeutic range: 3-5 ug/ml
o Toxic range:
- More than 4.5 ug/mL: anticholinergic effects such as dry
mouth and constipation
- 10 ug/mL: cardiac effects of bradycardia and
atrioventricular node blockage

ANTIBACTERIALS
AMINOGLYCOSIDES
o Gentamicin
CARDIOACTIVE AND ANTIBACTERIAL DRUGS o Tobramicin
o Drugs taken by patients with Cardiac arrhythmia o Amikacin
o Also known as Cardiac glycosides o Kanamycin
o Treatment of infections with Gram-negative bacteria
DIGOXIN o Measurement: chromatography and immune assay
o Used as treatment of patients with congestive heart failure
o Serum concentration at the therapeutic range is 0.8-2 ng/ml TOXIC EFFECTS
o Inhibits membrane Na-K ATPase o Nephrotoxicity
o Increases Calcium - Impairs proximal tubule function
o Improve cardiac contractility (inotropic effect) - Electrolyte imbalance
o Toxic range is more than 3 ng/ml - Proteinuria
o Causes decreased rate of ventricular depolarization - High levels of exposure (tissue necrosis and kidney failure)
o Controls tachycardia o Ototoxicity
- Disrupts the inner cochlea of the ear and vestibular membranes
TOXIC EFFECTS CARDIAC EFFECTS - Hearing and balance impairment
o Nausea o Premature ventricular
o Vomiting contractions (PVCs) VANCOMYCIN
o Visual disturbances o Atrio-ventricular node blockage o Treat infections against Gram-positive cocci and bacilli
o Measurement: Chromatography and immunoassay
HALF LIFE
o 38 hours in an average size adult SERUM CONCENTRATIONS
o Therapeutic range
PEAK ASSESSMENT - 5-10 ug/ml: Red-man syndrome or erythmic flushing of the
o Conducted after 8 hours extremities, renal and hearing effects
o Measurement of digoxin is by immunoassay o Toxic range
- More than 10 ug/ml: nephrotic effects
- More than 40 ug/ml: ototoxic effects
-

[SARANILLO, KA.]
 PSYCHOACTIVE DRUGS & BRONCHODILATORS IMMUNOSUPPRESIVE
LITHIUM TACROLIMUS
o Treat manic-depression o Also known as FK-506
o Bipolar disorder o Immunosuppressive drug
o 100x more potent than cyclosporine
SERUM CONCENTRATIONS o Toxic effects: nephrotoxicity and thrombus formation
o Therapeutic range o Specimen of choice: whole blood
- 0.8-1.2 mmol/L o Measurement:
o Toxic range - Conducted by High Performance Liquid Chromatography (HPLC)
- 1.2 to 2 mmol/L: apathy, lethargy, speech difficulties and - Original method of detection
muscle weakness - Immunoassay
- At more than 2 mmol/L: muscle rigidity, seizures and
possibly coma CYCLOSPORINE
o Used in the suppression of host-versus-graft rejection
TRICYCLIC ANTIDEPRESSANTS o Heterotropic transplanted organs
o Imipramine o Serum concentration
o Amitriptyline - Cardiac
- Desipramine and nortriptyline as metabolites - Liver
o Doxepin - Pancreas transplants
o Treat depression, insomnia, extreme apathy and loss of libido - 300 ng/ml
o Total tricyclics are measured by chromatography and immunoassay o Toxic range: 350-400 ng/ml
o Toxic effects: o Toxic effects:
- Drowsiness - Renal tubular dysfunction
- Constipation - Glomerular dysfunction leading to hypertension
- blurred vision o Measurement: Chromatography
- memory loss o Specimen of choice: Whole blood
o At higher levels, it causes:
- Seizure SIROLIMUS (RAPAMYCIN)
- Cardia arrhythmia o Antifungal agent with immunosuppressive activity
- Unconsciousness o Approved for patients receiving kidney transplants
o Steady-state levels o Adverse events include
- After 2-4 weeks of initiation of therapy - Thrombocytopenia, anemia, leukopenia, infections and
hyperlipidemia
CLOZEPINE o Commonly used in conjunction with cyclosporine or tacrolimus
o Refractory schizophrenia o Rapidly absorbed after once-daily oral administration
- Negative symptoms: suicidal tendencies & various types of o Peak blood levels at about 1 hour
cognitive deficiencies o Oral bioavailability is 15% when taken in conjunction with
o Not a well-established clinical serum concentration, cyclosporine
o beneficial effects: 350-420 ng/ml o Half-life of 62 hours
o To check for compliance and in patients with altered o Binds more highly to lipoproteins than to serum protein → whole blood
pharmacokinetics o Therapeutic range
o Symptoms of toxicity and overdosing: seizures - 4-12 g/L when it is used in conjunction with cyclosporine
- 12-20 g/L if cyclosporine therapy is discontinued
OLANZAPINE o Assayed using chromatography
o Thienobenzodiazapine derivative
o Treats schizophrenia, acute manic episodes & recurrence of MYCOPHENOLIC ACID
bipolar disorders o Mycophenolate mofetil (MMF)
o Intramuscular injection: 2.5-10 mg per injection - Prodrug
o Orally: 85% absorbed, approximately 40% inactivated by first- - Active form of Mycophenolic acid (MPA)
pass metabolism o Lymphocyte proliferation inhibitor
o Women and non-smokers tend to have lower clearance and thus o Most commonly as supplemental therapy with cyclosporine and
a higher serum concentration tacrolimus in renal transplant patients
o Therapeutic range: 20-50 ng/mL o Low trough levels: ↑ risk of acute rejection
o High levels: toxicity
BROCHODILATORS o Orally administered drug
o Used for the treatment of: o Absorbed under neutral pH conditions in the intestine
- Asthma o 95% Protein bound
- Chronic obstructive pulmonary diseases o Therapeutic serum concentrations = 1.0-3.5 g/mL
o Therapeutic range: 10-20 ug/ml o Plasma: sample of choice when using chromatography
o Toxic range o Immunoassay is a less specific but common
- More than 20 ug/ml causes nausea, vomiting and diarrhea - Cross-reactivity between MPA & its active metabolite (AcMPAG)
- More than 30 ug/ml causes cardia arrythmia and seizures
ANTI-NEOPLASTIC DRUGS
METHOTREXATE
o Anti-neoplastic drug
o Inhibits DNA synthesis in all cells
o Toxic effects: cytotoxicity affects most cell types
o To control the period of cytotoxicity of Methotrexate, a drug
called Leucovorin is also administered to the patient after
dosage of Methotrexate
o Leucovorin rescue ensures that cytotoxicity is not deleterious by
inhibiting the action of Methotrexate

[SARANILLO, KA.]