CC LAB-MODULE 1 Allosteric Site- cavity other than active site &

regulator molecules binds in the allosteric site;

ENZYMES DISCUSSIONS
TERMS ASSOCIATED WITH ENZYMES
ENZYMOLOGY
 Substrates
ENZYMES
 Cofactors
- Primary Structure Protein  Isoenzyme
 Apoenzyme
 A specific biologic protein that catalyzes  Holoenzyme
biochemical reaction without altering  Proenzyme
the equilibrium point of the reaction or
consumes or changes in composition. SUBSTRATES
 They are measured in terms of their
inactivity and not in terms of their  Substances that acts upon enzymes,
absolute values and they are specific for each of their
 These are catalysts that can be used in particular enzymes
the monitoring and diagnosis of disease
e.g. triglycerides-substrate is lipase;
and their remarkable properties makes
them sensitive indicators of pathologic starch-substrate is amylase
change.
 COMPONENTS: Active Site and COFACTORS
Allosteric site
 Non-protein substances added in the
enzymes substrate complex to manifest
the enzyme activity.

ISOENZYMES

 Enzymes with similar enzymatic activity

but differ in their physical, biochemical,
and immunologic characteristics.
 Helps in understanding of organ specific
patterns of metabolism.
NOTE: Catalytic activity of enzymes depends on  Usually tissue specific or organ specific
the integrity of its structure. When active and e.g. lactate dehydrogenase & creatine
allosteric site is filled no further binding can kinase (CK) – exist due to prescence of
occur until an active site discharges its contents. multiple genes.

Active Site- water-free cavity/ substrate APOENZYMES

interact w/ particular charge amino acid
residues; substrate interacts  Protein portions of enzymes subject to
denaturation in which enzyme loses its
activity.
  Minimal denaturation may be reverse 4th number- Serial number/Enzyme number
by removal of denaturing reagent. within sub-subclass
 Affected by extreme pH temperature
and chemical addition. Classification of Enzymes- “OTHLIL”

HOLOENZYMES

 Active substance formed by

combination of a coenzyme and Apo
enzyme
 Apoenzyme +Prosthetic Group

PROENZYMES OR ZYMOGEN

 Inactive enzyme precursor

1. Oxidoreductases
e.g. coagulation factors & digestive
 Catalyze the removal or addition of
enzyme
electrons
ENZYME CLASSIFICATION NOMENCLATURE  Redox reaction
e.g. Dehydrogenases: Cytochrome
 Enzymes are classified according to oxidase, LDH, MDH, Isocitrate
their biochemical functions, indicating dehydrogenase, G-6-PD
substrate and class of reaction A+B→A+B
catalyzed, and are designated by
individual identification numbers. 2. Transferase
 Consists of 4 numbers separated by  Catalyze the reaction of a chemical
periods. (e.g. 1.1.1.27) group other than hydrogen from one
substance to another
 Transfer of a chemical group other than
hydrogen from 1 substrate to another
e.g. Kinases, Transaminases,
Aminotransferases: CK, GGT, AST, ALT,
OCT
A–X+B→A+B–X

Example: EC 2.7.3.2. 3. Hydrolases

 Catalyze hydrolysis or splitting of a
EC- Enzyme Classification
bond by the addition of water
1st number- Class e.g. Esterases: ACP, ALP, CHS, LPS
Peptidases: Trypsin, Pepsin, LAP
2nd number- Sub-Class Glycosidases:AMS, Galactosidases
A - B + H2O → OH – B - H
3rd number- Sub-subclass (Nitrogenous group or
acceptor)
 4. Lyases ENZYME THEORY
 Catalyze removal of groups from
substrates without hydrolysis. The 1) EMIL FISHERS / LOCK AND KEY THEORY
product contains double bonds.  Is based on the premise that the shape
of the key (substrate) must fit into the
e.g. Aldolase and Decarboxylase:
Glutamate decarboxylase, Pyruvate lock (enzyme)
decarboxylase, Tryptophan 2) KOCHLAND’S/ INDUCED FIT THEORY
decarboxylase  Is based on the substrate binding to the
ATP → cAMP + PP¡ active site of the enzyme.

5. Isomerases
 Catalyze the intramolecular
arrangement of the substrate
compound
e.g. Glucose phosphate isomerqase,
and Ribose phosphate isomerase
A→B
ENZYME KINETICS
6. Ligases
 A chemical reaction may occur
 Catalyze the joining of two substrate
spontaneously if the free energy or
molecules, couples with breaking of the
available kinetic energy is higher for the
pyrophosphate bond in the ATP or
substrate than the product
similar compound.
e.g. Synthases
 Enzyme catalyze physiologic reactions
AB + C → A - C + B
by lowering the activation energy level
ENZYME CLASSIFICATION NOMENCLATURE that the substrate must reach for the
reaction to occur
E + S → ES → E + P

 CATALYTIC MECHANISM
o Absolute Specificity
o Group Specificity
o Bond Specificity
o Steriosometric Specificity

Binding process transforms the substrates

molecule to its activated process transforms the
substrates molecule to its activated state. –
E.C. nomenclatures with red color are the 8
natawa ako parang riddle. -
enzymes to be discussed.
CATALYTIC MECHANISM GENERAL METHODS OF MEASURING
ENZYMATIC REACTION
ABSOLUTE SPECIFICITY
1) FIXED-TIME- the reactants are
 Enzyme combines with only 1 substrate combined; the reaction proceeds for
and catalyzes only 1 reaction designated time; the reaction is
GROUP SPECIFICITY stopped and measurement is made;

 Enzymes combines with all substrate in

a chemical group 2) CONTINOUS MONITORING/KINETIC
ASSAY- multiple measurements of
BOND SPECIFICITY changed in absorbance are made during
 Enzymes reacting with specific chemical reaction; preferred method.
bonds

STERIOISOMETRIC SPECIFICITY FACTORS AFFECTING ENZYMATIC REACTIONS

1) ENZYME CONCENTRATION
 Enzymes that combines with only 1
 The higher the enzyme concentration,
optical isomer
the faster is the reaction, because more
ENZYME REACTION enzyme is present to bind with the
substrate
 Zero-order reaction – the reaction rate
depends only on enzyme 2) SUBSTRATE CONCENTRATION
concentration; independent on  With the amount of enzyme exceeding
substrate concentration the amount of a substance, the reaction
rate steadily increases as more
 First-order reaction – the reaction rate substrate is added.
is directly proportional to substrate  However, when the substrate
concentration; independent on enzyme concentration reaches as maximal
concentration value, higher concentration of substrate
no longer result in increased rate of
reaction (saturation kinetics.)
ENZYME ACTIVITY
UNITS FOR EXPRESSING ENZYMATIC ACTIVITY:
Enzymes are measured in terms of:
1) INTERNATIONAL UNIT (IU or U)
1. Change in the substrate concentration
 1 micromole of substrate/minute
2. Change in the product concentration
3. Change in coenzyme concentration
2) KATAL UNIT (KU)
 1 mole of substrate/second
  Enzymes are quantitated based on
their activity rather than absolute A. COMPETITIVE INHIBITOR
values.  Physically binds to the active site of
 The units used to report enzyme an enzyme.
levels are activity units.  Reversible (Substrate > Inhibitor)
 The definition for activity unit must  Substrate concentration ↑ than the
consider change in pH, concentration of inhibitor will still
temperature, substrate, etc., proceed, called REVERSIBLE.

3) COFACTORS
 Non-protein entities that must bind
to particular enzymes before a
reaction occurs.(no cofactor, no
reaction)
A. COENZYMES- Organic compound;
 essential to achieve absolute
enzymatic activity and increasing its
concentration will increase the
velocity of an enzymatic reaction
B. NON-COMPETETIVE INHIBITOR
E.g. Nicotinamide Adenine
 It does not compete with the
Dinucleotide (NAD), Nicotinamide
substrate but look for areas other
Adenine Dinucleotide Phosphate
than the active site.
(NADP)
 Binds to the allosteric site (cofactor
site)
B. ACTIVATORS- Inorganic Ions;
 Irreversible
 alters spatial configuration of the
enzymes for proper substrate
E.g. Calcium, Zinc, Chloride,
Magnesium, Potassium
--sabi ni ma’am organic ions, sabi ni
rodriguez notes inorganic. --

C. METALLOENZYMES- Inorganic ion

attached to a molecule
E.g. Catalase and Cytochrome
oxidase

C. UNCOMPETITIVE INHIBITOR
4) INHIBITORS
 The inhibitor binds to the enzyme-
 Enzymatic reactions may not
substrate (ES) complex.
progress if an inhibitor interferes
 Increasing the substrate
with the reaction; (interferes with
concentration results in more
the enzymatic reactions)
 enzyme substrate complex to which there will be a two-fold increase in
the inhibitor binds thereby enzyme activity.
increases the inhibition  Repeated freezing and thawing tends to
↑Substrate concentration= ES = ↑ denature proteins and should be
Inhibitor/inhibition avoided.
 Low temperature renders enzymes
reversibly inactive-
refrigerated/freezing temperature.

7) HYDROGEN ION CONCENTRATION OR

Ph
 Most physiologic reaction occurs in the
pH range of 7-8
 Extreme pH level may denature an
enzyme or influence its ionic state
resulting in structure change or change
5) ISOENZYMES of amino acid residue in the active site
 These are enzymes (polypeptide chains)
having the same catalytic reactions but 8) STORAGE
slightly different molecular structures –  Low temperature
various forms occur because of (refrigeration/freezing) render enzymes
differences in the amino acid sequence reversibly inactive
of enzymes.  Repeated freezing and thawing to
 The importance of the total enzyme denature proteins and should be
activity is enhanced by fractionating the avoided
isoenzymes; (fractionation of  Enzymes: -20°C= for longer period of
isoenzymes) time
 Substrate and coenzymes: 2-8°C
6) TEMPERATURE  LDH(LD4 & 5): room temperature
 Enzymes are active at 25°C, 30°C, or
37°C 9) HEMOLYSIS
 37°C is the optimum temperature of  Mostly increases enzyme concentration
enzymatic activity
 Increasing temperature = Increasing 10) LACTESCENCE OR MILKY SPECIMEN
rate of chemical reaction (movement of  Decreases enzyme concentration
molecules
NOTES TO REMEMBER: *in enzymes*
 Increasing temperature = Increasing
rate of denaturation (40°C to 50°C)  An enzyme accelerates the rate of reaction,
 Inactivation of Enzymes: 60°C to 65°C) reducing the time required to reach
 Temperature Coefficient (Q10) means equilibrium.
for every 10°C increase in temperature
 A constant change in absorbance per unit ester with the contaminant production
time occurs only when the rate of the of an alcohol
reaction is zero order.  Major tissue sources: liver (sinusoidal
 A substrate concentration of >99 x Km is and bile canalicular membranes), bone
needed to achieve zero-order reaction. (osteoblasts), placenta and intestinal
 It is easier to measure small increases in  Reference values: 30-90 U/L
product than to measure small decreases in  Major isoenzymes:
a large amount of substrate. 1) Liver ALP – most anodal
 Enzymes do not alter the free energy or 2) Bone ALP
direction of a reaction, but it alters the 3) Placental ALP
energy of activation by forming a 4) Intestinal ALP- least anodal;
metastable intermediate, the ES complex. most cathodal
 Most enzymes are measured by monitoring  ALP comes in many forms and some of
the rate of absorbance change (kinetic which are true isoenzymes, encoded in
assay) at 340nm as NADH is reduced or separated gene loci (chromosome 1)
consumed, and it allows direct reporting 1) ALPL- found in liver, bone and
either by IU or KU. kidney
 In first-order reaction, the enzymes are 2) ALPI- found in intestine
used as reagents to measure a specific 3) ALPP- found in placenta
analyte.  Activator of ALP includes Mg, Co, Mn,
 In non-kinetic assay absorbance is made at and Zn.
10-seconds intervals for 100 seconds.  Inhibitor includes borates, oxalates, and
 Endpoint measurement determines the cyanide ions.
concentration of substrate or product at  Carcinoplacental ALP:
specific time after addition of the sample 1) Region ALP- is found in lung,
(beside glucose testing using strips.) breast, ovarian and
 Enzyme activity measurements may not be gynecological cancers; bone ALP
accurate if enzymes inhibitors are present, co-migrator; most heat stable
essential cofactors are not included in the ALP (65°C for 30 minutes);
assay, and improper specimen storage. inhibited by phenylalanine
reagent
SPECIFIC ENZYMES
2) Nagao ALP- found in
ALKALINE PHOSPHATASE (ALP) E.C.3.1.3.1 adenocarcinoma of the
pancreas and bile duct, pleural
 AKA: Alkaline Orthophosphoric cancer; variant of Regan ALP;
Monoester Phosphohydrolase/ inhibited by L-leucine and
E.C.3.1.3.1 phenylalanine. (56°C for 10-
 A nonspecific enzyme capable of 15mins.)
reacting with many different substrates
 It functions to liberate inorganic
phosphate from an organic phosphate
ALKALINE PHOSPHATASE METHODS METHODS SUBSTRATE END PRODUCTS
1. Bodansky
1) ELECTROPHORESIS Beta- glyceroPO4 InorganicPO4 +
2. Shinowara
 (+) Liver ALP – most anodal ← Bone 3. Jones Glycerol
ALP ← Placental ALP ←Intestinal 4. Reinhart
ALP – least anodal (-) 5. King and Phenylphosphate Phenol
Armstrong
2) HEAT FRACTIINATION/STABILITY TEST 6.Bessy, Lowry, P-nitro phenyl P-nitrophenol or
 It is performed at 56°C for 10-15 and Brock PO4 yellow
minutes 7. Bowers and (PNPP) nitrophenoxide
McComb ion
(▲ Stable) Regan ← Placenta ←
8. Huggins and Phenolphthalein Phenolphthalein
Intestine ← Liver ← Bone (▲Labile) Talalay diPO4 red
9. Moss Alpha naphthol Alpha-naphtol
3) CHEMICAL INHIBITION TEST PO4
 Uses different concentrations of 10. Klein, Babson, Buffered Free
phenylalanine, synthetic urea and and Read phenolphthalein phenolphthalein
levamisole solutions PO4
o Phenylalanine- Inhibits Regan,
Placental and Intestinal ALP ALP- CLINICAL SIGNIFICANCE
o L-leucine- inhibits Nagao ALP
o Levamisole- inhibits Liver and  Total ALP levels are increased, major
Bone ALP liver fraction that is most frequently
o 3M urea- Inhibits bone ALP elevated--especially in obstructive
jaundice
4) BOWERS AND MC COMB (SZASZ  Conditions in which ALP is ELEVATED-
MODIFICATION)-PNPP (substrate) o Hepatitis
 Considered as the most specific o Bone cancer
method; IFCC recommended o Osteomalacia
method o Paget’s disease
 It is a continuous-monitoring o Biliary obstruction
technique which requires a pH o Hyperthyroidism
environment of 10.15 and should o Hyperparathyroidism
be read at 405nm o Myocardial infraction
o Pregnancy
 Conditions in which ALP is LOWERED-
o Hypophophatasia
P-NITROPHENYLPHOSPHATE + H2O ←ALP→ P- o Pernicious Anemia
NITROPHENOL + PHOSPHATE ION o Aplastic Anemia
o Achondroplasia
OTHER ALKALINE PHOSPHATASE METHODS
o Cretinism
(4°C) Low temperature = Increased ALP o Wilson’s disease
o Post-menopausal women
 o Cardio pulmonary bypass PROCEDURE, RESULT, & CALCULATION
(blood transfusion)
 Placental ALP- tumor marker in serum  Reagent Preparation and Stability:
in CSF foremost germ cell tumors; CSF o Procedure 1 with reagent start
o Reagents are ready for use,
levels occur placental ALP are diagnostic
value in differentiating whether tumor stable even after opening up to
in pineal body. expiry date. Storage at 2-8°C.
Contamination of reagents
ALP-REFERENCE RANGE must be avoided.
o Procedure with sample start
 ALP (total), (37°C) o Working reagents is stable for 4
weeks at 2-8°C 5 days at 15-
MALES-FEMALES 4-15 Y/O 54-369 U/L
25°C,one must be kept light
MALES 20-50 y/o 53-128 U/L protected.
 Specimen:
>/= 60 y/o 56-119 U/L
o Serum or Heparinized plasma
FEMALES 20-50 y/o 42-98 U/L o Avoid hemolysis
>/=60 y/o 63-141 U/L o Loss of activity within 7 days:
0% at 4°C; 10% at 20-25°C
ALP- EXPERIMENT
 ASSAY:
METHOD, PRINCIPLE, & MATERIALS o Wavelength: Hg 405nm (400-
420nm)
 METHOD USE: Bessey – Lowry - Brock o Optical path: 1 cm
 Alkaline Phosphatase Liquicolor, o Temperature: 25°C, 30°C, 37°C
Orthophosphoric Monoester o Measurement: Read against air
Phosphohydrolase (increasing absorbance)
 PRINCIPLE OF TEST:  Pipette 1 ml from bottle of substrate
into one bottle of buffer. Mix
thoroughly.
 Warm the reagent and the cuvette to
P-NITROPHENYLPHOSPHATE + H2O ←ALP→ the desired temperature. Temperature
PHOSPHATE + P-NITROPHENOL must be kept constant (+/- 0.5°C) for
duration of the test.
 MATERIALS:  Procedure 1 with Reagent start
 BUFFER
 Diethanolamine Buffer (pH 10.35 +/- Pipette into cuvette 25°C, 30°C, 37°C
0.2)-----------1.2 mol/L Sample 20ul
 Magnesium chloride--------------- Buffer 1000ul
 Mix, incubate 1 min. at the desired
0.625mmol/L
temperature
 SUBSTRATE
 P-nitrophenyl phosphate--------------- Substrate 250 ul
50mmol/L
  Mix, Read the absorbance after 1 min. ALP-EXPERIMENT-REFERENCE RANGE:
and the same time start the stopwatch.
Read the absorbance again exactly after Temperature 25°c u/i 30°c u/i 37°c u/i
1, 2, and 3 minutes.
 Procedure 2 with Start Women 40-190 19-232 64-306

Pipette into cuvette 25°C, 30°C, 37°C Men 50-190 61-232 80-306
Sample 20ul
Working reagent 1000ul
(previously prepared) Children up to 15 up to 400 up to 488 up to 644
 Mix, read the absorbance after 1 min. y/o
and at the same time start the Children up to 17 up to 300 up to 366 up to
stopwatch. Read absorbance again y/o 483
exactly 1, 2, and 3 minutes.
 RESULT.CALCULATION:
NOTE: during the reaction-nitrophenol is
 CALCULATION: from the readings,
produced. This substance is poisonous when
calculate the mean absorbance change
inhaled, swallowed or when absorbed through
per minute (A/min.)
the skin. It comes into contact with skin or
 Calculate the alkaline phosphatase
mucous membranes, wash thoroughly with
activity in the sample using the
water and consult a doctor
following factor:
VIDEO: SLIDE NUMBER 31 (ALP VIDEO-5:37)
U//l = ▲ A/min. x 3433 (procedure 1w/ reagent
2:02:00-2:08:17
start)
ACID PHOSPHATASE (ACP) E.C.3.1.3.2
U/l = ▲ A/min. x 2757 (procedure 2 w/ sample
start)  AKA: E.C. 3.1.3.2 or Orthophosphoric-
monoester phosphohydrolase
 Conversion factor from traditional units
 It catalyzes the same reaction made by
(U/I) in SI-units (kat/I)
ALP, except that it is active at pH 5.0
 1U/I = 16.67 x 10-3 ukat/l
 Major tissue sources: prostate (major
 1 ukat/l = 60 U/I4
source), RBC, platelets, liver and bone.
 ACP is classified based on acidic pH it
works at and based on source:
BASED ON PH
o PH 6.0- found in RBC
o PH 5.0- found in prostate,
spleen, and kidney
 BASED ON SOURCE (ISOENZYME)  RBC ACP inhibited by cupric ions and
formaldehyde
o Prostatic ACP- found in
 REFERENCE RANGE:
prostate gland, strongly
inhibited by dextrorotary TOTAL ACP: Male: 2.5-11.7 u/L
tartrate ions. Function is mainly Female: 0.3-9.2 u/L
to preserve spermatozoa and PROSTATIC ACP: Male:0.2-5.0 u/L
protects them from phagocytic Female0.0-0.8 u/L
activity of female genital tract. ACP METHODS
o Non-prostatic ACP (TR-ACP)-
METHODS SUBSTRATES END PRODUCTS
found in other tissues, cannot
be inhibited by tartrate but 1. GUTMAN AND Phenyl PO4 Inorganic PO4
inhibited by formaldehyde and GUTMAN
cupric ions. 2. SHINOWARA PNPP p-nitrophenol
 Unstable in ph 7.0 or > 7.0; temperature
> 37°C 3. BABSON, READ, Alpha napthyl PO4 Alpha-naphtol
 To stabilize enzyme activity, acidify AND PHILIPS
serum specimen in ph below 6.5
 ACP used in forensic clinical chemistry 4. ROY AND Thymolphthalein Free
in investigation of great cases. HILLMAN MonoPO4 thymolpthalein
 ISOENZYME: Things to remember:

ACP ISOENZYMES TISSUE SPECIFICITY  Separate red cell to the serum asap to
prevent the leakage of erythrocyte and
Band 1 Prostate Platelet ACP
Band 2 Granulocyte  Serum activity is decreased within 1-2
Band 3 Plasma ( Platelets, hours if the sample is left at room
Erythrocytes and
temperature without the addition of
Monocytes)
preservatives. Results of increased ph
Band 4 Granulocytes
 Hemolysis should be avoided;
Band 5 Osteoclast
contamination from erythrocyte ACP;
 SUBSTRATES:
Low result of ACP due to improper
o Thymophthalein
anticoagulant
monosphosphate –quantitative
endpoint reaction ACP-CLINICAL SIGNIFICANCE
o A-naphthyl PO4 –continous
monitoring methods  Prostate cancer
 Prostatic ACP is inhibited by 20mM L-  Benign Prostatic Hypertrophy
tartrate ions while 1mM cupric sulfate  Paget Disease
and 2% formaldehyde ions inhibit red  Breast Cancer with bone metastases
cell ACP  Gaucher Disease
 Platelet Damage
  Idiopathic Thrombocytopenia 1-naphthol + FRTR-salt (ACP)→ azo dye

METHOD, PRINCIPLE, & MATERIALS

 Vaginal washings of victim can be used
for seminal fluid examination, for  MATERIALS:
detection of ACP within 4 days.  BUFFER: Citrate Buffer (pH 5.2)-----
OTHER CAUSES OF INCREASE SERUM ACP 100mmol/l
 SUBSTRATE:
 Urinary tract obstruction 1-naphthyl phosphate-------19umol/l
 Acute urinary retention FRTR-salt------------------------2umol/l
 Extensive prostatic massage  STABILISER: Acetic Acid ------0.7mil
 Prostatic inflammation  CALIBRATOR: AUTOCAL (for 5 ml
 Prostatic manipulations- immunobiopsy Lyophilised calibrator for HUMAN
and cystoscopy Clinical Chemistry reagents)
 CONTROL: Huma Trol N; Huma Trol P;
INCREASED ACP (METASTATIC BONE or SERODUS; SERODUS plus (Lyophilised
INVOLVEMENT): control serum for Human clinical
 Prostatic carcinoma Chemistry reagents
 Breast, Lungs, and Thyroid carcinoma
 Gaucher’s Disease PROCEDURE, RESULT, & CALCULATION
 Niemann Pick Disease
 REAGENT PREPARATION:
ACP- EXPERIMENT o Dissolve the contents of 1
bottle Substrate with exactly
 METHOD USE: Colorimetric Humazym
2ml of Buffer. (Mark label with
M-Test (Babson and Reed)
date of preparation.)
o 1-naphthyl phosphate is
hydrolyzed by acid phosphatase
 SPECIMEN: SERUM (No plasma)
to phosphate and 1-naphthol,
which is converted with FRTR-
 SAMPLE CONSIDERATIONS:
salt to an azo dye. The increase
of absorbance at 405nm is  PROCEDURE, RESULT, & CALCULATION
proportional to the total acid o Avoid hemolytic and icteric sera
phosphatase activity in the o Separate serum from RBC after
sample. centrifugation
o Analyze immediately (within 1-2
hours)
 REACTION PRINCIPLE:

 SAMPLE PREPARATION:
o Stabilize samples, AUTOCAL,
and Controls by addition of one
1-naphthyl phosphate + H2O (ACP)→phosphate
drop of stabiliser to 1 ml sample
+ 1napthol
immediately after
 separation/reconstitution.  REFERENCFE VALUE:
Samples will remain stable for 3
days at 2-8°C. 21 hours at 15- TOTAL ACID PHOSPHATASE
Assay temperature 37°C
25°C (room temperature)
Men up to (U/I) 6.6
 ASSAY:
Women up to (U/I) 5.5
o Wavelength: Hg 405nm
o Optical path: 1cm
o Temperature: 37°C (ACP ASPARTATE AMINOTRANSFERASE (AST)
active @ this temp.) E.C.2.6.1.1
o Measurement: against air ( ↑
 AKA: E.C. 2.6.1.1 OR L-Aspartate 2-
absorbance)
Oxoglutarate Aminotransferase or
 Warm working reagent and cuvettes up
aspartate transaminase. Formerly
to desired temperature (37°C).
known as SGOT (serum glutamic-
Temperature must be kept constant
oxaloacetic transaminase)
(+/- 0.5°C) for the duration of the test.
 Is involved in the transfer of an amino
 SEMI MICRO METHOD
group between aspartate and a-keto
 Pipette into Cuvette
acids with the formation of
SAMPLE 100ul oxaloacetate and glutamate
WORKING REAGENT 1000ul  It has 2 isoenzyme fractions, cytoplasm,
and mitochondrial ASTs – the
 Mix, read the absorbance A1 after 5 cytoplasmic isoenzyme is the
minutes and start the stopwatch at the predominant form in serum
same time.  Major tissue source: cardiac tissue,
 Read the absorbance A2 exactly after 3 liver, and skeletal muscle
minutes at 37°C  Other sources: kidney, pancreas, and
 A2 – a1 = a RBC
 REFERENCE VALUE: 5-37 U/L
 CALCULATION:  ISOENZYYME FRACTIONS:
 Calculate the total acid phosphatase o Cytoplasmic AST- predominant
activity in the sample using the form in serum
following factor. o Mitochondrial AST- 5-10% of
total AST
U/I 37°C  METHODS:
Total Acid 248 o Karmen Method/Couple
Phosphatase (A)
Enzyme assay
(multiply)
o Reitman and Frankei
 CONVERSION:
o 1 U/I = 16.67 X 10-3 UKAT/L
o 1 UKAT = 60 U/I
  Coupled enzyme assay (Karmen  AST is also used in monitoring therapy
Method) pH level 7.5 with wavelength with potentially hepatotoxic drugs, a
340nm result 3x of upper border of normal
o It uses malate dehydrogenase sensation of therapy.
(MD) and monitors the change
in absorbance at 340 nm
ASPARTATE AMONITRANSFERASE-
EXPERIMENT

METHOD, PRINCIPLE, & MATERIALS

Aspartate + a-ketoglutarate ←AST→  METHOD USE: LiquiUV Test (KARMEN

Oxaloacetate + Glutamate METHOD)
o Kinetic method for the
Oxaloacetate + NADH + H4 ←MD→ Malate + determination of AST activity
NAD+ according to the
reccomendations of the Expert
---Karmen method uses the principle AST
Panel of the ifcc (Int4ernational
catalyzes the transamination.
Federation of Clinical
o Reitman and Frankel is also used using Chemistry) without
dinitrophenylhydrazone (color pyridoxalphosphate activation.
developer) which produces blue color at
505nm due to diazonium salt coupled  REACTION PRINCIPLE
with keto acid (+ NaOH – color
intensifier)

AST- CLINICAL SIGNIFICANCE

 Viral Hepatitis (100x)

2-Oxoglutarate + L-aspartate ←GOT→ a L-
 Infectious Mononucleosis (20x)
glutamate + oxaloacetate
 Cirrhosis (4x)
 Skeletal Muscle Disorder (8x) Oxaloacetate + NADH + H+ ←MDH→ L-malate +
 Pulmonary Embolism (3x) NAD
 Acute Pancreatitis
 MATERIALS
 Myocardial Infarction
 BUFFER/ ENZYME REAGENT
 Acute Myocardial Infarction (AMI)- AST
o TRIS Buffer (pH 7.9) 100mmol/l
levels begin to rise 6-8 hours, peak after
o L-aspartate 300mmol/l
24 hours, and normalize within 5 days;
o LDH 1.13kU/I
at the onset of Myocardial infarction
o MDH 0.75kU/I
AST level increased
o Sodium Azide 0.095%
 Evaluation of myocardial infarction,
 SUBSTRATE
hepatocellular disorders and skeletal
o 2-oxoglutarate- 60mmol/l
muscle involvement.
 o NADH - 0.9mmol/l  For a ▲ A/min within 0.06-0.08 (Hg 365
o Sodium Azide - 0.095% nm) or 0.12-0.16 (Hg 334nm, 340nm)
 Use only measurements from the first 2
 SPECIMEN: Serum, Heparinized plasma minutes for calculation (1minute
or EDTA plasma incubation, 2 min. measurements).
o Avoid hemolysis
o Loss of Activity within 3 days at U/I + ▲ A/min. SAMPLE START
Wavelength 25°C, 30°C 37°C
+4°C: 8%; at 20-25°C: 10%
Hg 334nm 971 1780
 Test tubes, cuvettes, parafilm,
340 nm 952 1745
micropipettes, pipet tips, centrifuge Hg 365 1765 3235
 Conversion factor from traditional
 ASSAY unit (U/I) in SI units (kat/I)
 Wavelength: Hg 365nm; 340nm; or Hg o 1U/I = 16.67 x 10 ^-3 ukat/l
334nm o 1ukat = 60 U/L
 Optical path: 1cm
 Temperature: 25°C, 30°C, 37°C  Note: If the absorbance change per
minute or activity exceeds: dilute
PROCEDURE, RESULT, & CALCULATION
0.1 ml of the sample with 0.9ml
PROCEDURE physiological saline (0.9%) and
repeat the assay using this dilution.
 Procedure with sample start Multiply the result by 10.
 Reagent Preparation:
 Pipette 2 ml from bottle of Substrate VIDEO: SLIDE NUMBER 49 (AST VIDEO -4:50)
into a Buffer. Mix thoroughly 2:44:45-2:49:17
 Stable for 4 weeks at 2-8°C
ALANINE AMINO TRANSFERASE (ALT) E.C.2.6.2
 Warm the reagents and the cuvettes to
the desired temperature. Temperature  AKA. E.C 2.6.1.2 or L-alanine: 2-
must be kept constant for the duration oxoglutarate aminotransferase or
of the test alanine transaminase.. Formerly known
as SGPT (serum glutamic-pyruvic
Pipette into 25°C, 30°C 37°C transaminase)
cuvettes
 It has enzymatic activity similar to AST
Sample 200ul 100ul
Working reagent 1000ul 1000ul  It catalyzes the transfer of an amino
group from alanine to a-ketoglutarate
 Mix, read the absorbance after 1
with the formation of glutamate and
minute and at the same time start the
pyruvate
stop watch. Read the absorbance again
exactly 1, 2, and 3 minute.  The highest concentration is in the liver,
more liver-specific than AST
CALCULATION OF RESULTS  Major Tissue Source: Liver
 Other resources kidney, pancreas, RBC,
heart, skeletal muscles, lungs.
  Reference value: 6-37 U/L at 37°C
 Coupled Enzymatic reaction: using pH
ALT- INCREASED TRANSFERASES:
7.5; reading at 340nm
1. Toxic hepatitis
2. Acute Myocardial Infraction – AST
3. Wolff-Parkinson White Syndrome
4. Trichinosis – AST
Alanine + a-ketoglutarate ←ALT→ Pyruvate 6 5. Chronic Alcoholism
Glutamate 6. Dermatomyositis – AST
7. Hepatic Cancer
Pyruvate + NADH + H+ ←LD→ Lactate + NAD+
8. Reye’s syndrome
AST/GOT ALT/SGPT 9. Viral Hepatitis
10. Muscular Dystrophy – AST
Major Organ Heart Liver
11. Acute Pancreatic - AST
affected
Substrate Aspartic Alpha Alanine Alpha THINGS-TO-REMEMBER:
Ketoglutaric Acid Ketoglutaric Acid
End products Glutamic Acid + Glutamic Acid +  The highest elevations of
Oxoloacetic Acid Pyruvic Acid
transaminases is seen in acute
Color developer 2.4 DNPH 2.4 DNPH
hepatitis
Color intensifier 0.4N NaOH 0.4NaOH  Severe viral or toxic hepatitis may
Methods Reitman and Reitman and produce elevations of transaminases
Frankel Frankel up to 20x the normal limits.
 Moderate elevation of transaminases
in chronic hepatitis, hepatic cancer,
ALT- CLINICAL SIGNIFICANCE
and infection mononucleosis.
 Hepatocellular disorder (hepatitis and  End stage cirrhosis, AST and ALT level
cirrhosis) are not elevated but as low as massive
 Shock Liver tissue destruction
 Infectious Mononucleosis
ALANINE AMINOTRANSFERASE- EXPERIMENT
 Polymyositis
 Severe Pancreatitis METHOD, PRINCIPLE, & MATERIALS

Most important cause for increase  LiquiUV Test

transaminase activity is liver disease
METHOD:
If liver is affected both AST and ALT is increased
 Kinetic method for the determination of
ALT more liver specific ALT activity according to the
recommendations of the Expert panel
Alcoholic hepatitis, liver cirrhosis, and liver
of the IFCC (International Federation of
neoclasia, ↑ASP
Clinical Chemistry) without pyridoxal
phosphate activation
 REACTION PRINCIPLE RESULTS AND CALCULATIONS:

 For ▲ A/min. within 0.06-0.08 (Hg

365nm) or 0.12-0.16 (Hg
334nm/340nm) use only measurements
from the first 2 minutes
2-Oxoglutarate + L-alanine ←GPT→ L-glutamate  For calculations (1 minute incubation, 2
+ pyruvate minutes measurement)

Pyruvate + NADH + H+ ←LDH→ L-lactate + NAD U/L = ▲ a/min. x Sample start

MATERIALS:
Wavelength 25°C, 30°C 37°C
 BUFFER/ ENZYME REAGENT Hg 334nm 972 1780
o TRIS Buffer (pH7.4) 125mmol/l 340nm 952 1745
o L-alanine 625mmol/l Hg 365nm 1765 3235
 Conversion factor from traditional
o LDH 1.5kU/I
units (U/I) in SI-units (kat/I)
o Sodium azide 0.095%
1 U/I=16.67X10^-3ukat/I 1ukat/I = 60U/I
 SUBSTRATE
o 2- oxaglutarate 75mmol/l VIDEO: SLIDE NUMBER 49 (AST VIDEO -4:50)
o NADH 0.9mmol/l 2:44:45-2:49:17 – same lang ang AST at ALT
o Sodium Azide 0.095% process, watch mo ulet if u want, only the
difference is the substrate because diffuse
 SPECIMEN
 Note: if the absorbance change per
o Serum, heparinized plasma or EDTA
minute (A/min.) or the activity exceed,
plasma
dilute 0.1ml of the sample with 0.9ml
o Avoid Hemolysis
physiological saline (0.09%) and repeat
o Loss of activity within 3 days at 4°C:
the assay using this dilution. Multiple
10% at 20-25°C: 17%
the results by 10.

 Test tubes, cuvettes, micropipette, REFERENCE VALUE:

pipet tips, centrifuge
 Procedure with sample start Temperature 25°C 30°C 37°C IFCC*
Men up to 22 U/I 30 U/I 42 U/I 45 U/I
Pipette into 25°C, 30°C 37°C Women up to 17 U/I 23 U/I 32 U/I 34 U/I
cuvettes
Sample 200ul 100ul
Working reagent 1000ul 1000ul
 Mix, read the absorbance after 1
minute and at the same time, start the
stop watch. Read the absorbance again
exactly after 1, 2, and 3 minutes.
AMYLASE (AMS/AMY) E.C.3.2.1.1 peak at 24 hours, and normalize within
3-5 days.
 AKA: E.C. 3.2.1.1 OR alpha 1-4-glucan-4  In AP, Increased AMS blood levels are
glucohydrolase accompanied by increased urinary
 These enzymes catalyzes the hydrolysis excretion.
of 1,4-a-glucosidic linkages in  AMS in urine remains elevated for up to
polysaccharides. 7 days.
 The enzyme is small enough to pass  In acute renal failure in the absence of
through the glomerulus, and is the only AP (acute pancreatitis), increased
enzyme that is normally found in urine. plasma AMS is accompanied by
 It is the earliest pancreatic marker decreased urine AMS
 The magnitude of AMY levels does not
 Major Tissue Source acinar cells of the reflect the severity of pancreatic
pancreas and the salivary glands involvement, but higher levels increases
the probability of acute pancreatitis.
 Other Tissue Source: adipose tissue,  Biliary tract diseases such as
fallopian tubes, small intestine and cholecystitis cause up to 4x increase in
skeletal muscles serum P-AMY activity as a result of
Primary or Secondary pancreatic
 Isoenzymes: involvement.
1. Salivary amylase (ptyalin or S-  Hyperamylasemia also occurs in
type) - found in salivary glands neoplastic diseases.
2. Pancreatic amylase (amylopsin  Salivary gland inflammation (parotitis)
or P-type) – found in pancreas
due to mumps can also release AMS
 S1 – slowest salivary gland (near the into the circulation causing an elevated
cathode) serum AMS.
o S bands are strongly
inhibited by a protein Things-to-remember in amylase detection:
isolated from wheat
 Heparin may inhibit the activity of
 P2
amylase. (using some, but not all,
 P3 – increased in pancreatitis; most
methods)
predominant pancreatic AMS
 Triglycerides may inhibit serum
isoenzyme in AP
amylase activity
REFERENCE RANGE:  Sample is w/ high activity of
amylase, should be diluted with
 Adult: 23-85 U/L sodium chloride to prevent
CLINICAL SIGNIFICANCE: inactivation.
 Many endogenous inhibitors of
 In acute pancreatitis (AP), AMs levels AMS, such as wheat germ are
rise 2-12 hours after onset of attack, present in serum.
  The administration of morphine and 5-glucose-6-phosphates + 5 NAD ←G-6-PD→
other opiates for pain relief blood 5,6-phosphogluconolactone + 5 NADH
sampling will lead to falsely
Notes-to- remember:
elevated serum AMS levels.

SUBSTRATE FOR ALL THE METHODS: STARCH  Salivary amylase is inhibited by

wheat germ lectin.
1. SACCHAROGENIC  Enzyme molecules are too large to
 It is the classic reference method pass through the healthy
expressed in Somogyi units glomerulus of the kidneys, thus,
 It measures the amount of reducing urinary excretion is not a major
sugars produced by the hydrolysis route for elimination except
of starch by the usual glucose amylase; Elimination of amylase is
methods. not in urinary excretion

AMYLASE- EXPERIMENT
2. AMYLOCLASTIC
 It measures amylase activity by METHOD, PRINCIPLE, & MATERIALS
following the decreases in substrate
concentration (degradation of METHOD: Caraway Method, Modified
starch)
MATERIALS/RAGENTS:

3. CHROMOGENIC  Amylase Substrate

 It measures amylase activity by the  Test Sera/N – AB controls
increase in color intensity of the  Color developer
soluble dye-substrate solution  Deionized water
produced in the reaction.  Test tubes, cuvettes, paraffin,
micropipette, pipet tips, centrifuge
4. COUPLED- ENZYME
 It measures amylase activity by a PROCEDURE, RESULT, & CALCULATION
continuous-monitoring technique
PROCEDURE:

Reagents Reagent Blanks Sample/Controls

AMS substrate 250ul 250ul
Test Sera/N-AB ---- 10ul
Maltospentose ←AMS→ Maltrotriose + Maltose controls
 Incubate test and control for 5 minutes
Maltotriose + Maltose ←glucosidase→ 5- at 37°C
glucose
Color developer 250ul 250ul
5-glucose + 5 ATP ←hexokinnase→ 5-glucose-6- De-ionized water 2000ul 2000ul
phosphates + 5 ADP
  Invert to mix and read % T/absorbance  Minor Source: Gastric mucosa,
at 610/640nm against water blank. Intestinal mucosa, and adipose tissue
(dH2O)  Hemolysis should be avoided because
haemoglobin inhibits lipase activity
RESUTLTS/CALCULATION OF AMYLASE
ACTIVITY: (read by: absorbance mode) CLINICAL SIGNIFICANCE:

 Factor use: 750 ul  In acute pancreatitis (AP), LPS levels rise

6hours after onset of attack, peak at 24
hours, remains elevated for 7 days, and
normalize in 8-14 days
 In chronic pancreatitis, acinar cell
MINUS THEN DIVIDED degradation occurs resulting in loss of
amylase and lipase production
 Calculate the given readings using the
formula and interpret the value REFERENCE VALUE:
obtained.
 0-0.1 U/ml
Blank Abs = 0.495
Things-to-Remember in Lipase degradation:
Sample Abs = 0.420
 It uses olive oil as the substrate
 For Statfax: Use absorbance mode at because other esterase can hydrolyze
600nm against water blank (dH2O) TAG and synthetic diglycerides
 NORMAL RANGE: 60-180 U/I  Addition of colipase (protein secreted
by the pancreas) and bile salts will
VIDEO: SLIDE NUMBER 67 (AMS/AMY VIDEO
make assay more sensitive an specific
3:42 )3:18:20-3:21:50
for AP detection.
LIPASE (LPS/LIP)  Hemoglobin inhibits the activity of LPs
leading to falsely low values.
 Is an enzyme that hydrolyzes the ester  Triolein (more pure form of TAG) is
linkages of fats to produce alcohol and used also as a substrate for LPS assay
fatty acid
 It catalyzes partial hydrolysis of dietary LIPASE –EXPERIMENT
TAG in the intestines to the 2-
METHOD, PRINCIPLE, & MATERIALS
monoglyceride intermediate, w/ the
production of long chain fatty acids. METHOD:
 It is the most specific pancreatic
1. CHERRY CRANDAL (reference method)
marker – secreted exclusively in the
pancreas; not affected by renal  Principle: Hydrolysis of olive oil after
disorders. incubation for 20 hours at 37 °C and
titration of fatty acids using NaOH
 Major Source: Pancreas
 Substrate: 50% olive oil
 End Product: Fatty acids
 MATERIALS:

BUFFER:

Triglycerides (olive oil) + 2 H2O ←LPS→ 2-  Goods Buffer pH8.0

monoglyceride + 2 fatty acids  Taurodesoxysholate 1.0mmol/l
 Desoxycholate 1.0mmol/l
2. TIETZ AND FLEREC
 Calcium ions 1.0mmol/l
3. PEROXIDASE COUPLING –most
 Colipase 2mg/l
commonly used method; does not use
50% olive oil  Sodium Azide 0.095%

METHOD USE: Enzymatic Colorimetric Test for SUBSTRATE/SUBSTRATE REAGENT:

the Quantitative Determination of Lipase  Tartrate buffer pH 4.0
REACTION PRINCIPLE:  Lipase Substrate 0.1mmol/l
 Sodium azide 0.095%
 Lipase catalyses the reaction
 Synthetically produce lipase substrate CALIBRATOR:
is added to micro emulsion w/c is
 AUTOCAL/SERODS
specifically split by lipase in the
presence of colipase and bile salts. MATERIALS:
Combination of Colipase and Bile salts
ensure the specific determination of  Test tubes, cuvettes, parafilm,
pancreatic lipase w/o any reaction due micropipettes, micropipette tips,
to lipolitic enzymes/esterase centrifuge

SPECIMEN: SERUM OR HEPARINIZED PLASMA

STABILITY: 7 days at 20-25°C; 3 weeks at 4-8°C;

1 year at -20°C
1,2-0 dilauryl-rac-glycero-3-glutaric acid – 196-  Discard contaminated samples
methylresorufin) ester ←LIPASE/COLIPASE→  Reagents are ready for use and stable
1,2-0-dilauryl-rac-glycerin + Glutaric Acid – (6- until expiration date stated on the label
methylresorufin) ester when stores at 4-8°C.
Glutaric Acid –(6-methylresorufin) ester 2  Avoid freezing and contamination.
←spontaneous degradation→ Glutaric Acid  Mix at room temperature before use.
+Methylresorufin
PROCEDURE, RESULT, & CALCULATION
 End color red PROCEDURE:

 Pipetting scheme

Pipette into Blank Sample/Calibrato

 cuvettes r Precipitation may also appear on
Calibrator/Samples --- 20ul storage, especially if stored lower than
the recommended temperature, it does
Dist. Water 20ul ---
not affect the analysis. It just necessary
Buffer 1000ul 1000ul to slightly rotate the vial to resuspend
before the analysis.
 Mix carefully (do not shake, incubate
3. Sodium azide is a preservative so extra
for 5 minutes Start reaction by adding
careful not to swallow. Avoid in contact
substrate
to skin and the mucous membranes.
Substrate 250ul 250ul
VIDEO: SLIDE NUMBER 73 (LPS/LIP
 Mix, incubate for 2 minutes at 37°C,
VIDEO )3:18:20-3:21:50
read absorbance again and start stop
watch. LACTATE DEHYDROGENASE E.C. 1.1.1.27
 After exactly 1 and 2 minutes Read
absorbance again and then calculate  AKA E.C. 1.1.1.27 or L-lactate; NAD
A/min. oxidoreductase
 It is a n enzyme that catalyzes the
RESULT CALCULATION: interconversion of lactic and pyruvic
acid
 it is a zinc-containing enzyme that is
part of the glycolytic pathway and is
found in virtually all cells in the body.
A/min = [ A/min sample or calibrator ] – [ A/min blank ]
 It is a hydrogen-transfer enzyme that
Lipase [ U/ I ] = A/min (sample) x concentration calibrator uses the coenzyme nicotinamide
[ U/I ] dinucleotide (NAD+)
 It is a tetrameric molecule containing
Reference value: < 60 U/I four subunits of two possible forms (H
heart and M muscle)
REFERENCE VALUE:
 In plasma, the majority of LD comes
 </= 60 U/I from breakdown of erythrocytes and
platelets, with varying contributions
IMPORTANT PROCEDURAL NOTES: from other organs
1. As many other clinical chemistry  Tissue Source: heart, RBC’s kidneys (LD-
reagents, specially reagents for 1 & LD-2); lungs, pancreas, spleen (LD-
determination of triglycerides, HDL – 3); skeletal muscles, liver, intestine(LD-4
and LDL – cholesterol contain lipase or & LD-5)
high concentrations of detergents carry  Reference value: 100-225 U/L (Forward
over should be avoided by clearing the reaction) ; 80-280 U/L (Reverse
glassware thoroughly. reaction)
2. SUB is a turbid orange colored micro-
emulsion, if it becomes red discard,
LD ISOENZTME as a percentage of total LD: disorders. Include megaloblastic
anemia
LD-1 (HHHH or H4) = 17-27%
 In AMI, LD levels begin to rise
LD-2 (HHHM or H3M = 27-37% within 12-24 hours, peak levels
within 48-72 hours and remains
LD-3 (HHMM or H2M2) = 18-25% elevated for 10-14 days.
 Hepatic carcinoma and toxic
LD-4 (HMMM or HM3) = 3-8%
hepatitis will have 10-fold
LD-5 (MMMM or M4) = 0-5% increased.
 Viral hepatitis and cirrhosis would
 RBCs and cardiac tissues contain high give LD slightly increased values (2-
levels of LD-1 3x URL
 LD-1 is relatively abundant in cardiac  LD-1 > LD-2 also known as “flipped
muscle, whereas LD-5 is more abundant pattern” is seen in myocardial infarction
in skeletal muscle and hemolytic anemia
 LD-1 is not found in the skeletal muscles  LD-2, Ld-3, and LD-4 = LD cancer
and Liver; LD-2 is never found in the markers (predominantly LD-30; acute
skeletal muscles leukemia, germ cell tumors and lung
 LD-2 is the majority isoenzyme in the cancers
sera of healthy persons  LD-5 is moderately increased in acute
 LD-2 is greater the LD-1 is seen in viral hepatitis, and cirrhosis and
healthy sera. markedly increased in hepatic
 LD-5 has undetectable level in heart, carcinoma and toxic hepatitis.
RBCs and renal cortex.
 LD-6 represents the alcohol INCREASED LDH:
dehydrogenase enzyme, 6th band in
1) Anemias- pernicious, hemolytic,
electrophoresis; elevated in drug
megaloblastic
hepatoxicity and obstructive jaundice;
2) Myocardial Infarction
it is responsible for the metabolic
3) Leukemia
conversion of methanol and ethylene
4) Renal infarction
glycol to toxic compounds; present in
5) Hepatitis and Hepatic cancer
patients with arteriosclerotic failure.
6) Muscular dystrophy
 Another form of LD composed of four C
7) Delirium tremens
subunits in spermatozoa and in semen
8) Malignancy
but has never been detected in serum,
9) Pneumocystis jerovecii pneumonia
even in individuals with seminoma
METHOD:
CLINICAL SIGNIFICANCE:
 Lactate is a more specific substrate
 Highest serum levels are seen in
compared to pyruvate.
pernicious anemia and hemolytic
 LD-1 prefers forward reaction, whereas
LD-5 prefers the revers reaction.
  LD is stable at room temperature for 48  Modified method base on the
hours. recommendations of the SCE
1. WACKER METHOD (FORWARD/DIRECT (Scandinavian Committee on Enzyme)
REACTION) – reaction is at pH 8.8
REACTION PRINCIPLE OF THE TEST
Ps. Sinasabi ni ma’am sa recording WRECKER
pero kay rodriguez WACKER desisyon ka na
lang ano gusto mo. – ynor qt-

 It is the most commonly used Pyruvate + NADH + H+ < ----LDH------> Lactate +

method because it produces a NAD+
positive rate (NADH) and not
affected by product inhibition MATERIALS/REAGENTS
 Most widely method;
BUFFER:
 less contamination in NAD and
inhibiting products.  TRIS Buffer (pH 7.35) 62.5mmol/l
 Pyruvate 1.5mmol/l
 Sodium Azide 0.095%
Lactate + NAD ----LD--→ Pyruvate + NADH @
SUBSTRATE:
340nm

2. WORBLEUSKI LA DUE  NADH 0.75mmol/l

(REVERSE/INDIRECT REACTION)-  Sodium Azide 0.095%
reaction is at pH 7.2
MATERIALS:
 It is about 2x faster as the
forward reaction.  Test tubes, cuvettes, parafilm,
 It is the preferred method for micropipettes, pipet tips, centrifuge
dry slide technology
REAGENT PREPARATION
 It uses a less costly cofactor and
it has a smaller specimen  Pipette 2 ml from bottle Substrate into
volume requirement one bottle Buffer. Mix thoroughly, the
working reagent is stable at 2-8°C for 3
weeks and must be protected from
light.
Pyruvate + NADH -----LD---→ Lactate + NAD
SPECIMEN: Serum, Heparinized or EDTA plasma
LACTATE DEHYDROGENASE- EXPERIMENT
 Avoid Hemolysis
METHOD, PRINCIPLE, & MATERIALS  Loss activity within 3 days at 8% at +4°C

LiquiUV Test SPECIMEN CONSIDERATION:

METHOD USE: WORBLEWSKI LAUDE

  Erythrocytes contains LD contains LD 1 U/l = 16.67 x 10-3 ukat/l ; 1 ukat/l = 60U/l
concentration 100-150 time that found
in serum, thus immediate separation is  Factor to convert results to the new
advised. Store at 25°C and examined IFCC recommended method:
within 24 hours after collection. U/l (LDH SCE ) x 0.4796 = U/l ( LDH IFCC)
ASSAY: VIDEO: SLIDE NUMBER 84 (LD VIDEO 4:04)
PART 2 15:51-21:37
 WAVELENGTH: Hg 334nm; 340nm;
Hg365nm CREATININE KINASE E.C. 2.7.3.2
 OPTICAL PATH: 1cm
 TEMPERATURE: 25°C, 30°C, 37°C  AKA E.C.2.7.3.2. or ATP creatine, N-
 MEASUREMENT: against air phosphotransferase
(decreasing absorbance)  it catalyzes the transfer of transfer of a
phosphate group between creatine
PROCEDURE, RESULT, & CALCULATION phosphate and adenosine diphosphate
 It is involves in the storage of high-
PROCEDURE:
energy creatinine PO4 in the muscles
Pipette into cuvettes 25°C, 30°C, 37°C  It is a dimeric molecule with small
molecular sixe, composed of a pair of
Sample 20 ul 10 ul two different monomers called M and B
Working Reagents 1000 ul 1000 ul  It is found in small amounts throughout
 Mix and Read the absorbance after 1 the body, but is found in high
minute and at the same time start the concentrations only in muscle and
stop watch. Read the absorbance again brain, although CK from brain virtually
exactly after 1, 2, and 3 minutes never crosses the blood-brain barrier to
reach plasma.
RESULT & CALCULATION  Major Tissue Sources: brain tissue,
smooth and skeletal muscles and
 Using the absorbance readings,
cardiac muscles
calculate the mean absorbance change
 Reference Values: Male= 15-160 U/L;
per minute (a/min.)
Female 15-130 U/L; CK-MB= <6% of
 Calculate the LDH activity in the sample
total CK
by multiplying A/min. using the
 CK is released due to hypoxia and other
following factors.
muscle injuries this a sensitive but not
Hg 334nm 340nm Hg 365nm
specific for MI
U/l(250C,300C)= 8250 8095 15000
A/min. x  Optimal pH both forward and reverse is
U/l ( 370C) = 16345 16030 29705 9 and 6.7; Magnesium is an obligate
A/min. x activator of CK, however excessive
amount of Magnesium can also act as
 Conversion factor of the traditional inhibitor.
units (U/I) in SI units (ukat/I)
 Other metal ions; manganese, calcium,  Direct muscle trauma, as seen in
zinc and copper. contact sports, surgery, strenuous
exercise, and intramuscular
ISOENZYMES: injections, are common causes of
mild elevations of serum CK ( up to
 CK-BB or CK-1 – Found primarily in
about 5-6 times reference limits)
brain, GI tract muscle and urinary
 Intramuscular injections are known
bladder muscle
to increase CK (<5x URL)
 CK-MB or CK-2 - found primarily in
 Bedridden patients may have
heart and smooth muscles
decreased CK activity
 CK-MM or CK-3 – found in skeletal
muscles, and heart , found in
smooth muscles in small amounts OTHER FORMS OF CK INCLUDES THE MACRO-
 CK-MT – located at chromosome 15 CK IT EXIST IN 2 TYPES
constitutes 15% of total heart CK
activity 1) MACRO-CK TYPE 1 – complex of CK-
BB and non-pathologic origin but
 CK-BB- Most Anodal; BRAIN TYPE can interfere with other CK tests
 CK-MB- myocardium (20%); HYBRID 2) MACRO CK TYPE 2 – found in
TYPE severely ill with malignancy or liver
 CK-MM - least anodal, skeletal and disease. Detected by abnormally
smooth muscles (major 94-100%); migrating bands towards the
MUSCLE TYPE cathode.
 CK-1 is the most anodal and labile
CLINICAL SIGNIFICANCE:
enzyme; CK-3 is the least anodal
(cathode)  It is very sensitive indicator of acute
 CK-BB is the dominant isoenzyme myocardial infraction (AMI) and
of CK found in brain, intestine, and Duchenne disorder.
smooth muscle  Highest elevation of total CK is seen in
 Serum of adults rarely contains CK- Duchenne’s muscular dystrophy (50x
BB of brain due to its high URL)
molecular size; it may be normally  CK-MB is found mainly in myocardial
present in neonatal sera. tissue – it is used as a serodiagnostic
 Cardiac tissues contains significant test for AMI.
amount of CK-MB (20%) –  Following AMI, the CK-MB levels begin
myocardium is the only tissue from to rise within 4-8 hours, peak at 12-24
which CK-MB enters the serum in hours and normalize within 48-72
significant quantities. hours.
 Physically well-trained individuals  CK-MB is not elevated in angina
tend to have elevated baseline  Injury both cardiac and skeletal muscle
levels of total CK accounts for the majority of CK-MM
elevations.
  Total CKis markedly elevated after TROPONIN T 3-12 hrs. 12-48 hrs 5-14 dys
trauma to skeletal muscle from crush
LDH 12-24 hrs. 24-48 hrs 10-14 dys
injury, convulsions, tatany, surgical
AST
incisions or intra muscular injections. 12-24 hrs. 24-48 hrs 10-14 dys

METHODS:
NOTES-TO REMEMBER:
1) TANZER-GILBARG ASSAY
(FORWARD/DIRECT METHOD) – pH  Adenylate Kinase (AK ) released after red
9.0;340nm cell lysis interferes with CK assay
particularly with hemolysis of > 320 mg/L
 Liver cells and RBC do not contain CK
 To increase both the sensitivity and the
specificity of CK-MB in the diagnosis of
acute AMI, it has been found necessary to
perform serial determinations of MB
Creatine + ATP --CPK--→ Creatine PO4 + ADP fraction (at 3- to 4- hour intervals over a 12-
to 16 hour period) that show a progressive
ADP + phosphoenolpyruvate ←--PK--→ Pyruvate + ATP rise that reaches a peak, followed by a fall
to low levels.
Pruvate + NADH ←- LD-→ Lactate + NAD
 Adenosine monophosphate (AMP) is added
2) OLIVER-ROSALKI to the reverse method to inhibit AK which
(REVERSE/INDIRECT METHOD) may be present in the serum from
hemolysis – AK hydrolyzes ADP
 N-acetylcysteine is added to CK reagent to
activate the enzyme (aside from Mg+2) and
partially reversed the inhibition of oxidized
sulfhydryl groups.
 Imidazole serves as a buffer; urate and
Creatine PO4 + ADP ←- CPK--→ Creatine + ATP cysteine are potent CK inhibitors.
 CK is light and pH sensitive; it is also lost
ATP + glucose ←- *HK--→ ADP + glucose-6-PO4
with excessive storage.
Glucose-6-PO4+NADP ←-G6PD-→ ^-phosphogluconate +  Cleland’s reagent and glutathione – partially
NADPH restore lost activity of CK
 CK mass units assay are more sensitive than
AMI MARKERS: electrophoresis, but electrophoresis is still
the reference method for CK
AMI ONSET PEAKS AT NORMALIZE
MARKER AT
METHOD, PRINCIPLE, & MATERIALS
CK-MB 3-12 hrs. 18-24 hrs 48-72 hrs
MYOGLOBIN 2-3 hrs. 9-12 hrs 24 hrs CK-MB NAC ACTIVATED

TROPONIN I 3-12hrs. 24 hrs 5-10 dys METHOD:

  IMMUNOINHIBITION METHOD  Test tubes, parafilm, cuvettes,
(OLIVER-ROSALKI) micropipettes, pipet tips, centrifuge.

REACTION PRINCIPLE TEST:

PROCEDURE, RESULT, & CALCULATION

PROCEDURE:

Creatine phosphate + ADP <--CK------> Creatine  Reagent Preparation: Reconstitute one

+ ATP vial ENZyme with exactly 3ml Buffer
Swirl gently and incubate for 5 minutes
ATP + d-GLUCOSE <---HK---------> ADP + D-
at room temperature prior to use. This
glucose-6-phosphate (G6P) working solution is stable for 5 days at
G6P + NADP <----G6P-DH------> 6-phospho-D- 2-8°C
glucono-lactone + NADPH + H+ SPECIMEN: Serum, heparinized plasma or EDTA
MATERIALS/REAGENTS plasma

BUFFER:  Loss of activity within 1 day at 2-8°C <

10%
 Imidazole buffer (pH 6.7) 0.10mmol/l
 Glucose 20mmol/l ASSAY:
 Mg. acetate 110mmol/l  WAVELENGTH: Hg 334nm; 340 nm; Hg
 EDTA 2.00mmol/l 365nm
ENZYME: enzyme and antibody reagent  OPTICAL PATH: 1cm
(Lyophilized) 1 VIAL contains  TEMPERATURE: 25°C, 30°C, 37°C
 MEASUREMENT: against air (increasing
 ADP 2.00mmol/l absorbance)
 AMP 2.00mmol/l
 Diadenosine IMPORTANT PROCEDURAL NOTES:
pentaphosphate 10.00mol/l  Prior to determining CKMB activity. It is
recommended to measure total
 HK > 2.50 U/ml Activity. It is recommended to measure
 NADP 2.00mmol/l total CK activated method (CK-NAC
 G6P-DH >1.50U/ML LIQUIUV in order to ensure correct
 N-Acetylcysteine 20mmol/l diagnostic interpretation of he results
 Creatinine Phosphate 30mmol/l  Warm reagents and cuvettes to the
 Polyconal Antibody to CK-M subunit desired temperature. Temperature be
 CK-MB controls kept constant for the duration of the
 CK-MB calibrators test:

MATERIALS: Pipette into cuvettes

 Sample/CAL 40u/l method can be employed. Only control
Working reagent 1000il sera with human CK can be used
 Mix and incubate at the desired
temperature for 10 minutes VIDEO: SLIDE NUMBER 97 (CK VIDEO 4:57)
 Read the absorbance A, Exactly PART 2 43:00-48:35
after 5 minutes read the
absorbance A.

RESULTS AND CALCULATION:

 Calculate the absorbance change per

minute (A/min.) according to A/min. =
(A2-A1):5
 The CK-MB activity is calculated using
the following factors:

CK-MB ACTIVITY (U/I)

SEMI-MICRO
Hg 334nm 8414 x A/min
340nm 8254 x A/min.
Hg 365nm 14858 x A/min.
 Conversion factor units (U/I) in SI units
(kat/I)

1 U/l = 16.67 x 10-3 ukat/l ; 1ukat/l = 60 U/l

REFERENCE RANGE: MYO CARDIAL

INFARCTION (MI)

 The likelihood of myocardial damage is

high if the following 3 criteria are met

25°C 30°C 37°C

1.Total CK
MEN >80U/I >130U/I >195U/I
WOMEN >70U/I >110U/I >170U/I
2. CK-MB >10U/L >16U/L >25U/I
3.CK-MB activity Ranging between 6% and 25%
of the total CK activity

QUALITY CONTROL

 All control sera with CK-MB values

determined by this method by this
CC2 MODULE 2-LABORATORY central role in maintaining the
normal distribution of water and
ELECTROLYTES osmotic pressure in the ECF
 Electrolytes are ions that carry an compartments
electric charge.  It is also known as “natrium”
 Classified as anions and cations.  most abundant cation in the ECF
 Anions have a negative (-) charge and representing 90% of all extracellular
move toward the anode (+). cation.
 Cations migrate toward the cathode (-)  Na+ determines the osmolality of
because cations are positive (+) charge. plasma
 Electrolytes are essential component of  Na+ ATP ion pump moves 3 sodium
numerous processes: ions out of cell exchange to 2
o Volume and osmotic regulation potassium ions moving in to cell as
o Myocardial rhythm and ATP is converted as ADP.
contractility  Regulations for Sodium per plasma
o Cofactors in enzyme regulation sodium concentrations depend
o Regulation of adenosine greatly in the intake and excretion
triphosphatase (Atpase) ION of water.
PUMPS
3 process of sodium regulation:
o ACID-BASE BALANCE
o BLOOD COAGULATION 1. Intake of water in response to thirst can
o NUEROMUSCULAR be stimulated or suppressed by plasma
EXCITABILITY osmolality.
o PRODUCTION AND USE OF ATP 2. Excretion of water, it is greatly affected
FROM GLUCOSE by AVP- which release in the response
 Extracellular fluid (ECF) 1/3 total body to changes to either volume or
water(16 liters) osmolality.
 Intracellular fluid(ICF) 2/3 of total body 3. Blood volume status, affects Na+
water; fluid inside the cell (24 liters) secretion through aldosterone,
 40-75% is the average water content of angiotensin II and atrial natriuretic
the human body (advanced age and peptide (ANP)
obesity – decrease values
HORMONES AFFECTING SODIUM LEVELS:
 Normal plasma is composed of 93%
water and 7% solutes (glucose lipids, 1) ALDOSTERONE
proteins, NPN, amino acids, and ions)  It promotes absorption of
 Water content of plasma is 12% higher sodium in the distal tubule.
than that whole bloods  It promotes sodium retention
and potassium excretion.
SODIUM (Natrium, Na+)

 it is the major cation in the

extracellular fluid (ECF), plays a
 2) ATRIAL NATRIURETIC FACTOR (ANF) HYPONATREMIA W/ NORMAL RENAL FUNCTION
 it is an endogenous CAUSE Serum Na Urine Na 24-hr
antihypertensive agent Una
secreted from cardiac atria. 1.Overhydration Low Low Low
 It blocks aldosterone and renin 2.Diuretics Low Low High
secretion, and inhibits the 3.SIADH Low High High
action of angiotensin II and
4.Adrenal failure Mildly Elevated Normal
vasopressin.
5.Bartter’s Low Low High
 It causes natriuresis
Syndrome
6.Diabetic Low Normal Normal
Hyperosmolarity
ELECTROLYTE ABNORMALITIES:

HYPONATREMIA:
HYPONATREMIA W/ NORMAL RENAL FUNCTION
 low-serum sodium level; below normal CAUSE Urine Serum K
value Osmolality
1.Overhydration Low Normal or Low
 the most common electrolyte disorder,
2.Diuretics Low Low
 defined as reduced plasma sodium 3.SIADH High Normal or Low
concentration to a value less than 135 4.Adrenal failure Highi High
mmol/L 5.Bartter’s Syndrome Low Low
6.Diabetic normal High
CAUSED BY: Hyperosmolarity
 over hydration, use of diuretics,
syndrome of inappropriate ADH (SIADH) HYPERNATREMIA:
 Aldosterone deficit secondary to
Addison’s disease.  Serum sodium concentration above the
 BARTTER’S SYNDROME: It is a rare upper limit of the reference interval
condition wherein sodium chloride  Caused by loss of water, gain of sodium,
(NaCl) gradients cannot form in the loop or both
of Herle causing the ret4ention of  It usually results from excessive water
chloride ion that is not available for the loss
countercurrent; hyponatremia is not  ELEVATION IN CONDITIONS:
corrected with fluid restriction. dehydration; diabetes insipidus;
 PSEUDOHYPONATREMIA: condition hyperaldosteronism;
characterized by falsely elevated serum hyperadrenocorticism
Sodium level usually cause by the
presence of excess lipids in the serum;
reduction in serum concentration
caused by a systematic error in
measurement
.
ANALYTICAL METHODS: o Another electrode- measuring
electrode
SPECIMEN: serum, plasma, and urine; whole o The difference in potential
blood (depends on analyzer) between the reference and
PLASMA- Lithium heparin, ammonium heparin, measuring electrodes can be
and Lithium oxalate for anti-coagulant used to calculate the
“concentration” of the ion in
Haemolysed sample can cause decreased solution.
sodium level  Most analyzers use a glass ion-
exchange membrane in its ISE system
URINE must be 24-hr collection
for Na+ measurement.
Sweat can also be used in the analysis
Types Of ISE Measurement
 ION SELECTIVE ELECTRODE (ISE)- most Direct Method Provides an undiluted
-Commonly used- sample to interact with
routinely used method in clinical
the ISE membrane
laboratories
 ATOMIC ABSORPTION Indirect Method Diluted sample is used
SPECTROPHOTOMETRY (AAS) for measurement
 FLAME EMISSION No significance in result except if the sample is
SPECTROPHOTOMETRY (FES)/FLAME hyper lipidemic or hyper proteinemia; excess
EMISSION PHOTOMETRY (FEP) protein and lipids present in the serum because
 CHEMICAL METHODS outdated due to displace water that can cause decreased result
lack of precision
 COLORIMETRY (Albanese and Lein)

REFERENCE LEVEL:

 SERUM, PLASMA: 136-145mmol/L

 URINE (24-h): 40-220 mmol/day; varies
with diet
 CSF: 136-150 mmol/L

ION SELECTIVE ELECTRODE POTASSIUM (Kalium, K+)

 Uses a semipermeable membrane to  MAJOR INTRACELLULAR CATION- only

develop a potential produced by having 2% of the body’s total potassium
different ion concentrations on either circulates in plasma
side of the membrane.  Potassium is > inside the cell than extra
 Two Electrodes are Used: cellular fluid, 20x > potassium
o One electrode has a constant concentration inside the cell
potential- making in the o Functions include regulations
reference electrode of: Neuromuscular excitability;
contraction of the heart;
 intracellular fluid volume; failure, and reduced distal delivery of
hydrogen ion concentration sodium.
o Potassium concentration has a  Chronic Hyperkalemia
major effect in skeletal and  Severe Hyperkalemia: loss of muscle
cardiac muscle excitability
 Concentration in RBC is 105 mmol/L  Plasma K: 6-7 mmol/L alters ECG
 Reciprocal relationship with H+  Plasma K: 10mmol/L cardiac arrest
 Crump is signs of low potassium level. (fatal)
 Factors that influence the distribution  Acidosis: increased by 0.2-1.7 mmol/L
of potassium between the cell and per unit pH reduction
extracellular cell fluid: potassium loss  Low insulin: increased Digitalis;
frequently occurs whenever the heparin, captopril, spironolactone,
potassium and sodium potassium cyclosporine
ATPase pump is inhibited by conditions
such as: hypoxia, digoxin overdose, EFFECTS OF HYPERKALEMIA TO CARDIA
hypomagnesemia and insulin promotes MUSCLE
acute entry of potassium in skeletal
 Hyperkalemia decreases the resting
muscle, and insulin promotes also acute
membrane potential (RMP) of the cell.
entry of potassium in liver by increasing
 Severe hyperkalemia can ultimately
sodium potassium ATPase activity
cause a lack of muscle excitability
 Cathecolamines such as epinephrine
(8mmol/L)
promotes cellular entry of potassium to
the cell, and beta blockers/propranolol (↓)HYPOKALEMIA seen in
impairs cellular entry of potassium
 In potassium level determination, avoid  Infusion of insulin to diabetics: alkalosis;
hemolysis. Haemolysed sample will vomiting; over hydration; use of loop
result in hyperkalemia diuretics; syndrome of inappropriate
 Potassium removes in dialysis; in ADH (SIADH) secretion; Barter’s
hazing, potassium are syndrome (it is a condition whose
decrease/remove. primary cause is the excess excretion of
potassium)
ELECTROLYTE ABNORMALITIES:  Arrhythmia and paralysis
 Hyperaldosteronism: K wasting
(↑)HYPERKALEMIA seen in
 Alkalosis: K decrease by 0.4mmol/L per
 Dehydration; diabetes insipidus; unit pH increase
hypoadrenalism; acidosis hemolysis  Hypomagnesia: K excretion
 Is almost always due to impaired renal
SPECIMEN:
excretion.
 3 major diminished renal potassium  Serum, plasma, and urine (24-h). Whole
excretion: reduced aldosterone or blood (depends on analyzers)
sldosterone responsiveness, renal  Anti-coagulant for plasma is heparin
  Urine is a 24-hr sample to avoid diurnal conjunction with sodium in the
variation. proximal tubule and the excess Cl is
excreted in urine and sweat; excess
METHODS OF K+ DETERMINATION sweating stimulates aldosterone
secretion.
 FLAME EMISSION
SPECTROPHOTOMETRY (FES)  Aldosterone acts on sweat glands to
o Violet- end color conserve sodium and potassium levels.
 ATOMIC ABSORPTION ELECTROLYTE ABNORMALITIES:
SPECTROPHOTOMETRY (AAS)
 ION SELECTIVE ELECTRODE (clinical significance)
o Current methos of choice
 HYPOCHLOREMIA: seen in conditions
o Valinomycin membrane is used
salt-losing nephritis; prolonged
to selectively bind K, causing an
vomiting; metabolite alkalosis
impedance change that can be
 HYPERCHLOREMIA: seen in conditions;
correlated to K concentration
dehydration; renal tubular acidosis;
o KCI is the inner electrolyte
acute renal failure; metabolic acidosis
solution
associated with prolonged diarrhea;
 COLORIMETRY
diabetes insipidus; primary
 Lock head and Purcell
hyperparathyroidism; salicylate
REFERENCE VALUE: intoxication

 SERUM: 3.5-5.1 mmol/L ANALYTICAL MEHTODS:

 PLASMA: Males: 3.5-4.5 mmol/L;
SPECIMEN: serum, plasma, sweat
Females: 3.4-4.4 mmol/L
 URINE (24-h): 25-125 mmol/day  anti-coagulant Lithium heparin
 hemolysis does not cause significant
CHLORIDE (Cl-)
change in serum or plasma value as a
 It is the major extracellular anion result of decrease level of intracellular
 With sodium, they represent the chloride
majority of the osmotically active  mark hemolysis decrease chloride level
constituent of the plasma. because of dilutional effect
 FUNCTIONS: Chloride also maintains
osmolality and blood volume and  ISE (ION SELECTIVE ELECTRODE)
electric neutrality  MERCURIMETRIC TITRATION (SCHALES
 Most of the process Cl shifts secondarily AND SCHALES METHOD) –it involves
to the movement of sodium or titration Cl with a solution of Mercury
bicarbonate and Cl is ingested in the (Hg) forming soluble but nonionized
diet and completely absorbed by mercuric chloride. The endpoint is
intestinal tract and filtered by reached when excess Hg forms a
glomerulus—possibly reabsorbed in complex with an indicator, such as
 diphenylcarbazone, producing a violet- FUNCTIONS:
blue color
 Important in skeletal mineralization
 Plays vital role in: blood coagulation;
neural transmission, enzyme activity;
2Cl + Hg HgCl2 + diphenylcarbazone (indicator) maintenance of normal tone;
→ violet blue color excitability of skeletal and cardiac
muscle
 CUOLORIMETRIC METHOD: mercuric
thiocyanate and ferric nitrate to form a  It is involved in glandular synthesis and
reddish colored complex with a peak at regulation of exocrine and endocrine
480nm glands
 It preserves the cell membrane’s
 CUOLOMETRIC-AMPEROMETRIC integrity and permeability, particularly
TITRATION (COTLOVE in terms of sodium and potassium
CHLORIDOMETER)- it is a method using  According to ringer, calcium is essential
coulometric generation of silver ions for myocardial infarction
(Ag) which combine with Cl to  99% is part of bones and 1% is in the
quantitate Cl ion concentration. When blood and ECF
all Cl ions are bound to Ag ions, excess
DISTRIBUTION:
free Ag ions are used to indicate the
end point of the reaction. As Ag ions  Free or ionized form (45%);
accumulate, the coulometric generators  Bound to plasma protein (40%);
and timer are turned off. The elapsed  Complex form (15%); bicarbonate,
time is used to calculate the citrate, phosphate, and lactate.
concentration of the Cl ions in the o Ionized calcium is sensitive and
sample. The Cotlove chloridometer uses specific marker for calcium
this principle in chloride analysis. disorders.
o For every 1g/dL serum albumin
Ag + Cl → AgCl
decreased, there is 0.8 mg/dL
REFERENCE VALUE: decrease in total Ca+2—
hypocalcemia can be a
 PLASMA/SERUM: 98-107 mmol/L consequence of reduced plasma
 URINE (24-h): 11.-250 mmol/Day; varies albumin
with diet o Ionized calcium concentration
in the blood is 5000 to 10x
CALCIUM (Ca2+)
higher than cytosol of cardiac or
 Fifth most common element and the smooth muscle cells.
most prevalent cation in the human
body
FACTORS AFFECTING PLASMA CALCIUM  It inhibits PTH and vitamin D3—
LEVELS: hypocalcemic hormone
 It inhibits bone resorption
1) 1,25 Dihydroxycholecalciferol (1,25-
 It promotes urinary excretion of
(OH)2-D3)
calcium
 It increases intestinal
absorption of calcium HOMEOSTASIS:
 It increases reabsorption in the
kidneys.  Ionized Ca concentration of the
 It increases mobilization of extracellular fluid (ECF) is kept constant
calcium from bones through the action of parathyroid
hormone (PTH) & 1.2.5
 Enhances the effect of
parathyroid hormone on bone dihydroxyvitamin D3
resorption.  PTH, which secreted by the parathyroid
gland, acts on the bones and Ca into the
2) Parathyroid Hormone ECF when plasma ionized calcium levels
 It conserves calcium by decreases. It also acts on the kidney and
stimulates increased phosphate
increasing reabsorption in the
kidneys secretion and calcium reabsorption
returning the ionized calcium
 It increases the level by
concentration to normal.
mobilizing bone calcium
 Vitamin D almost exclusively activated
 It activates the process of bone
by the kidney stimulates calcium
resorption; activated osteoclast
absorption
breakdown, subsequently a
release of Ca to the ECF.  Calcitonin possible plays a role in
regulating process. It has a lowering
 It suppresses urinary loss of
effect on calcium.
calcium
 It stimulates the renal CLINICAL SIGNIFICANCE:
production conversion of
inactive vitamin D to active  INCREASED CALCIUM LEVELS seen in:
vitamin D3 in the kidneys periods of rapid growth in children;
 PTH Secretion in blood is pregnancy; lactation
stimulated by the decrease in  DECREASED CALCIUM LEVELS seen in:
ionized calcium. Secretion stop old age
by increase ionized calcium.
FACTORS THAT INFLUENCE CALCIUM LEVEL:

3) Calcitonin A. INCREASED CALCIUM ABSORPTION

 Is thyroid hormone, secreted by  Vitamin D (major stimulus)
the parafollicular C cells of the  Growth hormone
thyroid gland.  Increased dietary protein
 HYPOCALCEMIA:
B. DECREASED CALCIUM ABSORPTION
 Severe hypocalcemia leads to tetany
 Formation of insoluble salts
with phosphorus
 CAUSES: Hypoparathyroidism;
 Phytic acid
Pseudohypoparathyroidism; deficiency
 Dietary oxalate
in Vitamin D; chronic renal failure;
 Fatty acids
hypomagnesemia; acute pancreatitis
 cortisol
 CHARD (Calcitonin,
Hypoparathyroidism, Alkalosis, Renal
C. INCREASED URINARY CALCIUM
Failure, vitamin D deficit)
EXCRETION
 Hypercalcemia
 Phosphate deprivation ANALYTICAL METHOD:
 Acidosis
 Glucocorticoids A. TOTAL CALCIUM
a. Spectrophotometric analysis
D. DIMINISHED URINARY CALCIUM with the methaliochromic
EXCRETION: indicators
(Orthocresolphthalein
 PTH
complexone & arsenazo III)
 Certain diuretics
b. Titration of fluorescent calcium
 Vitamin D
complex with ethylene diamine
ABNORMALITIES: tetra acetic acid (EDTA_ or
ethylene glycol tetra acetic acid
HYPERCALCEMIA (EGTA)
c. AAS
 Associated with anorexia, nausea,
d. Clark and Collip method (redox
vomiting constipation, hypotonia,
titration method)
depression and coma

B. IONIZED CALCIUM: ISE (ION SELECTIVE

 CAUSES: primary hyperthyroidism;
ELECTRODE) FOR CALCIUM
multiple endocrine neoplasia; familial
hypocalciuric hypercalcemia; Vitamin D REFERENCE LEVELS:
intoxication
 Thyrotoxicosis: hypoadrenalism;  TOTAL CALCIUM - SERUM, PLASMA
multiple myeloma o Child: 12 years 2.20-2.70
 CHIMPS (Cancer, Hyperthyroidism, mmol/L (8.8-10.8 mg/dL)
Iatrogenic Cause, Multiple Myeloma, o Adult: 2.15-2.50 mmol/L (8.6-
HyperParathyroidism, and Sarcoidosis) 10.0 mg/dL)
  IONIZED CALCIUM – SERUM FACTORS AFFECTING PLASMA MAGNESIUM
o Child: 1.20-1.38 mmol/L (4.8- LEVEL:
5.5 mg/dL
o Adult: 1.16-1.32 mmol/L (4.6-
1. Parathyroid Hormone
5.3 mg/dL
 It increases renal reabsorption
of magnesium.
 IONIZED CALIUM – PLASMA
o Adult: 1.03-1.23 mmol/L (4.1-  It increases intestinal
4.69mg/dL) absorption of magnesium

2. Aldosterone and Thyroxine

 IONIZED CALCIUM – WHOLE BLOOD
o Adult: 1.15-1.27mmol/L (4.6-5.1  It increases renal excretion of
magnesium
mg/dL)

 TOTAL CALCIUM – URINE (24h) ABNORMALITIES:

o 2.50-7.50 mmol/day (100-300
mg/day); varies with the diet HYPERMAGNESEMIA:

MAGNESIUM (Mg2+)  Increased in blood that is rare and

usually iatrogenic
 It is essential for the function of cellular  Elderly and patients with bowel
enzyme and energy metabolism. disorder and renal insufficiency at risk.
 Has an important role in membrane  Clinical manifestation includes,
stabilization, nerve conduction, ion hypotension, bradycardia, respiratory
transport and calcium channel activity. depression, depressed mental status
 Role in maintenance of intracellular K and electrocardiographic (ECG)
concentration. abnormalities.
DISTRIBUTION:  Common cause renal failure
 Severe result combine effects decrease
 4th most abundant cation in the body renal function and increase intake of
and the 2nd most prevalent intracellular commonly prescribe magnesium
cation. containing medication such as antacids,
 Magnesium is found in bones and in enemas or catartics
soft tissue.
HYPOMAGNESEMIA:
 In serum, 50% Magnesium is in the form
of a free ion (ionized or free  Seen in magnesium in the GI tract as in
magnesium) chronic diarrhea and malabsorption
 30% associated with protein, primarily steatorrhea
albumin.  Diabetes mellitus secondary to
 15% is complexed with phosphate, glycosuria and osmotic diuresis.
citrate and other anions  Alcohol
  Stress REFERENCE INTERVAL:
 Frequently observe in hospitalized
 TOTAL MAGNESIUM: 0.75-0.95nmol/L
patients
(1.7-2.2 mg/dL or 1.5-1.9
 Very rare for non-hospitalized
mmol/L)
patients/out-patients
 IONIZED MAGNESIUM: 0.44-0.60
ANALYTICAL METHOD: mmol/L

A. TOTAL MAGNESIUM
a. AAS (Reference method) – PHOSPHOROUS* other book uses phosphate*
routinely done in the clinical
laboratory  An important constituent in nucleic
b. Photometric method on acid, phospholipid and
automated analyzers phosphoproteins.
c. These methods employ  It forms high energy compounds such as
metallochromic indicators or ATP and cofactor (NADP) and is involved
dye like calgamite, formazan in intermediary metabolism and
dye, magon, and titan yellow various enzyme system
dye.  Essential for muscle contractility,
neurologic function, electrolyte
B. IONIZED (FREE) MAGNESIUM: ISE FOR transport, and oxygen carrying by
MAGNESIUM hemoglobin.
 In blood, organic phosphate is located
C. INTRACELLULAR MAGNESIUM in the red cells, while plasma contains
a. Flourescence measurement the inorganic ones.
using Furapate (Magnesium  In serum, 65% is free, 10% is bound to
binder) protein such as albumin and 35% is
b. Nuclear magnetic resonance complexed with sodium, calcium, and
Spectroscopy magnesium
c. Electro probe microanalysis  Only organic phosphorus is measured
d. ~Intracellular magnesium routinely
measurement are not currently
DISTRIBUTION:
employed in clinical lab;
research purposes  85% of the total body phosphorus is
present in the skeleton and remaining
DYE BINDING METHODS 15% is in the extracellular fluid (ECF)
and soft tissue.
Calgamite ( reddish violet complex at 532 nm)
 The skeleton contains primarily
Formazan ( Blue complex at 660nm) inorganic phosphates.
 Soft tissues contain primarily organic
Methylthymol Blue ( 600nm) phosphates
O-cresolpthalein complexone ( 570 nm)
  In blood, phosphate is located in red Phosphate + ammonium molybdate →
cells; plasma contains organic phosphomolybdate complex (measured at
phosphate direct UV absorption @ 340 nm)

ABNORMALITIES:  Reduction of phosphomolybdate to

molybdenum blue measured at 600-700
HYPERPHOSPHATEMIA: nm spectrophotometrically
CAUSES:  Enzymatic method

 Decreased level excretion in acute and FISKE-SUBBAROW METHOD

chronic renal failure  Most commonly used method to
 Increased in intake with excessive oral, measure serum organic phosphate
rectal, intravenous administration  Most common reducing agent: pictol
 Increase extracellular load due to (Amino Napthol Sulfonic Acid)
transcellular shift in acidosis  Other reducing agents: elon (methyl
 Secondary to over medication with amino phenol), ascorbic acid, senidine
Vitamin D and production of Vitamin D (N-phenyl-p-phenylene diamine
by granulomatous tissue hydrochloride)
 End product: ammonium-molybdate
HYPOPHOSPHATEMIA:
complex (unstable)
SEEN IN:  The unreduced complex at 340nm is the
most accurate measurement of
 Alcohol abuse inorganic phosphorus in serum
 Intestinal loss due to vomiting, diarrhea,  The pH must be maintained in the acid
and use of phosphate binding antacids. range because higher range (alkaline)
 Induced by a shift of phosphorus from can result in reduction of the complex.
extracellular fluid in cells.  The reduced form of the end product
 Increased urinary excretion, secondary yields a blue color and determined
to hyperparathyroidism, renal tubular between 600nm to 700nm; pag di
defects and diuretic therapy, reduced 340nm accurate
Avitaminosis D)
 D deficiency and steatorrhea REFERENCE LEVEL:

PHOSPHOROUS HAS INVERSE RELATION SHIP  Adult: 2.3-4.7 mg/dL (0.74-1.52

WITH CALCIUM mmol/L)
 Children: 4.0-7.0 mg/dL (1.29-2.26
ANALYTICAL METHOD: mmo/L)

 Reaction of phosphate with Ammonium OTHERS: BICARBONATE

Molybdate (Fiske-Subbarow)
 Quantitatively second most important
anion fraction in serum.
  Production in the body results from the  Abnormal anion gaps in sera from
dissociation of H2CO3 produced from healthy person indicate an instrument
the formation of CO2 during problem
metabolism  NO TRUE ANION GAPS JUST A
 Reconverted to H2CO3 and hence to CALCULATED MEASURE.
H2O and CO2 ad the blood perfuses in  NO/NEVER ACTUAL GAP BETWEEN
the lungs. CATIONIC AND ANIONIC CHRGES.

MEASUREMENT: THERE ARE TWO COMMOLNY USED MTHODS

FOR CALCULATING ANION GAP (AG):
 Direct titration with an acid.
 Indirectly using PCO2 and pH (H) in an 1. AG= Na^+ - (Cl^- + HCO3-)
equation or nomogram REFERENCE VALUE: 7-16 mmol/L
 As total CO2 since 89-90%of all CO2 in
the serum is in the form of HCO3 2. AG= (Na^+ + K^+) – (Cl^- + HCO3-)
 Total CO2 determinations are useful REFERENCE VALUE: 10-20 mmol/L
along with pH and PCO3 to evaluate
acid-base disorder

REFERENCE VALUE: 22-32 mmol/L

ANION GAP

 It is difference between sodium and the

sum of chloride and bicarbonate which
is around 26 mEq/L
 Used to monitor recovery from diabetic SWEAT CHLORIDE
ketoacidosis
 An indirect measure of the molar SWEAT TEST is performed for the diagnosis of
concentration of acid added to the cystic fibrosis
system.
 Pilocarpine is introduces into the skin
 An increase anion gap is due to by iontophoresis to stimulate locally
replacement of bicarbonate with anion increased sweat secretion.(sweat
organic acid. inducer) (Gibson and Cooke)
 The change in anion gap should equal  The resulting sweat is absorbed by filter
the change in bicarbonate. paper, weighed, diluted with water and
 The presence of a widened anion gap analyzed for sodium and chloride
signifies the presence of metabolic  Concentration of over 60 mmol/L of
acidosis due to a non-chloride sweat in least 2 occasions is diagnostic
containing acid. of cystic fibrosis in children
 Low anion gap signifies the presence of  Levels of 50-60 mmol/L are suggestive
high levels of basic proteins, often a of cystic fibrosis.
myeloma protein
CYSTIC FIBROSIS (mucoviscidosis) of the
pancreas is an autosomal recessive disease
characterized by abnormal secretion of the
various exocrine glands including the sweat
glands. In cystic fibrosis, there is an increased
demonstration of sodium and chloride in the
sweat.

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RECORDING START NG 2:20:00

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BITCHES!!!
MODULE 3- LABORATORY  THYROGLOBULIN – building blocks for
the active thyroid hormones Thyroxine
THYROID HORMONES
(T4) and Triiodothyronine (T3)
 T4 has half-life of one week in
THYROID GLAND
circulation; T3 has half-life of one day
 (T4) N.S. 5.5-12.5 ug/dL ; T3 100-200
ng/dL
 T3 exerts majority of thyroidal hormone
effects
 Both T3 and T4 are bound in serum
proteins mostly with thyroxine binding
globulin (TBG)
 3 binding proteins: thyroxine-binding
globulin (TBG), thyroxine-binding
POSTERIOR VIEW OF THYROID GLAND prealbumin (transthyretin), thyroxine-
 Located near the thyroid capsule binding albumin (TBA).
(region of the thyroid gland);  TBG- transports the majority of T3;
sometimes within the thyroid gland transports 70-75% of total T4.
 May also be found outside their normal  TBP/Transthyretin- transports 15-20%
anatomic site- between the hyoid bone total T4; and it has no affinity for the T3
in the neck and mediastinum  TBA- transports T3 and 10% of the T4
 Butterfly shaped gland BIOSYNTHESIS OF THYROID HORMONES
 Located in the lower part of neck and (formation)
just below the voice box or larynx
 Connected by a narrow band called  Thyroid gland synthesizes its
isthmus hormone from iodine and essential
 11 weeks of gestation, hormone amino acid tyrosine
produce---the measurable hormone or  Iodine enters through the
the thyroid hormone alimentary tract as iodide (I), lungs
 Follicle - fundamental unit of thyroid and skin.—also enters thyroid
gland gland.
 2 types:  1/3 (of iodine) enters thyroid gland,
o FOLLICULAR CELLS or 2/3(of iodine) leaves body in urine.
SECRETORY (T3 & T4)  Enzymes oxidize iodide to organic
o PARAFOLLICULAR or C CELLS iodine into monoiodotyrosine (MIT)
(CALCITONIN)--polypeptide and diodotyrosine (DIT).
 THYROGLOBULIN – acts as preformed  Iodination of tyrosine residues in
matrix containing tyrosyl group; thyroglobulin results in formation of
glycoprotein- stored in the follicular MIT and DIT
colloid of the thyroid gland
TYROSINE + IODINE = MIT & DIT
 T3 = MIT + DIT

T4 = DIT + DIT

 99.97% of T4 circulates in bound form

with TBG
PITUITARY – HYPOTHALAMIC RELATIONSHIP
 99.7% of T3 circulates in bound form
with TBG  Thyroid gland responds with the
 Main stimulus of biosynthesis of thyroid production of thyroxine on stimulation
hormone is when TSH stimulates the of anterior pituitery hormone TSH
synthesis of thyroid hormone. (thyrotropin)
 Note: TSH stimulates synthesis of  TSH follows stimulation of the gland by
thyroid hormone the Hypothalamic peptide TRH –
 T4 secreted 100% in the thyroid gland responds to active levels of T3 and T4
 Conversion of T4 to T3 takes place in  Low hormone levels TRH provokes TSH
many tissues particularly liver and secretion thus, increased thyroidal
kidney. iodine metabolism and hormone
 Protein bound hormone are production
metabolically inactive and free  The Hypothalamic-pituitarry-thyroid
hormones (FT4 and FT3) are axis (HPTA) is neuroendocrine system
physiollogically active portions of the that regulates the production and
thyorid hormones. secretion of thyroid hormones
 Protein bound hormones do not enter
cells, thus considered to be biologically FUNCTION OF THYROID HORMONE
inert and functions as storage sites of
1. For tissue growth
circulating thyroid hormones.
2. For development of the central nervous
 Reverse T3 (rT3) produces by removal
system (CNS)
of one iodine from the inner ring of T4,
3. Elevated heat production
metabolically inactive product of T4
4. Control of oxygen consumption
metabolism
5. Influences carbohydrate and protein
 Iodine is the most essential in the
metabolism
thyroid hormone synthesis
6. For energy conversation
 recommended daily intake of iodine is
150 ug/day; EFFECTS OF THYROID HORMONE
 Iodine intake has a below 50 ug/day it is
 Control of oxygen consumption
an indicative of defiency on hormone
secretion.  Measured by basal metabolic rate
 Thyroid hormone is very important  Influence CHO and protein metabolism,
because it affects synthesis, mobilization of electrolytes, conversion
degradation, and intermmediate of carotene to vitamin A
metabolism of adipose tissue and  Essential for development of CNS-
circulating lipids. deficient in infant causes irreversible
 mental damage CRETINISM; in adults 14 years old -105-245 ng/dL or 1.8-3.8
slows deep tendon reflexes, diffuse nmol/L
psychomotor retardation
 Thyroid hormone can be a treatment on
Hormone Replcement Therapy (HRT)

TRIIODOTHYRONINE (T3)/ 3,5,3’

TRIIODOTHYRONINE
HINDI KO ALAM KUNG SERYOSO SI MA’AM
 Also known as 3,5,3’ PAREHO ANG DEFINITION NG T3 AT T4 SA PPT
TRIIODOTHYRONINE NIYA , NALOKO NA. yung nasa lower part nang
 Principal secretory product, originates T3 pang 6th bullet ayun mga sinabi niya, di ko
in the thyroid gland inalis ang mga informations from ppt niya at
 Major fraction of organic iodine in the baka malintikan pero information/definition
circulation yun for T4 TALAGA– YNOR QT  ♥
 A prohormone for T3 production
 Serum T4 - indicator of thyroid TETRAIODOTHYRONINE (T4)/ 3,5,3,5’
secretory rate TETRAIODOTHYRONINE
 Elevated Thyroxine causes inhibition of
 Also known as 3,5,3,5’
TSH secretion and vice versa.
TETRAIODOTHYRONINE/THYROXINE
 Major/most active thyroidal hormonal
 Most abundant thyroid hormone
activity
 Principal secretory product, originates
 Almost 75-80% is produced from the
in the thyroid gland
tissue deiodination of T4 – conversion
 Major fraction of organic iodine in
of T4 to T3 takes place in many tissues,
circulation
particularly the liver and the kidneys
 A prohormone for T3 production
 The principal application of this
 Serum T4 - indicator of thyroid
hormone is in diagnosing T3
secretory rate
thyrotoxicosis
 Elevated Thyroxine causes inhibition of
 T3 is a beta indicator of recovering from
TSH secretion and vice versa.
Hyperthyroidism and reccurence of
 All circulating T4 hormone originates in
hyperthyroidism
the thyroid gland – it is secreted 100%
 T3 is helpful in confirming the diagnosis
in the thyroid gland.
of Hyperthyroidism, especially with the
 Amount of Serum T4 is a good insicator
patients with no or minimally elevated
of the thyroid secretory rate.
levels of T4
 REFERENCE VALUE: ADULT: 5.5-12.5
 Increase in the plasma level of T3 is the
ug/dL or 71-161 nmol/L; NEONATE:
first abnormality seen in cases of
11.8-22.6 ug/dL or 152-292 nmol/L
hyperthyroidism.
 REFERENCE VALUE: ADULT- 60-160 THYROID AUTOANTIBODIES RESPONSIBLE FOR
ug/dL or 0.9-2.46 nmol/L; CHILDREN- 1- AUTOIMMUNE THYROID DISORDERS:
 1. Thyroperoxidase (TPO)  Thyroid hormones affects synthesis,
-is involved in the tissue destructive degradation and intermediate
process (Hashimoto’s disease- metabolism of adipose tissue and
autoimmiune thyroid disease) circulating lipids.
2. Thyroglobulin (Tg)  Hyperthyrodism- degradation and
3. TSH receptor (TR) excretion
-it is involved in Grave’s disease  Increase than synthesis, resulting to low
levels of cholesterol, phospholipids and
FACTORS THAT DECREASE THYROID ACTIVITY
triglycerides
 Enzyme deficiencies may interfere with  Hypothyroidism slow catabolism more
iodine metabolism causing congenital then affecting synthesis,
goiter (enlargement of thyroid gland) hypercholesterolemia and
 Exogenous thyroid hormone suppresses hypertriglyceridemia—high levels of
hormone production by depressing TSH cholesterol and triglyceride
levels.  Increased serum cholesterol levels may
 Thiocyanate and Perchlorate interfere indicates impending hypothyroidism
with iodide concentration  Cholesterol levels drops to normal
 Thiourea and Thiouracil prevent within 2-3 weeks after successful
incorporation of thyroidal iodine into therapy for hypothyroidism
organic compounds.  Severe hypothyroidism serum levels of
 Anti-thyroid effects of iodine includes muscle-associated enzymes tend to rise.
inhibition of iodine binding and CK and LDH. isoezyme partition reveals
hormonal release skeletal muscle as the source
 Radioactive iodine (RAI) has the
EFFECTS OF THYROID DYSFUNCTION ON
additional effect of selectively
NONTHYROID TEST
irradiating hormonally active tissue,
owing to its uptake by the actively HYPOTHYROIDISM
functioning gland.; also use for the
treatment of thyroid cancer.  Increased serum cholesterol,
triglycerides
NON-SPECIFIC INDICATORS OF THYROID  Increased serum carotene (yellow skin
ACTIVITY discoloration)
 Increased serum levels of muscle
 All thyroidal effects is to stimulate
enzyme: CPK, AST, LDH
overall metabolism
 Increased serum prolactin
 Earliest test for thyroid function was
 Increased capillary fragility
Basal Metabolic Rate (BMR )- measures
 Increased spinal fluid protein
oxygen consumption.
 Normochromic anemia, hemoglobin
BLOOD CHEMISTRY VALUES around 10 g/dL
 Decreased urinary excretion of 17KS,
17—OHCS
HYPERTHYROIDISM  Iodine is also a component of
radioactive machines; CT-Scan,
 Increased skin temperature, pulse rate,
pulse pressure 3. THYROXINE (T4)
 Decreased serum cholesterol,  Radioimmunoassays (RIA)/ Enzyme-
triglycerides Linked immunoassay (ELISA)
 Increased serum level of  Widely available as antibody specifities
aminotransferases and alkaline have no significant cross activity
phophatase between T4 and T3 in assays for each
 Increased retention of BSP hormone.
 Altered glucose insulin relationship  Uses TBG as the specific binder in place
 Increased proportion of lymphocytes in of an anti-T4 antibody
differential white count  Measurement of T4 is the first round
 Increased urinary calcium excretion test in screening thyroid disease.

MEASURING THYROID ACTIVITY TRIIODOTHYRONINE (T3)

IODINE LEVELS  Most active thyroid hormonal activity

 75-80% produced from tissue
 Iodine plays one physiologic role in
deiodination of T4
serum- as a vital constituent of thyroid
 Useful in the confirmation of a
hormones, thus, iodine measurements
suspected thyroid abnormality (T3
were used as indicator of thyroid
thyrotoxicosis)
function
 Indicator of recovery from
hyperthyroidism, recognition of
1. PROTEIN BOUND IODINE (PBI)
recurrence
 Earliest measurement of iodine, but
 Triiodothyronine levels are necessary to
severely affected by organic and
monitor thyroid replacement therapy
inorganic iodine contaminants
consist of T3
 Useful to document nonhormonal
iodine, physical damage to the thyroid THYROXINE- BINDING GLOBULIN & FREE
gland liberating iodine to the blood HORMONE
stream.
 Diagnosis of iodine overload or toxicity  Evaluated when T3 and T4 values are
like burn victims. abnormal
 Use RIA or T3 uptake test.
2. BUTANOL-EXCTRACTABLE IODINE o For HYPERTHYOIDISM, both T3
(BEI) and T4 have HIGH values.
 Technically more difficult o For HYPOTHYROIDISM, both T3
 Does not offer great accuracy and T4 have LOW values.
 High levels of iodinated radiographic  Increase in the plasma level seen in
contrast dyes causes interference cases of hyperthyroidism
  REFERENCE VALUES: 80-200 ng/dL or T3 uptake occupied by the
1.2-3.1 nmol/L (ADULT); 105—245 nonhormonal agents.
ng/dL or 1.8-3.8 nmol/L (CHILDREN 1-14  Administration of heparin causes
yrs. Old) elevation of free fatty acids from
breakdown of triglycerides by post-
heparin lipoprottein lipase activation.
 False High T3 uptake
 Lithium also afects thyroid hormones

STIMULATION OFTHYROID-STIMULATING
TETRAIODOTHYRONINE/THYROXINE (T4) HORMONE (TSH)

 Principal secretory product, originates  Thyroid activity is regulated by the

in the thyroid gland body’s perceived need for hormone.
 Major fraction of organic iodine in the  If in-adequate thyroid hormone
circulation circulates in free fraction,
 A prohormone for T3 production hypothalamus produces, TRH that
 Serum T4 – indicatorr of thyroid provokes rising TSH levels to stimulate
secretory rate thyroid output.
 Elevated Thyroxine causes inhibition of  Immunoassays
TSH secretion and vice versa  VALUES: 0-10 ulU/ml
 REFERENCE VALUES: 5.5-12.5 ug/dL or  SIGNIFICANCE:
71-161 nmol/L (ADULT); 11.8-22.6 o TSH is increased in primary
ug/dL or 152-292 nmol/L (NEONATES) hypothyroidism.
o TSH levels distinguish true
FACTORS AFFECTING THYROXINE- BINDING
hypothyroidism from euthyroid
GLOBULIN (TBG)
condition with low thyroid
 Estrogens- increase the amount of TBG hormone levels
 Androgens and glucorticoids-
CLINICAL DISORDERS
decrease/depress TBG
 Pregnant women and patients taking  SCREENING: recommended at the age
exogenous estrogens, or endocrine of 35 and every 5 years thereafter
secreting tumors,- T3 uptakes are low
due to increase TBG concentration THYROID DISORDERS
 Low TBG concentration, high T3 uptake, HYPOTHYROIDISM
protein levels are low.
Example: Liver disease,  Hypometabolism called myexedema
nephrotic syndrome Example: Lethargy ,
 Drugs like salicylates and phenytoin, constipation, dry skin, and hair,
anticonvulsants binds TBG, competes premenopausal women
with thyroxine. Thus gives hish residual (excessive bleeding)
  SYMPROTMS: Slowed tendon reflexes,  Thyroid cancer is compatible to life; also
coarse skin texture, facial puffiness, it is a friendly cancer.
cold intolerance, decreased sweating,
impaired memory, slowing of speech THYROIDITIS
and motor activity  Lymphocytic infiltration and fibrosis
 Systolic and distolic is high but slow occur
heart rate  Histologic appearance: that of
 CONGENITAL HYPOTHYROIDISM – mediated chronic inflammation
irreversible, severe mental retardation,  CONDITION: Lymphocytic
characteristic body changes, Cretinism. thyroiditis or Hashimo’s disease
All newborn are screened by heel-stick (thyroglobulin is the auto antibody).
blood sample for low T4, confirmed by Often with glandular enlargement.
TSH assay. High titer of thyroglobulin and
 Treatment- lifelong administration of microsomal antigen
thyroid hormone soon after birth
SUBACUTE THYROIDITIS
HYPERTHYROIDISM
 May indicate viral agent
 Thyroid produces excessive hormone  Disorder begins as sore throat with
from nodular areas of: fever, progresses involving one or both
o Hyperfunctioning – ex: lobe, large and tender on palpation or
adenomas and toxic nodular on swallowing
goiter.
 Thyroid Biopsy- inflammatory cell
o Overall hyperactivity – diffuse
infiltrates
toxic goiter. Ex: Grave’s disease
 Atrophy - leads to irreversible
 Manifest nervousness, fatigue, weight
hypothyroidism
loss, heat intolerance, increased
 Can recover with treatment of aspirin:
sweating
Steroids
 TOXIC NODULAR GOITER- excessive
prominence of eyes (exopthalmos), EUTHYROID SICK SYNDROME
widening of palpebral fissures.
 T3T4 are high T3 uptake and high free  Severe illness that indicates
T4 index. hypothyroidism. Patients with
neoplastic disease, diabetes mellitus,
 Subclass, T3 thyrotoxicosis. Normal T4
burn, trauma, cardiovascular
and Ft4 but increased T3.
conditions, liver disease, renal failure or
 STIMULATION OF CHRONIC
prolonged infection
GONADOTROPIN (same chemical
 2 changes underlie abnormal hormonal
structure with TSH): Px with high hCG
measurement. Serum albumin drops on
levels from hydatid form mole,
severe illness, fall of prealbumin
choriocarcinoma and embryonal
decreases hormone binding capacity.
carcinoma of the testis causes
TSH normal, reverse T3 elevated.
hyperthyroidism
CLINICAL DISORDERS
D. SUBCLINCAL HYPERTHYROIDISM
1. HYPERTHYROIDISM  Shows no clinical symptums BUT Low
 Excess circulating TH TSH, FT3 & FT4 normal
 Signs and symptoms/ Features:
Tachycardia, tremors weight loss, heat
intolerance, emotional changes,
menstrual changes.
 Primary Hyperthyroidism (PM Hy):
Elevated T3 and T4 decreased TSH; E. SUBACUTE
major hormone GRANULOMATOUS/SUBCACUTE
 Secondary Hyperthyroidism(Sec. Hy).: NONSUPPURATIVE THYROIDITIS/DE
QUERVAIN’ THYROIDITIS (PAINFUL
Increased TSH and FT4 (due to lesion in
pituitary gland or other gland) THYROIDITIS)
 SS/Features: neck pain, low grade
A. THYROTOXICOSIS fever, swings in thyroid function test.
 High levels of free TH in circulation  Thyroidal peroxidase (TPO) antibodies
 Is applied to a group of syndrome absent; ESR and thyroglobulin levels are
caused by high levels of free thyroid elevated
hormones in the circulation
 T3 thyrotoxicosis or Plummer’s 2. HYPOTHYROIDISM
disease: FT4 increased but FT4 normal  Develops whenever insufficient
amounts on thyroid hormone are
with low TSH
 T4 thyrotoxicosis: T3 normal or low but available to tissues.
T4 increased with low TSH  It is treated with thyroid hormone
replacement therapy (levothyroxine)
B. GRAVES’ DISEASE (DIFFUSE TOXIC  Signs and symptoms Features:
GOITER) bradycardia, weight gain, coarse skin,
 Common cause of thyrotoxicosis cold intolerance, and mental dullness
 Autoimmune disease in which
antibodies are produced that inactive 1. PRIMARY HYPOTHYROIDISM
the TSH receptor  Deficiency in elemental iodine (↓T3
 More in women; occurs 6x more and T4, ↑ TSH)
commonly in women than in men  Cause by destruction or ablation of
 SS/ Features: exophthalmos (bulging thyroid gland.
eyes), pritibial myxedema  Signs and symptoms/Features: surgical
 Diagnostic Test: TSH receptor antibody removal of gland, used of radioactive
test. iodine for hyperthyroidism treatment;
radiation exposure; drugs like lithium
C. REIDEL’S THYROIDITIS
 Thyroid turns to woody or stony hard
mass.
 a. HASIMOTO’S DISEASE  Defect in development or function of
(CHRONIC AUTOMIMMUNE the gland
THYROIDISTIS  Symptoms: physical and mental
 Most common cause of primary development of the child are retarded
hypothyroidism  Lab results: T4 decreased, TSH
 Characterized by a thyroid replaced increased
by a nest of lymphoid tissue –
sensitized T 5. SUBCLINICAL HYPOTHYROIDISM
lymphocytes/autoantibodies bind  Lab results: Tt3 and T4 normal but TSH
to cell membrane causing cell lysis is slightly increased
and inflammatory reaction.
 It is associated with enlargement if THYROID FUNCTION TEST
the thyroid gland (goiter) 1. TRH Stimulation Test
 METHOD OF TESTING: 2. TSH test
o TPO antibody= +result 3. Radioactive Iodine Update (RAIU
o TSH= increased 4. Thyroglobulin (Tg) Assay
5. Reverse T3 (rT3)
b. MYXEDEMA 6. Free Thyroxine Index (FTI or T7)
 Describes the peculiar nonpitting 7. Total T3 (TT3) Free T3, Free T4
swelling of the skin 8. T3 uptake test
 Skin becomes infiltrated by 9. Thyroxine binding globulin (TBG) test
mucopolysaccharides 10. Fine needle Aspiration
 Myxedema coma is the severe 11. Recombinant Human TSH
form of primary hypothyroidism 12. Tanned Erythrocyte Hemmaglutination
 Signs and Symptoms/ Features: Method
“puffy” face, weight gain, slow 13. Serum Calcitonin Test tumor marker for
speech, eyebrows thinned, dry and detecting residual thyroid metastasis in
yellow skin, andanemia Medullary thyroid carcinoma (MTC)
14. Pentagastrin (Pg) Stimulation test
2. SECONDARY HYPOTHYROIDISM
 Due to pituitary destruction or pituitary TRH STIMULATION TEST (THYROTROPIN
adenoma RELEASING HORMONE)
 Lab results: T3 and T4 low levels, TSH
decreased  Measures the relationship between the
TRH and TSH secretions
3. TERTIARY HYPOTHYROIDISM  Most specific and sensitive test for the
 Due to hypothalamic disease diagnosis of thyroid diseases
 Lab results: T3 and T4 low levels, TSH  Used to differentiate euthyroid and
decreased hyperthyroid patients who both have
undetectable TSH levels.
4. CONGENITAL HYPOTHYROIDISM/  May also be helpful in the detection of
CRETINISM thyroid hormone resistance syndromes
  Confirm borderline cases of euthyroid THYROXINE BINDING GLOBULIN (TBG)
Graves’s disease.
DOSE NEEDED: 500ug TRH by IV  Useful to distinguish between
INCREASED LEVEL: primary hyperthyroidism causing high thyroxine
levels and euthyroidism with increased
hypothyroidism
DECREASED LEVEL: hyperthyroidism binding by TBG and increased thyroxine
(↑ T4 + N TBG)
TSH TEST  Total serum T3 and T4 are dependent
on the amount of TBG
 Most important thyroid function test –
 Used to confirm results of FT3 and FT4
best screening test; the best method for
or abnormalities in the relationship of
detecting clinically significant thyroid
the total thyroxine (TT4) and THBR test
dysfunction
 Helps in the diagnosis of patients having
 Most clinically sensitive assay for the
elevated T3 and T4 levels but no
detection of primary thyroid disorders
correlation with the other thyroid
 Helps in the early detection of function tests, or not compatible with
hypothyroidism clinical findings
 Used to differentiate primary
 TBG excess leads to increased serum
hypothyroidism from secondary levels T3 and T4 but the unbound or
hypothyroidism free form of these hormones in the
 Used to monitor and adjust thyroid blood remain unchanged
hormone replacement therapy HORMONAL EFFECT: estrogen increases
 Sensitivity of the third-generation TSH TBG while androgens depresses TBG
assays has led to the ability to detect INCREASED LEVELS: hypothyroidism,
what is termed subclinical disease – or pregnancy, estrogen surge
mild degree of thyroid dysfunction (due DECREASED LEVELS: anabolic steroids
to the large reciprocal change in TSH nephrosis
levels seen for even small changes in REFERENCE VALUE: B13-39 ug/dL (150-
free T4) 360 nmol/L)
 REFEENCE VALUES: 0.5-5 uU/mL
FINE-NEEDLE ASPIRATION
INCREASED TSH DECREASED TSH
 Is the most accurate tool in the
1. Primary Hypothyroidism 1. Primary hyperthyroidism
evaluation of thyroid nodules
2. Hashimoto’s disease 2.Secondary/Tertiary
Hypothyroidism RECOMBINANT HUMAN TSH
3. thyrotoxicosis due to 3.Treated Grave’s disease
pituitary tumor  Used to test patients with thyroid
4. TSH antibodies 4.Euthyroid sick disease cancers for the presence of residual or
5. Thyroid hormone 5.Over replacement of thyroid recurrent disease
hormone in hypothyroidism
TANNED ERYTHROCYTE HEMMAGLUTINATION

 Measure of antithyroglobulin antibodies

 SERUM CALCITONIN  High uptake indicates metabolically
active gland (active hormone
 Tumor marker for det4ecting residual production)
thyroid metastasis in medullary thyroid
 High uptake + TSH deficiency =
carcinoma (MTC)
autonomous thyroid activity
 Should be measured before and 6
months after surgery THYROGLOBULIN (Tg) ASSAY

PENTAGASTRIN (Pg) STIMULATION TEST  Normally used as a postoperative

marker of thyroid cancer
 Used for the diagnosis of MTC
 Used in monitoring the course of
 PROCEDURE: an intravenous Pg (0.5 metastatic or recurrence of thyroid
ug/kg body weight) is given within 5 cancer (a well-differentiated tumors
seconds; blood samples are collected at typically display a 10-fold increase in Tg
baseline and 1, 2, 5, an 10 minutes after in response to a high TSH)
the start of the infusion
 When checking Tg as a tumor marker
DISORDERS T3 T4 TSH FT rT3 Tg TBG for thyroid cancer, Always check a
4 simultaneous sample for thyroglobulin
GRAVE’S DISEASE ↑ ↑ ↓ ↑ ↑ ↑ N antibodies
 Differentiates subacute thyroiditis
PRIMARY N/ ↓ ↑ ↓ ↓ N N
HYPOTHYROIDISM ↓ / (↑Tg) from thyrotoxicosis factitia (↓
↓ Tg)
HASHIMOTO N/ N/ ↑ N/ ↓ N N  INCREASED: untreated and metastatic
THYROIDITIS ↓ ↓ ↓ / differentiated thyroid cancer, nodular
↓ goiter and hyperthyroidism
NONTHYROIDAL ↓ N/ V V N/ N N
 DECREASED: infants with goitorous
ILLNESS ↓ ↑
hypothyroidism and thyrotoxicosis
THYROID HORMONE ↑ ↑ N/ ↑ ↑ ↑ N factitia
RESISTANCE ↑
 REFERENCE VALUE: (ADULT)= 3-42
NEONATAL ↓ ↓ ↑ ↓ ↓ N N
HYPOTHYROIDISM / ng/mL or ug/mL; (INFANT) = 38-48
↓ ng/mL or ug/mL
N-NORMAL V- VARIABLE  METHODS FOR TESTING: double-
antibody RIA, ELISA, IRMA,
MGA HINDI BINANGGIT SA PPT PERO KASAMA immunochemiluminescent assay (ICMA)
SA THYROID FUNCTION TEST:
REVERESE T3 (rT3)
RADIOACTIVE IODINE UPTAKE (RAIU)
 Formed by the removal of one iodine
 Used to measure the ability of the from the inner ring of the T4
thyroid gland to trap iodine  An end product of T4 metabolism; the
 Helpful in establishing the cause of 3rd major circulating thyroid hormone
hyperthyroidism  Identifies patients with euthyroid sick
syndrome (elevated rT3)
  Used to assessed borderline or DECREASED: hypothyroidism, oral
conflicting laboratory results contraceptives, pregnancy; acute
 REFERENCE VALUES: 38-44 ng/dL hepatitis
REFERENCE VALUE: 25-35 %
FREE THYROXINE INDEX (FTI OR T7)
TERMINOLOGIES
 Indirectly assesses the level of free T4 in
blood 1) AMENORRHEA
 Based on the equilibrium relationship of  Absence of menstruation; Primary-
bound T4 and FT4 never occurred
 Important in correcting euthyroid  Secondary absence of menses for 6
individuals months
 It is elevated in hyper thyroidism and
decreased in hypothyroidism 2) CARCINOID SYNDROME
 REFERENCE VALUES: 5.4-9.7  Constellation of symptoms like
diarrhea, flushing, tachycardia and
hypotension which are produced by
the release of amines (histamine,
kallokrein, and prostaglandine) into
TOTAL T3 (TT3) FREE T3, FREE T4 the circulation

 FT4 is used to differentiating drug

induced TSH evaluation and 3) CUSHING DISEASE
hypothyroidism  Metabolic disorder characterized by
 The value of TT3 or FT3 is in confirming abnormal increased secretion of
hyperthyroidism ACTH results to cortisol production
 DIRECT/REFERENCE METHOD:
Equilibrium dialysis 4) CUSHING’S SYNDROME
T3 UPTAKE TEST  Chronic excessive production of
cortisol by adrenal cortex or
 Measures the number of available administration of large doses of
binding sites of the thyroxine-binding glucocorticoids.
proteins, most notably TBG; a test for COMMON CAUSE: pituitary
TBG hormone
 Does not measure the level of T3 in
serum but it reflects the serum level of 5) GYNECOMASTIA
TBG  Development of breast tissue in
 Inversely related to TBG- decr4eased T3 males. Related balance of estrogen
uptake results to elevated TBG result to androgen
and vice versa
INCREASED: hyperthyroidism, 6) HIRSUTISM
euthyroid patients, chronic liver disease
  Excessive growth of hair with a
male distribution pattern in a
female. Endocrine disorder to
women

7) TURNER’S SYNDROME
 Short stature. “webbed neck” low
hairline on the neck and broad
shield-like chest
 Amenorrhea with increased levels
of FSH and LH

8) VIRLIZATION
 Secondary male sexual
characteristic are acquired by a
female. Result of adrenal
dysfunction or hormonal
medication

9) ZOLLIGER-ELLISON SUNDROME
 Severe peptic ulcer of the stomach
MODULE 4- LABORATORY or PP CELLS) – producing
pancreatic polypeptide (5%
PANCREATIC HORMONES of all islet cells)
 Islets of Langerhans play a crucial role in
PANCREAS
carbohydrate metabolism and so in a
plasma glucose concentration. It
involves:
o GLYCOLYSIS – the anaerobic
conversion of glucose to
lactate. Occurs in the red blood
cells, renal medulla and skeletal
muscles.
o GLYCOGENESIS – the synthesis
of glycogen from glucose.
 Digestive gland in the Glucose is stored (in liver,
gastrointestinal system. muscle) in the form of glycogen
 Both an endocrine and exocrine and this serves to maintain a
gland constant plasma glucose
o ENDOCRINE GLAND – concentration
insulin, glucagon, and o GLYCOGENOLYSIS- the
somatostatin breakdown of glycogen to
o EXOCRINE GLAND - glucose
amylase and Lipase; o GLUCONEOGENESIS- the
Digestive enzymes production of glucose from
 The specialized tissue called islets non-sugar molecules (amino
of Langerhans acids, lactate, glycerol)
 Islets of Langerhans represent o LIPOLYSIS- the breakdown of
approximately 1-2% of the pancreas triacylglycerols into glycerol and
 TYPES OF CELLS ARE RECOGNIZED fatty acids.
IN THESE ISLETS: o LIPOGENESIS- the synthesis of
o A CELLS (alpha) – Producing triaglycerols.
glucagon (25% of all islets
FUNCTIONS OF PANCREAS
cells)
o B CELLS (beta)– producing  Pancreatic hormones are responsible
insulin (60% of all islets for storage of fat and glucose, as
cells) glycogen, after meal
o D CELLS (delta)– producing  Enables the mobilisation of energy
somatostatin (10% of all reserves as a result of food deprivation,
islet cells) stress, physical activity
o F CELLS (known now as  Maintain the constant plasma glucose
PANCREATIC POLYPEPTIDE concentration.
  Promote growth

PANCREATIC HORMONE

 Insulin – primary hormone responsible

for decreasing the glucose levels;
synthesize by beta cell of islets of
Langerhans; release when there is a
high glucose levels; only hormone that
HYPOGLYCEMIC; stored in the liver, fat
and muscles; reciprocal relationship
with glucagon
 Glucagon – primary hormone
responsible for increasing glucose
levels; HYPERGLYCEMIC AGENT;
synthesize by the alpha cell; release
during stress and fasting state
 Somatostatin- produce by the delta
cells of islets of Langerhans; primarily
inhibits the action of insulin growth
hormone and glucagon

PANCREATIC ENZYMES

 Amylase
 Lipase

Note: Please see your notes in ENZYMES

LECTURE DISCUSSION the AMYLASE and LIPASE
to be exact; MODULE 1
MODULE 5- LABORATORY  synthesized by the Leydig cells of the
testis from progesterone,
GONADS
 controlled primarily by FSH and LH
REPRODUCTIVE HORMONES/ SEX HORMONES  FUNCTIONS: growth and development
of the reproductive system, prostate
 Testes and Ovaries: Produces sex and external genitalia
hormones- Androgens and Estrogens  Demonstrate circadian pattern: peak at
from cholesterol time of awakening (8am), lowest at
 OVARIES- converts testosterone to 8pm
estradiol and androstenedione to  Gradual reduction of testosterone at
estrone the age of 30, average decline of about
 Peripheral tissues converts or reduce 110 ng/dL every decade
testosterone to dihydrosterone (DHT)  Obesity may cause decrease plasma
hydroxylate estradiol to estriol, convert testosterone concentration
adrenal androgens to testosterone and  After age 50, men experience a
androgens to esterone and estradiol decrease in secretion rate and
 Major transport proteins: Sex concentration of testosterone, and
hormone-binding globulin (SHBG) 60%, women have an increase in pituitary
Corticosteroid-binding globulin (CBG) gonadotropins, especially follicle-
and albumin 40% stimulating hormone.
 Concentration of binding protein  Infertility testing: semen analysis,
determines the level of total testosterone, FSH,LH
testosterone but not free testosterone  Reference value: 3.9-7.9 ng/mL (serum)
level during laboratory estimation
 TYPES OF TESTICULAR INFERTILITY
SEX HORMONES
(HYPOGONADISM)
 SHBG: transports androgens and o PRETESTICULAR INFERTILITY
estrogens; (SECONDARY
 CHG: delivers progesterone and HYPOGONADISM):
glucocorticoids HYPOTHALAMIC/ PITUITARY
 1-2% sex steroids are unbound or free. LESIONS- Normal to decrease
Remaining is bound to proteins levels in testosterone, FSH and
(ALBUMIN, SHBG, AND CHG) LH levels
o TESTICULAR INFERTILITY
 Free fractions are active, and only the
(PRIMARY HYPOGONADISM) :
ones can diffuse into the vascular
may be congenital
system and interact with the target cells
(cryptorchidism, Kinefelter’s
TESTOSTERONE syndrome and 5-a-reductase
deficiency) or acquired
 Principal androgen hormone in the (varicocele, tumor, orchitis).;
blood -- Most potent male androgen, Decreased testosterone levels
 and increased FSH and LH o ASSOCIATED DEFECTS: anosmia
levels. (inability to smell) and midline
o POST-TESTICULAR defects (cleft palate and lip)
INFEERTILITY: due to disorders
DHEA: DEHYDROEPIANDROSTERONE/
of sperm transport and function
Normal blood levels: ESTROGEN
testosterone, FSH and LH  DHEA
OTHER DISORDERS: o formed by adrenal cortex;
weak androgen;
 TESTICULAR FEMINIZATION o Androgen is derived from
SYNDROME adrenal gland.
o most severe form of androgens o Valuable in the assessment of
resistance syndrome resulting adrenal cortical function
in lack of testosterone action in
target tissues.  ESTROGEN
o Physical development pursues o Carbon-18 steroid hormone
the female phenotype, with that have a phenol A ring.
fully developed breast and o structural alteration of
female distribution of fat and testosterone molecule;
hair o produce by ovaries after
o No utility or response to menopause
administration of exogenous o FUNCTION: promotion of breast
testosterone development, maturation of the
o Lab tests: normal levels of external genitalia, deposition of
testosterone with elevated FSH body fat and termination of
and LH levels linear growth. (secondary
sexual characteristic in the
 SERTOLI-CELL-ONLY SYNDROME female)
o lack of germ cells, o PROGESTERONE: function in
o men with small testis, high FSH uterine growth and regulation
levels, azoospermia and normal of menstrual cycle and
testosterone levels maintenance of pregnancy
o testicular biopsy is the only o DEFICIENCY: irregular and
procedure to confirm this incomplete development of the
diagnosis endometrium
o PRECURSOR: acetate,
 KALLMANN’S SYNDROME cholesterol, progesterone, and
o Result of inherited, x-linked testosterone
recessive trait that manifest
hypogonadism during puberty
ESTROGEN o promotes uteroplacental blood
flow as potent as estrogen;
 Estrone and Estriol are metabolites of o used as marker for Down
intraovarian and extra glandular syndrome (together with AFP
conversion and HCG)
3 FORMS: o PREFFERED SPECIMEN: plasma

PROGESTERONE
 ESTRONE (E1):
o most abundant estrogen in  Carbon-21 compound in the steroid
post-menopausal women family
 Produced by the Lutein cells of the
 ESTRADIOL (E2): corpus luteum in the female
o most potent estrogen secreted  Prime secretory product of ovary.
in ovary, major estrogen;
 Dominant hormone responsible for the
o most abundant in
luteal phase cycle among females.
premenopausal women; low
 Single best hormone in the
levels in menopausal stage;
determination of ovulation if occurred.
o synthesized from testosterone
 Prepares the uterus for pregnancy and
then diffuses out of the thecal
lobules of breast for lactation
cells of the ovaries in female;
 Intermediate in the synthesis of adrenal
o precursors of E1 and E3
steroids and androstenedione.
o used to assess ovarian
 DEFICIENCY: failure of implantation of
functions; serves as negative
embryo
feedback for FSH
 METABOLITES: pregnanediols (easiest
o TRANSPORT PROTEIN: albumin
measured metabolite), pregnanediones,
(60%); SHBG (38%)
pregnanalones
o Free form of E3 is
approximately 2% SPECIFIC TESTS:

 ESTRIOL (E3)  Test of menstrual cycle dysfunction

o metabolite of estradiol; and anovulation: Estrogen,
o found in maternal urine; progesterone, FSH and LH
o major estrogen secreted on  Test for female infertility: HCG (human
fetal and placental during Chorionic Gonadotropin), PRL, FT$, TSH,
pregnancy-formation in a FSH, LH, ESTRADIOL, and
pregnant women is dependent PROGESTERONE
on fetal and placental function  Test of infertility male comes first.
o Used to assess fetoplacental
unit postdate gestations and
intrauterine retardation;
MODULES IN THE CANVAS makes use of two highly specific monoclonal
antibodies: A monoclonal antibody specific for
THYROID GLAND TSH is immobilized onto the microwell plate and
another monoclonal antibody specific for a
Thyroid Function Test different region of TSH is conjugated to horse
radish peroxidase (HRP). TSH from the sample
and standards are allowed to bind
simultaneously to the plate and to the HRP
conjugate. The washing and decanting steps
remove any unbound HRP conjugate. After the
washing step, the enzyme substrate is added.
The enzymatic reaction is terminated by
addition of the stopping solution. The
absorbance is measured on a microtiter plate
reader. The intensity of the colour formed by
Introduction the enzymatic reaction is directly proportional
to the concentration of TSH in the sample. A set
The thyroid gland is a butterfly-shaped of standards is used to plot a standard curve
endocrine gland that is normally located in the from which the amount of TSH in patient
samples and controls can be directly read.
lower front of the neck. The major thyroid
hormone secreted by the thyroid gland is Materials:
thyroxine, also called T4 because it contains
Manufacturer: Labor Diagnostika Nord GmbH
four iodine atoms. To exert its effects, T4 is
converted to triiodothyronine (T3) by the TSH TEST ELISA
removal of an iodine atom. This occurs mainly in
the liver and in certain tissues where T3 acts, 1. Precision pipettes to dispense 50, 100, 150
and 300 µl
such as in the brain. The amount of T4
2. Disposable pipette tips
produced by the thyroid gland is controlled by 3. Distilled or deionized water
another hormone, which is made in the 4. Plate shaker
pituitary gland located at the base of the brain, 5. Microwell plate reader with a filter set at
called thyroid stimulating hormone 450nm and an upper OD limit of 3.0 or
(abbreviated TSH). The amount of TSH that the greater*
pituitary sends into the bloodstream depends
Procedure:
on the amount of T4 that the pituitary sees. If
the pituitary sees very little T4, then it produces 1. Prepare working solutions of the anti-TSH-
more TSH to tell the thyroid gland to produce HRP conjugate and wash buffer.
more T4. Once the T4 in the bloodstream goes 2. Remove the required number of microwell
above a certain level, the pituitary’s production strips. Reseal the bag and return any
of TSH is shut off. unused strips to the refrigerator.
3. Pipette 50 µl of each calibrator, control and
Principle of the test: specimen sample into correspondingly
labelled wells in duplicate.
The principle of the following enzyme 4. Pipette 100 µL of the conjugate working
immunoassay test follows a typical one-step solution into each well. (We recommend
capture or ‘sandwich’ type assay. The assay using a multichannel pipette).
5. Incubate on a plate shaker (approximately  changes in binding protein levels don’t
200 rpm) for 90 minutes at room affect free T4levels, many healthcare
temperature. professionals prefer to measure free T4.
6. Wash the wells 3 times with prepared wash
buffer (300 µL/well for each wash) and tap c. T3 test
the plate firmly against absorbent paper to
ensure that it is dry (The use of a washer is  If your health care professional thinks you
recommended). may have hyperthyroidism even though
7. Pipette 150 µL of TMB substrate into each your T4 level is normal, you may have a
well at timed intervals. T3 test to confirm the diagnosis.
8. Incubate the plate on a plate shaker at  Sometimes T4 is normal yet T3 is high, so
room temperature for 15-20 minutes. (or measuring both T4 and T3 levels can be
until Calibrator A attains dark blue colour useful in diagnosing hyperthyroidism.
for desired OD).
9. Pipette 50 µl of stopping solution into each d. Thyroid antibody tests
well at the same timed intervals as in step
7. 10. Read the plate on a microwell plate  Measuring levels of thyroid antibody (Links
reader at 450 nm within 20 minutes after to an external site.)may help diagnose an
addition of the stopping solution. autoimmune thyroid disorder such as
Grave's disease—the most common cause
of hyperthyroidism—and Hashimoto's
disease the most common cause of
hypothyroidism.
 Thyroid antibodies are made when your
immune system attacks the thyroid gland
a. TSH test
by mistake.

 A high TSH level most often means you Pancreatic Hormones

have hypothyroidism, or an underactive
thyroid.
 This means that your thyroid isn’t making
enough hormone. As a result, the pituitary
keeps making and releasing TSH into your
blood. A low TSH level usually means you
have hyperthyroidism, or an overactive
thyroid.
 This means that your thyroid is making too
much hormone, so the pituitary stops
making and releasing TSH into your blood.
Introduction
b. T4 tests
Pancreas is both exocrine and endocrine gland.
 A high blood level of T4may mean you have The exocrinal part secretes pancreatic fluid into
hyperthyroidism. the duodenum after a meal. The endocrinal part
 A low level of T4may mean you have secretes various types of hormones. These are
hypothyroidism. produced by a specialized tissue in the pancreas
and then released to the capillary system and
reached the liver by the portal venous
circulation. The specialized tissue is called islets Procedure:
of langerhans. Islets of Langerhans represent
approximately 1-2 % of the pancreas. 1. Samples and standards were prepared off
line and 50 µL of each was pipetted
Principle of the test: manually into the assay plate.
The human insulin assay used is a sandwich 2. After loading samples and standards, 50 µL
ELISA. The solid phase microplate is coated with of assay conjugate was added to all
a blend of monoclonal antibodies directed wells. The plates were incubated at room
against distinct epitopes of the human insulin temperature for 30 minutes on a
peptide. Samples, standards and controls are microplate rotary shaker.
pipetted into these wells, along with a 3. After incubation the plates were
conjugate monoclonal antibody against human transferred to ELx405 Automated
insulin, which has horseradish peroxidase (HRP) Microplate Washer and washed three times
covalently linked to it. During the incubation with 400 µL of washer buffer with a 15
the insulin antigen binds to the immobilized second soak period between wash
capture antibodies. At the same time the cycles. After washing, 100 µL of substrate
conjugate antibody is binding to the insulin solution was added.
present in the well forming the sandwich. After 4. Plates were returned to the microplate
washing to remove unbound materials, a shaker and the color development allowed
chromogenic-substrate for HRP, consisting of for 7.5 minutes.
tetramethylbenzidine (TMB) and hydrogen 5. After color development, 100 µL of Sulfuric
peroxide (H2O2) is added. The substrate is acid stop solution was added and the
converted to a colored compound by the action absorbance of each well at 450 (650 nm
of HRP. The reaction is halted by the addition of reference wavelength) was determined
H2SO4 and the microplate is read using Synergy™ Mx Multi-Mode Microplate
spectrophotometrically. The absorbance of the Reader (BioTek Instruments).
solution is proportional to the amount of insulin
present in the original sample. Results of the test:

Materials Normal values for Insulin Determination

Optimal: 3-8 uIU/mL (18-48 pmol/L)

1. BioTek (Machine and commercially
prepared reagent) Low: <3 uIU/mL (<18 pmol/L)
2. Serum sample
3. Pipettes High: >8 uIU/mL (>48 pmol/L)

Low Insulin

Many labs cannot detect insulin lower than 2

uIU/mL. In such cases the result is reported as
“below detectable limits.” If there is no
detectable fasting insulin ( <2 uIU/mL, or <12
pmol/L) and blood sugar is significantly elevated
(ex HgbA1C >6.4%, or >46 mmol/L), the patient
could have type 1 diabetes or Latent
Autoimmune Diabetes of Adulthood (LADA) and
should be immediately assessed.
High Insulin Principle of the test

High insulin on a fasting blood test indicates a

chronically anabolic state – effectively storing
sugars while building fat and muscle. Practically,
high insulin results in central obesity and high
blood pressure. High fasting insulin is rare in a
thin or frail person. Future risk of type 2
diabetes begins to go up in those with fasting
insulin above 8 uIU/mL (48 pmol/L).

Sex Hormones

A. Total testosterone

Generally draw blood for testosterone lab tests

between 7 a.m. and 10 a.m. Total testosterone
measures free, albumin bound and SHBG bound
testosterone in the blood and is the most
commonly used measure of testosterone levels.
It is the test usually used to assess testosterone
levels in men with suspected hypogonadism or
Introduction
testosterone deficiency. There are several
Sex hormone, a chemical substance produced methods available to measure total
by a sex gland or other organs that has an effect testosterone.
on the sexual features of an organism.
B. Free Testosterone
Progesterone, testosterone, androgens,
estrone, and estradiol are steroid hormones A test for free testosterone measures only the
synthesized either in the testes, ovaries, or 2% of testosterone which remains unbound to
adrenal glands. Each of these steroid hormones proteins in blood. Free testosterone levels may
has cholesterol as its precursor. be measured in men whose total testosterone
test results showed testosterone levels which
are on the borderline of levels considered to
indicate testosterone deficiency.

Materials

1. Morning blood sample for men

2. reagents for immunoassays

Procedure

A. Total testosterone

Automated immunoassay systems

Automated platform immunoassay systems are
testosterone tests in which most steps (e.g.
adding the blood sample, adding reagents) are
performed automatically by a machine, rather
by people in the laboratory. These tests are the
fastest method of assessing testosterone levels.
They are considered accurate enough for
assessing testosterone levels in men but not in
women.

B. Free Testosterone

Direct immunoassay test kits

Direct immunoassay test kits are the easiest and

fastest available methods of assessing free
testosterone. They are test kits in which
reagents containing testosterone antibodies
and tracer particles (either radioactive or non-
radioactive) which bind to free testosterone in
the sample. The tracer particles are easily
identifiable and can be counted to measure the
concentration of free testosterone in the
sample. The results are less accurate than other
methods though and lower results are typically
obtained compared to equilibrium dialysis.

Results of the test

Testosterone level range:

MEN: 300 to 1,000 nanograms per deciliter

(ng/dL).

WOMEN: 15 and 70 ng/dL.

Testosterone levels can decrease naturally due

to your age or other health conditions. After the
age of 40, men’s testosterone levels decrease
by an average of at least 1 percent every year.
Some symptoms of low testosterone,
particularly erectile dysfunction, are commonly
seen in men over 40. Low testosterone levels
have often been observed in people with
obesity, no matter their age. The most common
testosterone-related problem in men is
hypogonadism, also called low testosterone.
CC2-MODULE 6- LABORATORY  REGULATORY TOXICOLOGY
o Combined data from
TOXICOLOGY AND THERAPEUTIC DRUG mechanistic and descriptive
MONITORING studies are used to establish
standards that define the level
TOXICOLOGY
of exposure that will not pose a
 Absorption of toxins in the GIT is by risk to public health or safety
passive diffusion – this process requires
SPECIALTIES WITHIN TOXICOLOGY
that the substance cross cellular
barriers  FORENSIC TOXICOLOGY
 Toxins that are not absorbed from the o Is primarily concerned with the
GIT not produce systemic effects but medical and legal consequences
may produce local effects- diarrhea, of exposure to chemicals or
bleeding and malabsorption of drugs; used in medico legal
nutrients o Used in establishing and
 In cases of drug overdose, CBC, serum validating the analytic
electrolytes, BUN, glucose, urinalysis performance of test method
and blood gas must be determined
 The study of the adverse effects of  CLINICAL TOXICOLOGY
xenobiotics in humans o Focuses on the relationships
o XENOBIOTICS are chemicals between xenobiotics and
and drugs are not normally disease states; not only for
found in or produced by the diagnostic but also for
body therapeutic intervention

THREE MAJOR DISCIPLINES:

 ENVIRONMENTAL TOXICOLOGY
 MECHANISTIC TOXICOLOGY o Includes the evaluation of
o Elucidates the cellular, environmental chemical
molecular, and biochemical pollutants and their impact on
effects of xenobiotics human health
o Effect of a toxin
TERMINOLOGIES

 DESCRIPTIVE TOXICOLOGY  POISONS- are any substances that

o Uses the results from animal affect biological function in other
experiments to predict what organism.
level of exposure will cause
harm in humans (risk  TOXINS- are biologically produce
assessment) chemical substances that affect
biological functions of programs
  TOXICANT- synthesized chemical POISONS
substances that affects other organism
 Exogenous agents that have an adverse
on a biological system
 ACUTE TOXICITY- single, short term
exposure to a substance  More often used when describing
substances from an animal, plant,
mineral, or gas.
 CHRONIC TOXICITY- repeated exposure
for extended period of time  EXAMPLES: venoms from poisonous
snakes or spiders, poison hemlock,
arsenic, lead, and carbon monoxide
 TD50- is the dose that would be
predicted to produce a toxic response in TOXINS
50% of the population
 Endogenous substances biologically
 LD50- is the dose that would predict synthesized either in living cells or in
death in 50% of the population microorganisms
 EXAMPLE: botulinum toxin produced
 ED50- is a dose that would be predicted from clostridium botulinum,
to be effective or have a therapeutic hemotoxins produces from venomous
benefit of 50% of the population snakes, wasps, and bees, and
mycotoxins produces from fungus;
 POISONOUS ORGANISM- secretes Plants- Psilosybin- mushrooms, cacti
chemical substances that affect
POISONIG CASES (WHO, 2012)
biological function of other organism

 VENOMOUS CREATURES- injects

chemical substances that gives impact
to biological function in other organism

XENOBIOTICS, POISONS, AND TOXINS

XENOBIOTICS

 Define as exogenous agents that can

have an adverse effect on a living
organism ROUTES OF EXPPOSURE
 More often used to describe  Toxins can enter the body via several
environmental exposure to chemical or routes
drugs o Ingestion
 EXAMPLES: antibiotics and o Inhalation
antidepressants; chemical (per o Transdermal absorption
fluorinated and brominated
compounds)
 ***For most toxins to exert systemic effect, o Can be used to evaluate the
they must be absorbed into circulation doses of therapeutic drugs
 The ED50 is the dose that would be
***Toxins that are not absorbed from the
predicted to be effective or have a
gastrointestinal tract do not produce systemic
thera peutic benefit in 50% of the
effects, but may orduce local effects (diarrhea,
population; effective dose
bleeding, and malabsorption) whichc may cause
 The therapeutic index is the ratio of
systemic effects secondary to toxin exposures
the TD50 (or LD50) to the ED50
DOSE-RESPONSE RELATIONSHIP o Drugs with a large therapeutic
index demonstrate fewer toxic
 The concept that all substances have adverse effects when the dose
the potential to cause harm, even of the drug is in the therapeutic
water is a central theme in toxicology range.
 Paracelus (1493 to 1591) pionereed
the use of chemicals in medicine and
coined the term “the dose makes the
poison”
 Fundamental and essential to modern
toxicology
 To enable assessment of substances’
potential to cause pathololgic effects, it
is necessary to establish an index of the
relative toxicities of the substancs

TOXICITY RATING SYSTEM

DOSE-RESPONSE RELATIONSHIPS MAY APPLY
TOXICITY RATING LETHAL ORAL DOSE IN
TO AN INDIVIDUAL OR A POPULATION
AVERAGE ADULT
Super toxic <mg/kg  INDIVIDUAL DOSE-RESPONSE
Extremely toxic 5-50 mg/kg RELATIONSHIP relates the individual’s
Very toxic 50-500 mg/kg health status as well as changes in
xenobiotic exposure levels
Moderately toxic 0.5-5 g/kg
 QUANTAL DOSE-RESPONSE
Slightly toxic 5-15 g/kg RELATIONSHIP describes the change in
P:ractically nontoxic >15 g/kg health effects a defined population
based on changes in the exposure to
 The TD50 is the predicted dose that
the xenobiotic
would produce a toxic response in 50%
of the population; toxic dose ACUTE AND CHRONIC TOXICITY
 The LD50 is the predicted dose that
would result in death in 50% of the Are terms used to relate the duration and
population; lethal dose frequency of exposure to observed toxic effects.
  ACUTE TOXICITY is usually associated  Interference with enzymatic
with a single, short-term exposure to a actions
substance in which the dose is sufficient  Formation of metabolites which
to cause immediate toxic effects are more toxic than the parent
 CHRONIC TOXICITY is generally poison
associated with repeated and frequent  Effects on DNA.
exposure for exrtended periods of time
(monthe to years) at doses that are CLASSIFICATION OF TOXIC AGENTS
insufficient to cause an immediate
 According to the target organ they are
acute response
acting upon. E.g hepatoxic, nephrotoxic
***Dose-response relationship may differ for  According to their use: E.g. food
acute and chronic exposures for the same additives, drug, pesticides
xenobiotic  According to source: animal or plant
 According totheir effefct: Carcinogen or
SITES OF TOXIC ACTION mutagen
 According to physical state: gas or liquid
 LOCAL (NON-SPECIFIC) : wherever the
 According to their chemistry: amines or
poison contacts the biological system it
hydrocarbons.
starts its harmful effects, does not
 According to their poisoning
require specific site or receptos to elicit
potentiality: extremely toxic, slightly
its effect as toxicity by acids or alkaline
toxic
 According to their biochemical
 REMOTE ( SYSTEMIC) : the poison
mechanisms of action: alkalating agent
affects a system far from portal of entry
FACTORS AFFECTING ACCTION OF POISON
 LOCALL AND REMOTE : the poison has
the capacity of acting locally and FACTORS RELATED TO THE POISON:
systematiclally; EXAMPLe: Oxalic Acid
 Dose- basic principle of toxicology;
TYPES OF TOXIC MECHANISM interference/increase dose, will
increase severity of toxicity
A. DIRECT: poison itself can cause toxic  Physical status- gaseous state is more
effects as in corrosives toxic than liquid state than solid state
B. INDIRECT: Toxicity results from the  Purity- this depends on the impurity of
interaction of the poison with the the poison. If the impurity is more toxic
biological activity of the poison within than the poison, the toxic will be more
the system and vice versa
 Binding to cell membrane to
change in their function or
structure thus affecting their
normality
 FACTORS RELATED TO THE screening tests that provide a
INDIVIDUAL: result of positive (drug is
present) or negative (drug is
 Age absent).
 Health  More specific method
 Sensitivity
 Gender QUALITATIVE METHODS

COLOR TESTS-rapid, easilyperformed but

FACTORS RELATED TO MODE OF not specific
EXPOSURE:
 Ferric chloride test for salicylates (pink
 Inhalation > IntraMuscular> purple)
ingestion> skin contact  Zwikker test for barbiturates (purple
color)
FACTORS RELATED TO ENVIRONMENT:  Formaldehyde-Sulfuric Acid test for
benzodiazipine (orange)
 Temperature pressure,
humidity, radiation can cuse  Mandalin Test for opioid 9brown)
alterations on poisons’ status CHEMICAL TEST
METHODS OF ANALYSIS  Reinsch Test- an initial indicator to
SCREENING TESTS detect presence of one or more of the
 Is a rapid, simple, qualitative following heavy metalas
procedure intended to detect  Antimony, Arsenic, Bismuth, Selinium,
the presence of specific Thalium, Mercury
substance or classes of QUANTITATIVE METHODS;
toxicants.
 Have good analytic sensitivity CHROMATOGRAPHY
by lack specificity
THIN LAYER CHROMATOGRAPHY TEST
 A negative result can rule out a
drug or toxicant and a positive  Mobile phase (a mixture of organic
result should be considered a solvents such as chloroform and
presumptive positive until methanol is run across a stationary
confirmed by a second, more phase) silica gel spread on a glass plate.
specific method  Stahl provided methods for 264 stains
CONFIRMATORY TEST or dyes that can be applied for the
required component such as ninhydrin
 Is generally quantitative and will react with amphetamine to give a
report the concentration of the pink color.
substance in the specimen in  Flourescent dye can be incorporated in
contrast to qualitative the solid phase, so that ultraviolet light
 can reveal the sample components as  Mass spectrometry: as sample
dark spots against the bright components exit the GCcolumn, they
background. are routed into a vacuum chamber
where they hit with a beam of
electrons, This knocks electrons off the
sample molecules, creating positive
electrons and breaking them into
fragments. These fragments are then
passed through an electromagnetic
field, which separates them by their
mass/charge ratio. The resulting
spectrum plotting the abundance and
mass/charge ratio of each fragment is
specific for a given substance

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY
GAS CHROMATOGRAPHY-MASS  Stationary phase is acolumn packed
SPECTROMETRY with solid particles and the mobile
 Stationary phase is a liquid and the phase is a liquid solvents.
mobile phase (carrier gas) is an inert gas  As the mobile phase is injected. A
like helium or nitrogen detector then identifies the
 Use 2 types of columns in GC: components a s they exit the column.
o Packed column: liquid is  Components are identified by their Rt,
coateed onto particles packed the length of time they take to pass
into a stainless steel or glass through the column and the results are
column compared with standards.
o Capillary column: the liquid is IMMUNOASSAYS;
coated onto the walls of the
column itself, which is narrow  Enzyme Multiplied Immunoassay
and made of glass. Technique/EMIT- drugs in URINE
 Samples are injected into a heated port,  Polarization Immunoassay
where they are vaporized and carried
into the column along with thae carrier TOXICOLOGY OF SPECIFIC AGENTS:
gas. A detector then produces a signal ALCOHOL
components exit the column.
 This is aplot of electronic signal versus  Common Central Nervous System
time (Rt- retention time) which shows depressants
series of peaks that corresponds to the  May cause disorientation, confusion,
components of the sample. euphoria, progresses to
 unconciousness, paralysis. High level INDICATORS OF ETHANOL BLUE
exposure may end with death. TEST COMMENTS
 Symptoms of alcohol intoxication begin
GGT > Increase can be seen before the onset of
when the concentration in > 0.05% w/v pathologic consequences
(>50mg/dL blood alcohol) >Increases in serum activity can occur in many
non-ethanol related conditions
AST >Increases in serum activity can occur in many
non-ethanol related conditions
AST/ALT > A ratio of >2.0 is highly specific for ethanol-
1. ETHANOL (GRAIN ALCOHOL) ratio related liver disease
 Inhibits ADH → Cause diuresis HDL > High serum HDL is specific for ethanol
consumption
 Most common abused drug; a CNS
MCV > Increase erythrocyte MCV is commonly seen
depressant
with excessive ethanol consumption
 Causes diuresis by inhibiting ADH > Increases are not related to folate or Vitamin
 Readily absorbed in GIT and diffuses B12 deficiency
easily in tissue. 2. METHANOL (WOOD ALCOHOL)
 Ethanol abuse causes acidosis through  Commonly used solvent and
accumulation of ketones and lactate contaminant of homemade liquors
and also through direct generation of  Formaldehyde to formic acid (alcohol
hydrogen ions as alcohol is ozidized dehydrogenase) severe acidosis
 Ethanol abuse: Lipid accumulation in  Formic acid causing optic neuropathy to
hepatocytes; alcoholic hepatitis; toxic blind ness
hepatitis; cirrhosis  Symptoms of intoxication: frank
 Intoxication SS: blurred vision; blindness (ocular toxicity) and
incoordination; slurred speech and metabolic acidosis
coma; hangover symptoms-due to the  Screening test: osmolal gap
effect of acetaldehyde  METHOD: GC-MS
 Antidote: Diazepam (for alcoholic  FATAL DOSE: 60-250 mL
mania)  TOXIC BLOOD LEVEL: >50 mg/dL
 SPECIMEN Precaution: spx must be
capped at all times to avoid evaporation 3. ISOPROPANOL (RUBBING ALCOHOL)
of alcohol; prior to blood collection,  Rapidly absorbed by GIT
alcohol-free skin cleanser must be used  Metabolized by hepatic alcohol
instead of isopropanol dehydrogenase to acetone (long life)
 SPECIMEN: serum severe acute phase
 MAJOR METABOLIC PATHWAY:  Depressant effect. Activated charcoal
conversion of ethanol to acetaldehyde  SYMPTOMS INTOXICATION: CNS
and acetyl coenzyme A by hepatic depression and hypertension
alcohol dehydrogenase  INDICATION OF TOXICITY: elevated
 METHOD: enzymatic using alcohol levels of acetone in the blood and urine
dehydrogenase reagent  METHOD: gas chromatography
  ANTIDOTE: activated charcoal  PHASES OF PROGRESSION OF
 FATAL DOSE: 250 mL ALCOHOLISM:
o Pre-alcoholic phase
4. ETHYLENE GLYCOL (1,2-ethenediol) o Prodomal phase
 Common component of hydraulic fluid o Crucial phase
and anti-freeze o Chronic phase
 Metabolism resulting to oxalic acid and  POSSIBLE OUTCOMES OF
glycolic acid causing severe metabolic ALCOHOLISM: (BAD)
acidosis o B- rain damage
 Complications: rapid deposition of o A- lcoholic hallucination
calcium oxalate crystals in renal o D- eath
tubules-renal tubular damage.  COMMON BEHAVIORAL PATTERNS OF
 SYMPTOMS OF INTOXICATION: ALCOHOLIC PATIENTS (5D)
metabolic acidosis, depressed reflexes, o Denial
anuria and necrosis o Dependency
 INDICATION OF TOXICITY: deposition o Demanding
of calcium oxalate crystals in renal o Destructive
tubules o Domineering
 MODE OF TREATMENT: inhibit the  COMMON WITHDRAWAL SIGNS AND
action of alcohol dehydrogenase SYMPTOMS (HITS)
 MAJOR METABOLITE: glycolic acid o Hallucination (visual or tactile)
(cause of acute toxicity and death) o Increased vital signs
 METHOD: HPLC o Tremors
 FATAL DOSE: 100g o Sweating and seizures
 COMMON DEFENSE MECHANISM BY
ALCOHOLISM ALCOHOLIC PATIENTS (DRIP)
o Denial
 A chronic disease or a disorder
o Rationalization
characterized by excessive alcohol
o Isolation
intake
o Projection
 Causes interferences in:
o The individual’s health TREATMENT:
o Interpersonal relationships
o Economic functioning  DRUG OF CHOICE: DISULFERAM
 LEVEL OF INTOXICATION (ANTABUSE)
o 0.1-0.2%- Low coordination  ACTION: delays metabolism of alcohol
o 0.2-0.3%- Presence of ataxia,  SIDE EFFECT: nausea, abdominal
tremors, irritability cramps, sweating, and more severe
o 0.3 and above- includes neuritis, and polyneuritis,
Unconsciousness drowsiness, skin eruptions
 IMPORTANT NOTE: instruct the patient to
avoid alcohol based substances for at least SYMPTOMS OF CARBOXYHEMOGLOBINEMIA
12 hours before giving Antabuse COHb (%) SYMPTOMS AND COMMENTS
0.5 Typical in non-smokers
 ETHANOL ENZYMATIC METHOD 5-15 Range of values seen in smokers
PRINCIPLE: 10 Shortness of breath with vigorous exercise
20 Shortness of breath with moderate
exercise
30 Severe headaches, fatigue, impairment of
40-50 Confusion, fainting on exertion
CARBON MONOXIDE 60-70 Unconsciousness, respiratory failure,
death with continuous exposure
 A colorless, odorless, tasteless gas; very 80 Immediately fatal
toxic substance CARBON MONOXIDE SPOT TEST
 Produced by incomplete combustion of  A qualitative test to identify the
carbon-containing substances like presence of Carboxyhemoglobin
gasoline.
 Odorless, colorless, incomplete
 Improperly ventilated furnaces, wood combustion
or plastic fire, and rubber. This includes
 Sources:
cigarette smoking o Tobacco or cigarette smoking
 CO binds with heme proteins o Grilling
(cytochromes, hemoglobin and o Vehicles
myoglobin) CO + Cytochrome A3 results  SIGNS AND SYMPTOMS OF CO
to inhibition of cellular respiration and POISONING: headache, dizziness,
electron transport
contusion, muscle pain, hallucination,
 CO + Hgb (myoglobin) reduces oxygen depression, impaired mental state. Fatal
supply to cardiac and skeletal muscles episode is manifested with vomitous
or direct muscle damage with whitish fluid
 CO + Hgb → COHb (200-225 x >  REACTION:
oxygen); Air 20% oxygen by volume o NaOH + Oxyhemoglobin →
 Causes decrease concentration of BROWN COLOR
oxyhemoglobin o NaOH + Carboxyhemoglobin
 High affinity for hemoglobin than does →CHERRY RED COLOR
oxygen (200x faster than oxygen)  PROCEDURE:
 SIGN OF TOXICITY: Cherry red colored 1. To 20 ml distilled water
face; affects brain and heart 2. Add 0.5 ml whole blood
 SAMPLE FOR TESTING: EDTA whole 3. Add 0.5 ml 1M NaOH
blood 4. Mix and observe
 DEFINITIVE METHOD FOR TESTING:  RESULT:
cooximetry (carboxyhemoglobin o BROWN COLOR: NORMAL
measurement) oxyhemoglobin is present
 o CERRY RED COLOR: HEAVY METAL TOXICITY
CARBOXYHEMOGLOBIN IS
PRESENT  All metals can be toxic if ingested in
 TREATMENT: Potassium Ferricyanide large quantities and absorbed in their
ionized forms.
“CLEAN AIR = INCREASE OXYGEN: CLEAR  Heavy metal poisoning can be acute or
MIND” chronic and may be caused by the
following:
NOTE: FOR THE DEGREE OF EXPOSURE AND
o Lead, Mercury, Iron, Arsenic
EFFECT
o Cadmium, Thallium, Bismuth
CYANIDE  Metals may enter the body by
ingestion, inhalation, absorption
 Exists as solid, gas or in solution through the skin or mucous membranes
 Super toxic; fast acting death may occur  Most common heavy metal poisoning:
in less than an hour Lead
 Components of insecticides and  Mercury can be found in the elemental
rodenticides; common suicidal agent state (dental amalgam, thermometers),
 Also a pyrolysis product- burning inorganic (industrial processes), and
plastics organic compounds (pesticides, wood
 Binds with iron (ferric or ferrous form) – preservatives, some medicines and
tissue or cellular hypoxia contaminated fish)
 Inhibits cellular respiration, electron
transport and ATP formation by ARSENIC
preventing reoxidation of cytochrome  Arsenic poisoning: depending on form
A3- inhibition of cellular respiration  3 MAJOR GROUPS OF ARSENIC
leads to metabolic acidosis due to
o Arsine gas
increased lactic concentration in the o Inorganic (trivalent or
blood pentavalent)
 Toxic effect is inhibition of electron o Organic form (arsenobetaine &
transport chain and cell death arsenocholine)
 INDICATION OF TOXICITY: Odor of  Found in sea foods as easily absorbed in
bitter almonds breath and altered GIT by passive diffusion.
mental status
 Component Ant poison, rodenticides,
 ANTIDOTE: sodium thiosulfate, amyl paints and metal alloys
and sodium nitrite  Common suicide and homicide agent;
 Toxic symptoms: tachypnea, common agent of heavy metal
convulsions and coma poisoning
 > 2 ug/MI Enough to be Toxic  Expresses its toxicity by high affinity
binding to the thiol groups in proteins.
  Inhibits sulfhydryl enzymes throughout  It may also accumulate in renal tubules
the body; crosses the placenta, high causing tubular damage
affinity to thiol groups in proteins  TOXIC INDICATOR: (+) GGT in urine
 King of poisons an poison of Kings- sample
bioaccumulation
LEAD
 SYMPTOMS: Headache, confusion,
severe diarrhea and drowsiness  A potent enzyme inhibitor- it blocks
 As the poisoning develops, convulsions delta aminolevulinic (ALA) synthetase,
and changes in fingernail pigmentation pyrimidine-5’-nucleotidase and Na-K-
called leukonychia may occur; use hair dependent ATPase
and nails as specimen for long term  Lead poisoning- Age- dependent
exposure “mees line” differences in the absorption: adult
 CHRONIC EXPOSURE: Vitamin A absorb 10% of a n ingested dose;
deficiency, heart disease, night Children 40%
blindness.  Initially distributed to the soft tissues
 INDICATION OF TOXICITY: “odor of and slowly redistributed to bone, teeth
garlic” breath and metallic taste and hair
 TOXIC EFFECTS: intravascular  Detected by x-ray
hemolysis, hemoglobinemia,  HALF-LIFE: 1-2 months in blood; 20-30
nephroxicity and multi-organ year in the bone;
involvement  CNS: headache, confusion, clumsiness,
 ANTIDOTE: British anti-Lewisite (BAL) insomnia, fatigue, impaired
 METHOD: AAS, Reinsch test concentration, disease progresses,
clonic convulsions and coma occur
CADMIUM
 Treated with chelation therapy
 Utilized In electroplating and  BLOOD: 5-20 ug/dl in children result to
galvanizing lower IQ levels:
 A significant environmental pollutant  TOXIC DOSE: >0.5mg/day
 Poisoning can result from ingestion of  FATAL DOSE: 0.5g
acidic foods stored or prepared in metal  CDC CUT-OFF LEVEL IN CHILDREN: <10
containers made up of cadmium ug/dL
 Significant environmental pollutant –  TOXIC BLOOD LEVELS: <10 ug/dL
pigment on piant and plastics  REQUIRES CHELATION THERAPY
 Poisoning can result from ingestion of (CHILDREN): <25 ug/dL
acidic foods stored or prepared in metal  LEAD CHELATORS: EDTA and
containers made up of cadium dimercaptosuccinic (DMA)
 Toxicity may result to destruction of  GIT: discomfort, constipation. Higher
type 1 epithelial cells in the lungs and exposures can produce painful
decreased resistance to bacterial intestinal spasms
infections
  BLOOD: hypochromic, microcytic  Consumption of food contaminated
anemia. Inhibit the heme synthesis with methyl-mercury (fish) cause
enzymes. organic mercury toxicity
 SOURCE: paints and gasoline  Visual disturbances, Parenthesias,
 MODE OF ACQUISITION: ingestion or ataxia, hearing loss, mental
inhalation deterioration, muscle tremors,
 INDICATION OF TOXICITY: urinary movement disorders, paralysis and
aminolevulinic acid, free RBC death
proporphyrin and presence of  SAMPLES: whole blood and 24-hour
basophilic stippling in RBC urine

MERCURY IRON

 Binds with sulhydryl proteins.  Iron is a vital mineral in the human

 A potent enzyme inhibitor- inhibits body
catecholamine-0-methyltransferase, an  Toxicity causes hemosiderosis and
enzyme essential in the metabolism of hemochromatosis
“catecholamines”  Iron toxic serum level:
 FORMS OF MERCURY: elemental or o Mild toxicity: 450-500 ug/dL
metallic mercury, mercurous, mercuric o Severe toxicity: 800-1000 ug/dL
and alkyl mercury  Toxic effect due to hemorrhagic
 MODES OF ACQUISITION: inhalation, necrosis in GIT
skin absorption, and ingestion
TREATMENT
 SYMPTOMS OF TOXICITY:
hypertension, tachycardia, and  Chelation therapy: Chelation agent
sweating-“cardinal signs” of plus metal = chelate
pheochromocytoma or mimics that  A chelating agent is a chemical
adrenal gland disorder compound or drug molecule capable of
 GENERAL TOXIC EFFECT: organ forming a heterocyclic ring with a metal
dysfunction- lungs, kidney, and CNS ion as its closing member.
 MAJOR TOXIC EFFECT OF ELEMENTAL
MERCURY: pink disease (acrodynia) and METALS ANTIDOTES
erethism LEAD Dimercapto succinic acid, Calcium
 MAJOR TOXIC EFFECT OF ALKYL disodium edetate
MERCURY: congenital Minimata disease MERCURY, Dimercaprol
 Ability to “Tremors, depression, ARSENIC, GOLD
memory loss, decreased verbal skills IROON Deferoxamine
and inflammation of the kidneys. Non- COPPER/MERCURY Penicillamine
selective pulmonary toxicity CYANIDE Sodium nirite, Sodium thiosulfate,
 An exposure to inorganic salts of Amyl Nitrite pearls
mercury (mercuric chloride) leads to
renal damage.
REINSCH TEST FOR HEAVY METALS  BISMUTH: Shiny Black is seen
with light
A qualitative screening test for the presence of
 MERCURY: Silver Gray
the following metals: Sb, As, Bi, Hg
THERAPEUTIC DRUG MONITORING
 Specimen:
o Gastric washing or lavage for  Must be monitored to determine what
acute poisoning doses are inadequate or excessive in
o Urine for chronic poisoning the treatment of the patient. Often, the
o Tissue biopsy (if observed that ingested drug (called the “parent” drug)
it has reach the tissues Reinsch is metabolized to form an active
test is not used) metabolite that produces an effect
 MATERIALS: similar to the parent drug
o 10cm COIL of Copper wire  Involves the analysis, assessment and
heavy gauge evaluation of circulating concentrations
o 2M Hydrochloric acid of drugs in serum, plasma or whole
o Water bath blood.
 PROCEDURE: (USING EARLY MORNING  Also it is a quantitative procedure
URINE/RANDOM) performed for drugs with a narrow
1. Using the 10cm coil of a heavy therapeutic index.
gauge of copper wire  It allows for the safe use of drugs that
2. Ad 10 ml of urine and 10 ml of would otherwise be potentially toxic.
2M HCI “acidification” to  Ensures that a given drug produces
enhance the reaction maximal therapeutic benefit and
3. Boil in the water bath for 10 minimal side effects; to achieve a
minutes and examine the constant serum level of the drug that
copper coil for any stain will be therapeutic
4. Remove from the water bath  Most drugs have a half-life independent
and let it stand for 1 hour. If no of their concentrations.
stain in the copper coil is found,  Half-life of the drug determines the
This could be due to delayed time to reach the steady-state or
reaction average concentrations
5. Detection: up to 5 microgram of
heavy metals in urine INDICATIONS FOR TDM
6. After the incubation period and
1. The consequences of overdosing and
there is no stain observed then
under-dosing are serious.
can be reported as NEGATIVE
2. There is a small difference between a
therapeutic and toxic dose
 RESULT:
3. There is a poor relationship between
 ANTIMONY: Blue/ Black Purple
the dose of drug and circulating
 ARSENIC: Flat Black
concentrations but a good correlation
 between circulations and therapeutic stream, and enter the extravascular
and toxic effect space, or they can migrate into various
4. There is a change in the patient’s tissues. This is referred to as
physiologic state that may be distribution, a process that typically
unpredictably affect circulating drug occurs between a period of 30 minutes
concentrations and 2 hours.
5. A drug interaction is or may be
occurring **the bioavailability of a drug is the amount of
6. It helps in monitoring patient drug that is absorbed into the system and is
compliance available for distribution.

D. METABOLISM is the process of

ROUTES OF ADMINISTRATION:
transformation of the parent drug
 Circulatory system is a convenient molecule to its metabolite(s).
route that can effectively deliver most Metabolites are usually water soluble
drugs to its site of action and can be easily excreted; most of the
 Intravenous metabolism occurs in the liver, where
 Oral -0.7 enzymes catalyze oxidation, reduction,
 Intramuscular or hydrolysis of the drug.
 Subcutaneous
 Inhalation E. ELIMINATION is the process of
 Suppository excretion of the drug from the body.
 Transcutaneous Drugs are typically excreted in the urine
but also can be eliminated in the feces,
PHARMACOLOGICAL PARAMETERS THAT sweat, expired air, and saliva
DETERMINES SERUM DRUG CONCENTRATION:
 ABSORPTION
A. LIBERATION – is the release of the
o Most drugs are absorbed by
ingredient, followed by the process of
passive diffusion
the drug passing into solution
Intestine → hepatic portal system (liver)
B. ABSORPTION – is the process by which →general circulation
the drug molecule is taken up into
systemic circulation. Following o Factors affecting absorption:
absorption through the intestinal changes in intestinal
mucosa, a drug traverses the hepatic movement, pH, inflammation,
system, where some drugs undergo and presence of food and other
substantial metabolism and elimination. drugs
This called first-pass elimination or
metabolism  DISTRIBUTION
o The location where drugs are
C. DISTRIBUTION- Drug molecules can be effective are in the body tissues
confined to the blood, leave the blood not generally in blood
 o Drugs diffuse widely throughout PHARMACOKINETICS is the mathematical
the body, frequently reaching interpretation of drug overtime to determine
higher concentrations in tissues proper dosing amounts of a therapeutic drug.
than in blood Pharmacokinetic responses are typically graphic
plots of blood concentration of the drug versus
 EXCRETION time, such as a dose-response curve.
o All drugs are excreted-either
Three kinetics processes are used to describe
unchanged in the urine or
the fate of drugs in the body over a period of
excreted as metabolite of the
time and can be illustrated in a dose-response
parent drug.
curve
o Rate at which a particular drug
is cleared from the circulation is 1) FIRST-ORDER KINETICS describe
dependent not only on the type absorption, distribution, and
of drug itself, but also on a elimination of drugs. This means that
patient’s capacity to metabolize the rate of change of concentration of a
and excrete it drug is dependent on the drug
concentration. It is represented by the
Basic principles. TDM measures drug
first phase of the dose-response curve.
concentrations during therapy with
pharmaceutical agents.
2) ZERO-ORDER KINETICS describes the
a) A steady-state drug level (complete rate of change of concentration of a
with peaks and troughs) exists for each drug that is independent of the
drug. When a single dose of a drug is concentration of the drug. That is a
administered orally, the blood level constant amount of drug is eliminated
changes markedly over time and, at per unit of time. This typically depends
some time, the concentration in the on the ability of the liver to metabolize
plasma reaches its peak (highest point) the drug. This is illustrated by the
and then declines. Immediately before second phase of the curve.
the next dose of medication, a trough
level occurs 3) MICHAELIS-MENTEN KINETICS state if a
1) For single-dose administration, the drug concentration in a system exceeds
rate of decline in concentration is the capacity of the system, the rate of
expressed in terms of half-life, change of concentration proceeds
which is the time required for the according to the Michaelis-Menten
concentration of the drug to equation.
decrease by 50%.
CAUSES OF DRUG TOXICITY
2) The half-life is different for each
drug. At steady-state levels, the 1) Elevated concentration of free drug
rate administration of the drug is 2) Abnormal response to drug after
allowing the drug level to remain administration
constant. 3) The presence of active metabolites
TERMINOLOGIES: o The mathematical expression of
the relationship between drug
 BIOAVAILABLE FRACTION (F) dose and drug blood level
o It is a fraction of dose that  THERAPEUTIC INDEX
reaches the blood
o Ration between minimum toxic
 Vd OF A DRUG and maximum therapeutic
o Represents the dilution of the serum concentration
drug after it has been  THERAPEUTIC RANGE
distributed in the body o the difference between highest
o It is used to estimate the peak and lowest effective dosages
drug blood level expected after
 TROUGH CONCENTRATION
loading dose is given
o The lowest concentration of a
o It is the principal determinant
drug obtained in the dosing
of the dose
interval
 FIRST-PASS HEPATIC METABOLISM
o Drugs that are transported to CARDIOACTIVE DRUGS
the liver lost a fraction of its
bioavailability before the drug  These drugs are divided into two
reaches the general circulation categories: the cardiac glycosides and
 FIRST-ORDER ELIMINATION the antiarrhythmic drugs. These agents
o Represents a linear relationship serve to maintain normal heart
between the amount of drug function.
eliminated per hour and the  Drugs are used for treatment of
blood level of a drug. arrhythmias and congestive heart
 HALF-LIFE (t1/2) failure.
o Time required to reduce a drug CLASSIFICATION OF CARRDIOACTIVE DRUGS:
level to half of its initial value
 PEAK CONCENTRATION  CLASS I – rapid sodium channel blockers
o The highest concentration of a o Quindine, Procainamide,
drug obtained in the dosing Lidocaine
interval  CLASS II - beta receptor blockers
 PHARMACODYNAMICS o Propanolol
o The relationship between the  CLASS III- K+ channel blockers
drug concentration at the target o Amiodarone
site and response of the tissues  CLASS IV- calcium channel blockers
 PHARMACOGENOMICS o Verapamil
o Refers to the study of genes
DIGOXIN
that affect the performance of a
drug in an individual  The major cardiac glycoside and alters
 PHARMACOKINETICS the force of contraction through its
 effect on the ATPase pump in heart The antiarrhythmic drugs are prescribed to
muscle treat irregular heart beat that produces
 Blood specimens must be collected 8 inappropriate ventricular contraction or
hours after a dose of digoxin is tachycardia (increased heart rate)
administered, because its peak
LIDOCAINE (XYLOCAINE)
concentration in tissue occurs 6 to 10
hours after administration  Used for the treatment of faulty
 Digoxin toxicity produces symptoms of ventricular contractions and
nausea, rapid heart rate, and visual arrhythmias. It binds to a1-acid
impairment. Digoxin is excreted as glycoprotein and is metabolized in the
digoxigenin in the urine. liver, producing two active metabolites
 Inhibits membrane Na-K-ATPase, thus it monoethylglycinexylidide and
decreases K+ and Mg+, and increases glycinexylide
Ca+ (cardiac contractility-inotropic  Used to correct ventricular arrhythmia
effect, 0.8-2 ng/mL) for treatment of acute myocardial
 25% is protein bound; free (nonbound) infarction
form is sequestered into muscle cells  Administered by continuous IV infusion
 Hyperthyroid individuals are resistant to after a loading dose
the action of digoxin  Can be used as a local anaesthetic
 Timing of blood collection after last  Bound to albumin and AAG
dose can be critical to interpretation of  Cannot be administered orally due to
the drug levels. almost complete hepatic removal of the
 HALF-LIFE: 38 hours (average adult) absorbed drug
 THERAPEUTIC LEVEL: 0.5-2 ng/mL  THERAPEUTIC LEVEL: 1.4-4.0 ug/mL
 TOXIC LEVEL: > 2 ng/mL  TOXICITY LEVEL: > 4.0 ug/mL
 The antiarrhythmic drugs are  CNS DEPRESSION: > 4-8 ug/mL
prescribed to treat irregular heart beat  SEIZURE AND DECREASED BP AND
that produces inappropriate ventricular CARDIAC OUTPUT: >8 ug/mL
contraction or tachycardia (increased  TOXIC EFFECT: CHF and heart block
heart rate)
 ELIMINATION: by hepatic metabolism;
 ELIMINATION: renal filtration of the changes in renal function have little
plasma free form effect
 PEAK SERUM LEVEL: 8 hours after an  PRIMARY PRODUCT OF HEPATIC
oral dose METABOLISM: monoethylglycinexy
 TOXIC EFFECTS: nausea, vomiting, visual lidide (MEGX); glycinexylide
disturbances, premature ventricular
contractions and atrioventricular node QUINIDINE
blockage.
 A myocardial depressant that decreases
that heart’s ability to conduct current. It
is metabolize in the liver to produce
 several active metabolite, including 3-  COMMON ROUTE: oral
hydroxyquinidine.  HEPATIC METABOLITE: N-acetyl
 If quinidine is added to digoxin therapy procainamide (NAPA)
regimen, an interaction occurs that
induces an increase in digoxin DYSOPYRAMIDE
concentration
 It stabilizes the heartbeat. It is both
 A naturally occurring drug for the excreted by the renal system as the
treatment of arrhythmia unchanged drug and is metabolized in
 85% protein-bound; GIT absorption is the liver to form an inactive metabolite
complete and rapid for the sulfate.  Used to treat cardiac arrhythmias; used
 PEAK SERUM LEVEL:2 hours after oral as substitute for quinidine
dose (sulfate); 4-5 hours (gluconate)  Administered orally; GIT absorption is
 THERAPEUTIC LEVEL: 2.3-5 ug/mL complete and rapid
 TOXIC LEVEL: >5 ug/mL  Has anticholinergic – dry mouth and
 TOXIC EFFECTS: cinchonism, blood constipation (> 4.5 ug/mL)
dyscrasia and hepatitis  THERAPEUTIC LEVEL: 3-5 ug/mL
 ELIMINATION: hepatic metabolism  TOXIC LEVEL: 10 ug/mL
 ROUTE OF DELIVERY: oral  TOXIC EFFECTS: bradycardia and
administration atrioventricular node blockage
 COMMON FORMULATIONS: quinidine  ELIMINATION: renal filtration
sulfate and quinidine gluconate
PROPANOLOL
PROCAINAMIDE (PRONESTYL)
 Prescribed for atrial and ventricular
 Used to treat inappropriate ventricular arrhythmias and hypertension. It is
contractions and tachycardia. This drug considered to be a beta-blocker
is metabolized in the liver to form an  A beta-receptor blocking drug; used in
active metabolite, N- the treatment of angina pectoris,
acetylprocainamide, which produces hypertension and coronary artery
the same effect as its parent drug. disease
Therefore, serum levels of both drugs
 Suppresses the conversion of T4 and T3-
must be analyzed
used in the treatment of throtoxicosis
 Administered orally
 THERAPEUTIC LEVEL: 50-100 ng/mL
 GIT absorption is rapid and complete
 TOXIC EFFECTS: bradycardia, arterial
 20% protein-bound; eliminated by renal insufficiency (Raynaud’s type), platelet
filtration and hepatic metabolism disorder and pharyngitis
 PEAK SERUM LEVEL: 1 hour after dose
 THERAPEUTIC LEVEL: 4-10 ug/mL AMIODARONE (CODARONE)
 TOXIC LEVEL: >12 ug/mL
 Blocks potassium channels in the
 TOXIC EFFECTS: reversible lupus-like
cardiac muscle and used for treatment
syndrome (ANA), nephrotic syndrome,
of ventricular arrhythmias
urticarial
  An iodine-containing drug which can  Toxic side effects occur in the
cause hyperthyroidism or therapeutic range (5-10 ug/mL)
hypothyroidism  Only the trough levels are monitored to
 THERAPEUTIC LEVEL: 1.0-2.5 ug/mL ensure the serum drug concentration is
 TOXIC LEVEL: >2.5 ug/mL within the therapeutic range.
 TOXIC EFFECTS: bradycardia, hepatitis,  TOXIC LEVEL: >1ug/mL- nephrotoxicity;
photodermatitis and thyroid >40 ug/mL - ototoxicity
dysfunction  TOXIC EFFECTS: red-man syndrome,
nephrotoxicity and ototoxicity
VERAPIMIL
 ELIMINATION: renal filtration and
 For treatment of angina, hypertension excretion
and supra ventricular arrhythmias
CHLORAMPHENICOL
 THERAPEUTIC LEVEL: 80-400 ng/mL
 TOXIC EFFECTS: hypotension, peripheral  It distributes to all tissues and it
edema and ventricular fibrillation concentrations in CSF
 50% protein bound; rapidly absorbed in
ANTIBIOTICS the GIT
AMINOGLYCOSIDES (GENTAMICIN,  TOXIC LEVEL: >25 ug/mL
TOBRAMYCIN, AMIKACIN, KANAMYCIN,  TOXIC EFFECTS: blood dyscrasia,
NEOMYCON, STREPTOMYCIN) cytoplasmic vacuolation (erythroid and
myeloid cells)
 Used for treatment of gram-negative
bacterial infections; not given to ANTICONVULSANTS/ANTIEPILEPTIC DRUGS
outpatient
 Function to alter transmission of nerve
 Administered IM or IV; not well
impulses within the brain to minimize
absorbed from the GIT
the seizures of epilepsy.
 May cause damage to the 8th cranial
 general seizure- both sides of brain;
nerve at toxic level (hearing loss)
petit mal/absence seizure- rapid
 TOXIC LEVEL: > 30 ug/mL (amikacin and blinking or few seconds of space; grand
kanamycin)- peak levels; 12-15 ug/mL
mal seizure- loss of consciousness
(gentamicin and tobramycin)- peak
levels PHENOBARBITAL
 TOXIC EFFECTS: nephrotoxicity and
 Long-acting barbiturate that controls
ototoxicity
grand mal tonic-clonic seizure and focal
VANCOMYCIN epileptic; not used for petit mal seizure;
 Used for treating withdrawal symptoms
 Glycpeptide effective against gram- in infants – mothers are addicted to
positive cocci and bacilli opiate or barbiturate
 Poor oral absorption; administered by
IV infusion
  Used to treat cases of congenital  Decreases sodium and calcium influx
hyperbilirubinemia – this drug enhances into hyperexcitable neurons
bilirubin metabolism  IV administered; GIT absorption is
 Absorption is slow but complete; 50% incomplete
orotein-bound; majority is stored in the  87-97% protein-bound; free ( unbound)
brain form is the biologically active portion
 Used to treat all types of seizures  INJECTABLE PERFORM: fosphenytoin
except absence seizures. It is effective  THERAPEUTIC LEVEL: 10-20 ug/mL; 1-2
in children and neonates. It is ug/mL (free form)
metabolized in the liver, and serum  TOXIC LEVEL: >20 ug/mL
concentrations increase during the  MAJOR TOXICITY: initiation of seizures;
administration of valporic or salicylic tetratogenic action (cleft lip and palate)
acid. andynystagmus
 INACTIVE FORM: primidone (mysoline)
 HALF-LIFE: 70-100 hours VALPROIC ACID (DEPAKENE)
 PEAK SERUM LEVEL: 10 hours after an
 Used for treatment of petit mal
oral dose
(absence seizure(, atomic seizure and
 THERAPEUTIC LEVEL: 20-440 ug/mL grand mal
(phenobarbital); 5-12 ug/mL
 Orally administered; Git absorption is
(primidone)
rapid and complete
 TOXIC EFFECTS: nystagmus, stupor,
 93% highly protein bound
ataxia and respiratory depression
 Hepatic dysfunction is observable which
**PRIMIDONE is metabolized in the liver to requires monitoring after 6 months of
form phenobarbital. Therefore, dual analysis therapy
must be performed to determine the proper  Prescribed for absence (petit mal)
dosage of this drug. It is used to treat both seizures. Valproic acid affects many
grand mal and complex-partial seizures. others anticonvulsants by inhibiting
their metabolism in the liver, thus
PHENYTOIN (DILANTIN) increasing serum concentration.
 THERAPEUTIC LEVEL: 50-100 ug/mL
 Corrects grand mal seizures. It is
 TOXIC LEVEL: >100 ug/mL (nausea,
metabolized by the liver and can
lethargy, weight gain); >200 ug/mL
interact with several drugs that induce
(pancreatitis, hallucinations and
increased serum concentration or
hyperammonemia)
increased metabolism or phenytoin.
 Controls seizures (tonic-clonic, simple CARBAMAZEPINE (TEGRETOL)
partial seizures); a short-term
prophylactic agent in brain injury  Typically used for treatment of various
 Not used for petit mal and atomic seizures and facial pain.
seizures  Tricyclic compound related to
imipramine (TCA)
  Effective for grand mal seizures and TOPIRAMATE
treating seizures accompanied by pain
 Used as an adjunct drug for partial
 Has antineuralgic action; 70-80%
seizures
protein-bound
 Has serious effects and not frequently LAMOTRIGINE (LAMICTAL)
used; orally administered
 IDIOSYNCRATIC EFFECT: rashes,  Used as an adjunct drug for partial
leukopenia, nausea, vertigo, febrile seizures
reaction
FELBAMATE
 THERAPEUTIC LEVEL: 4-16 ug/mL
 TOXIC LEVEL: >12 ug/mL  Monotherapy or adjunct in refractory
 TOXIC EFFECT: hematologic dyscrasias, partial seizures with or without
aplastic anemia, irregular pulse and secondary generalization
ataxia
PSYCHOACTIVE DRUGS
ETHOSUXIMIDE (ZARONTIN)
 Are used to treat psychotic patients
 Prescribed for the treatment of petit  They can be categorized in two classes:
mal seizures;orally administered lithium and the antidepressants
 Free serum and not protein bound
 THERAPEUTIC LEVEL: 40-100 ug/mL LITHIUM
 TOXIC LEVEL: >100 ug/mL  Treats manic-depressive illness (bipolar
 TOXIC EFFECT: GI disturbances, ataxia, disorders). The mechanism of action of
SLE, aplastic anemia and pancytopenia lithium as a mood stabilizer remains
GABAPENTIN (NEUROTIN) unknown, although effects on synaptic
neurotransmission are thought to be
 Chemically similar to neurotransmitter cause
gamma aminobutyric acid (GABA)  Lithium is filtered by the renal
 Used for partial seizures; for adjunctive glomerulus and eliminated as the
therapy unchanged drug.
 Administered orally; it is unbound to  Drug of choice of the prevention of
plasma proteins chronic cluster headache
 Excreted unchanged in the urine; not  Inhibits thyroid hormone synthesis and
metabolized in humans release- inhibits iodine uptake
 ADVERE EFFECTS: dizziness, ataxia,  Cationic metal that does not bind to
fatigue and nystagmus proteins
 THERAPEUTIC LEVEL: 2-15 ug/mL  Orally administered; absorption is rapid
 TOXIC EFFECT: somonolence and complete
(drowsiness) and ataxia  Subject to reabsorption
 Distribution of this drug is uniform
throughout the body water.
  THERAPEUTIC DRUG: 0.8-1.2 mmol/L  THERAPEUTIC LEVEL: 90-300 ng/mL
 TOXIC LEVEL: 1.2-2 mmol/L- apathy,  TOXIC EFFECTS: attempted suicide,
lethargy, speech difficulties; >2 mmol/L- decreased libido and sexual function
seizures, muscle rigidity and coma
BRONCHODILATORS
ANTIDEPRESSANTS, OR TRICYCLIC
ANTIDEPRESSANTS  Act to relax bronchial smooth muscle
for relief or prevention of asthma.
 Used to treat depression that has no Theophylline is the most common in
apparent organic or social cause. this category of therapeutic drugs and is
Antidepressants include imipramine, metabolized in the liver to produce
nortriptyline, amitriptyline, and several metabolites, including caffeine
desipramine, all of which are
metabolized by the liver to form active THEOPHYLLINE
metabolites.  belongs to methylated xanthine class
 The active metabolites include  action is to relax bronchial smooth
desipramine (parent is imipramine; muscle
major metabolite), nortriptyline (parent
 used for treatment of asthma and
is amitriptyline), and 2-hydroxy-
chronic obstructive pulmonary disease
desipramine (parent is desipramine)
 drug of primary apnea prematurity-
 Used for the treatment of depression,
absence of respiratory effort in
insomnia, extreme apathy and loss of
newborn infants
libico
 action is inhibitory to the release of
 Orally administered with variability in
histamine and other proinflammatory
absorption; highly protein-bound
agents
 PEAK SERUM CONCENTRATION: 2-12
 It crosses the placenta and may be
hours
tetratogenic in pregnant females
 THERAPEUTIC LEVEL: 100-300 ng/mL
 Best predictor of toxicity is the blood
 TOXIC EFFECTS: drowsiness, blurred level of the drug and not early signs or
vision, memory loss, seizure, cardia symptoms toxicity
arrhythmia, parkinsonian syndrome and
 THERAPEUTIC LEVEL: 10-20 ug/mL
unconsciousness
 TOXIC LEVEL: >20 ug/mL
FLUOXETINE (PROZAC)  TOXIC EFFECTS: GI bleeding, seizures,
tachycardia and syncope
 Not chemically related to the tricyclic
antidepressants, but has a similar effect IMMUNOSUPPRESSIVE DRUGS
by blocking serotonin uptake by nerve
CYCLOSPORINE
terminals in the CNS and by platelets
 Blocks the re-uptake of serotonin in  Inhibits the cellular immune response
central serotonergic pathways by blocking production of interleukin-2
 Used for treatment of obsessive-
compulsive disorders
  Used to prevent rejection of allogenic ANTINEOPLASTIC DRUGS
organ transplants
 Used in the management of certain
 Used for suppression of acute graft-
tumors, including those found in breast,
versus-host disease (GVHD)
testicular, pharyngeal, and sometimes
 Organs such as heart, liver and pancreas
lung cancer
require high dosage (300 ng/mL)
 These agents work by inhibiting DNA
 Marked affinity with RBC; RBC-
synthesis
cyclosporine is temperature dependent
 TOXIC LEVEL: > 500 ng/mL METHOTREXATE
 TOXIC EFFECTS: renal tubular and
glomerular dysfunctions, GI  Inhibits DNA synthesis in all cells by
disturbances, hirsuitism, and blocking dihydrofolate reductase
hematologic dyscrasias  Effective etherapy for a variety of
neoplastic conditions; also an
TACROLIMUS (PROGRAF/ FK-506) immunosuppressive agent
 Leucovorin is used to reverse the
 It is a molecule lactine antibiotic and
action-leucoverin rescue
100x more powerful than cyclosporine
 TOXIC LEVEL: 0.01 umol/L
 Macrolide lactone antibiotic; GIT uptake
 TOXIC EFFECTS: leukopenia, GI
is variable
ulceration, thrombocytopenia, cirrhosis
 Elevated levels are observed in
cholestasis BUSULFAN
 TOXIC EFFECTS: thrombus formation,
nephrotoxicity and neurotoxicity  Alkalyting agent used to treat leukemias
and lymphomas prior to bone marrow
RAPAMYCIN (SIROLIMUS) transplantation
 OVERDOSAGE: hepatic occlusive
 Similar to tacrolimus
disease
 Major side effects are lipid
abnormalities and thrombocytopenis ANTI-INFLAMMATORY/ANALGESICS

MYCOPHENOLATE MOFETIL SALICYLATES/ASPIRIN (ACETYLSALICYLIC ACID)

 Decreases renal allograft rejection  Commonly used analgesics, antipyretic

and anti-inflammatory drug
LEFLUNOMIDE (LFM)
 Direct stimulator of the respiratory
 Inhibits lymphocyte proliferation system and an inhibitor of the Kreb’s
 Treatment for rheumatoid arthritis cycle
 Acute aspirin intoxication- a common
cause of fatal drug poisoning in children
 An anticoagulant property (antiplatelet
activity( by inhibiting the action of
cyclooxygenase
  FUNCTION: Decreases thromboxane NEUROLEPTICS (ANTIPSYCHOTIC MAJOR
and prostaglandin formation through TRANQUILIZERS)
inhibition of cyclooxygenase
 Blocks the action of dopamine and
 SIDE EFFECTS: gastrointestinal
serotonin in limbic system
disturbance and interference with
platelet aggregation  Used for treatment of acute
schizophrenia
 THERAPEUTIC LEVEL: 5mg/dL
(treatment of headache)  Monitoring of these drugs in serum is
difficult due to abundant metabolites
 TOXIC LEVEL: >30 mg/dL
for each drug resulting to extensive
 TOXIC EFFECTS: mixed acid-base
metabolism in the liver
disturbances (metabolic acidosis and
 2 Classes: phenothiazines
respiratory alkalosis), Reye’s syndrome
(chlorpromazine) and butyrophenones
and hypoglycemia
(haloperidol)
 METHOD: Trinder assay: enzymatic
 TOXIC EFFECTS: cholestasis, orthostatic
assay (salicylate hydroxylase), HPLC and
hypotension, aplastic anemia, muscle
EMIT)
rigidity
ACETAMINOPHEN (TYLENOL)  EXAMPLES: risperidal, olanzapine
(Zyprexa), quetiapine (Seroquel),
 Inhibitor of prostaglandin metabolism ariprazole (abilify)
 Commonly used as analgesic and
antipyretic drug METHODS
 OVERDOSAGE: hepatoxocity
 SPECIMEN OFCHOICE: serum or plasma
 THERAPEUTIC LEVEL: 25 ug/mL
 CYCLOSPORINE AND TACROLIMUS
 TOXIC LEVEL: >50 ug/ml; 100-300
TEST: whole blood EDTA sample
ug/mL (hepatic necrosis)
 Sample for TDM should not be
 TOXIC EFFECTS: cyanosis due to
collected in SST or gel separators (some
methemoglobinemia, CNS depression
gels absorbs certain drugs (phenytoin,
and seizure
phenobarbital, lidocaine, quindine and
 METHOD: HPLC
carbamazepine) → false decreased
IBUPROFEN results
 SPECIMEN CONSIDERATIONS: through
 Analgesic and anti-inflammatory actions concentrations and peak concentrations
 Has a lower risk of toxicities than  No changes occurred in theophylline
salicylates and acetaminophen and salicylate levels when blood is
 THERAPEUTIC LEVEL: 10-50 ug/mL collected in serum separator tube (SST)
 TOXIC LEVEL: >100 ug/mL  Measurement of serum concentration
 TOXIC EFFECTS: nausea, vomiting, should be done only after steady state
blurred vision, abdominal pain and has been achieved
edema  Trough concentrations
 o Drawn immediately(or 30 CHROMATOGRAPHIC METHODS
minutes) before the next dose
 Peak concentrations  BEST SPECIMEN: urine
o Drawn one hour after orally a. Thin Layer Chromatography
administered dose (except  It qualitatively identifies drugs
digoxin) by means of their Rf values
o Determine after IV infusion is  Extraction of drugs is pH
completed dependent: acidic drugs (pH
4.5); alkaline drugs (pH 9.0)
COLORIMETRY b. High Performance Liquid
Chromatography (HPLC)
 Acetaminophen in urine is detected by
 It is a highly quantitative
boiling to form p-amphenol which
procedure
reacts with o-cresol to from indophenol
 It is ideal for separation of
blue
tricyclic antidepressants and its
 Trinder assay for salicylate using ferric
metabolites
nitrate forming a (+) colored complex
 Measurement depends on the
IMMUNOASSAY METHODS type of column used, the
solvent and detector systems
 Rapid analysis of blood and urine c. Gas Chromatography-Mass
samples Spectrometry (GC-MS)
 Detect drug levels in the nanomolar  Used for quantitation of many
range; sensitive and specific methods drugs
 Uses an antibody specifically reactive  Drugs must be volatile in form
with a particular drug- the drug must or can be chemically derivatized
bind to antibody before it is identified into valuable form.
a. Enzyme-Mediated (Multipled  MS can added onto effluent end
Immunologic Technique /EMIT of GC, enhancing its capability
 Enzyme activity is directly to quantify drugs
proportional to the amount of
drugs present in the sample
b. Fluorescence Polarization
Immunoassay (FPIA)
 The binding of the marker drug
to antibody can be quantitated
by the angle at which the
emissions occurs
 The drug is attached to a
fluorescent label or fluorophore
CC2-MODULE 7- LABORATORY  Tetrahydrocannabinol (THC)- most
potent components or psychoactive
DRUGS OF ABUSE
substance of Marijuana
 THC is a lipophilic substance, distributes
DRUGS OF ABUSE
in the adipose tissues; it is easily enters
 Almost all drugs of abuse are basic the brain; it induces a sense of well-
drugs (amine derivatives) which being and euphoria; it is a hallucinogen;
contain benzene rings; barbiturates are also associated with the impairment of
acidic drugs memory and intellectual functions
 Many of the abuse drugs directly act on  After a single use, THC-COOH can be
dopaminergic neurotransmitter system detected in urine for 3-5 days; up to 4
(limbic system; smell brain) weeks for chronic user
 A positive drug screening test cannot  Principal psychoactive component of
differentiate casual user from chronic or cannabis is delta 9-tetra-
habitual user, likewise detect the time hydrocannabinol (also referred to as
frame of using the drug dose of the delta—9THC)
drug taken  Urinary metabolite is 11-
 Designer drug- modified forms of nordeltatetrahydrocannabinol
established drugs of abuse  Symptoms- wide array
 Effects of low-moderate doses
PHARMACOLOGICAL CLASSSIFICATION OF
includes: loquacious euphoria, changes
DANGEROUS DRUGS of perception of time and space,
1. Hypnotics impaired coordination, judgement and
2. Sedatives and tranquilizers memory, increased visual and auditory
3. Hallucinogens and Psychotomimetic sensitivity, conjunctivitis and bronchitis
drugs  Psychologic effect: reddening of
4. Stimulants conjunctiva and increased pulse rate
5. Inhalants and Intoxicants  Higher dosage: illusions, delusions,
6. Cannabis/Cannabinoids depression, confusion, alienation and
7. Anabolic steroids hallucinations, impaired psychomotor
cognitive and endocrine functions.
CANNABIS/CANNABINOIDS  Toxic effects: paranoia, disorientation,
altered physical senses and
 Naturally occurring cannabinoids:
bronchopulmonary disorders
marijuana and hashish
INHALANTS
MARIJUANA
 Produces psychoactive vapors
 Also known as: Pot grass, Weed, Mary
1. Nitrous oxide - laughing gas,
Jane or MJ, Acapulco gold, :Damo”
whippets
 Hemp, plant (Cannavis sative), Hashish
2. Amyl nitrite - poppers,
(Hash), Hashish oil (Hash oil)
snappers
 3. Butyl nitrite – bolt, locker  Alkaloid salt (ecognine) that can be
room, bullet taken directly (insugfflation of IV) or by
4. Chlorohydrocarbon - aerosol inhalation/snorting
spray  Potent CNS stimulant that elicits a sense
5. Hydrocarbon - solvents of excitement and euphoria; increase
 It includes: aerosols, gasoline, some physical activity
glues, solvents, Butyl nitrites (room  Cocaine can be sniffed, smoked or
odorizers) injected
 Short time sniffing may cause: disturb  Smoking coca leaves
vision, impair judgement, reduce  A stimulating drug can induce euphoric
muscle and reflex control excitement and hallucinatory
 Death without warning due to experience, sense of being “super
suffocation, respiratory collapse or powerful” combined with paranoid
heart failure delusions and auditory visual and tactile
 Abused inhalants and solvents hallucination. When induced user can
includes: rugby, glue, gasoline, commit serious anti-social acts.
acetone, paint thinner  Used as local anaesthetic for
 SS: odor of substance in breath and nasopharyngeal surgery
clothing; excess nasal secretions and  Can cause sudden death due to direct
lacrimations, poor muscle control, toxicity on myocardium- it
slurring of speech vasoconstriction, platelet aggregation
 Deep inhaling of vapors causes and synthesis of plasminogen activator
disorientation and violent behaviors. inhibitor
 Long period of sniffing concentrated  Cause malformations in uterus
vapors: permanently damage the  Not considered as an addictive drug- it
central nervous system does not reflect the true dependence
commonly seen in abusers of
STIMULANTS
barbiturates and opiates
1. Cocaine- coke, flakes, white blow  Easily passes the placenta and
2. Crack or cocaine crack or rocks mammary glands (readily passed from
3. Amphetamines- speed, uppers, black mothers to infants) resulting mental
pep pills, bumble bees, co-pilot retardation, slow mental development
4. Methamphetamine- crack, crystal and drug dependence in newborns
meth, methedrine, speed or “shabu”  Overdosage: violent behavior; high
abuse potential
COCAINE (CRACKS)  Single use: can be detected in urine for
up to 3 days
 Derived from coca bush (Erythroxylon
 Chronic user: up to 20 days
coca) evergreen shrub grown in South
 Inhibitor: Prozac
America
 Treatment and addiction:
Benzodiazepine
  Toxic effects: hypertension, DEPRESSANTS
arrhythmias, seizures and myocardial
infarction  DEPRESSANTS: acts on central nervous
system. Close pharmaceutical relative
 Urine metabolite: benzoylecgonine
to sedatives that higher doses induce
AMPHETAMINE AND METHAMPHETAMINES sleeps.
o Examples: Demerol
 Therapeutically used for treatment of (meperidine hydrochloride)
narcolepsy and attention deficit cough syrups (codeine)
disorder 1. BARBITURATES: Downers, barbs, red
 Increases mental alertness and physical devils, yellow jackets, Nembutal,
capacity, and has no anorectic property seconal, amytal
 It is structurally related to dopamine 2. METHAQUALENE: Qualudes, Ludes,
and catecholamine Soppers, Mandrakes
 Have a capacity to elevate mood and 3. TRANQUILIZERS: Valium, Librium,
dispel fatigue Equanil, Miltown, Serax, Trnxene
 Cause the release (together with  Most abused: Barbiturates and
cocaine) of dopamine from the brain Tranquilizers
leading to a “pleasant feeling” (so called  SS: signs of alcoholic intoxication but no
“high”_ among users alcoholic breath, Drowsiness and
 Most cases taken for euphoric effect appears disoriented, difficulty of
 Used to be treatment for narcolepsy concentration, Pupils dilated; all vitals
(sleep episode) and to calm down are down/decreased
hyperkinetic children or overactive  Large doses: produce respiratory
children with attention deficit disorder depression, slurring of speech and
 3,4 methylenedioxymethamphetamine altered perception
(MDMA or ‘ecstacy’) - drug derivative  Taken with alcohol multiplies the risk.
of methamphetamine; designer drug Babies born to depressant drug
 Amphetamine like compounds: dependent mother may produce mental
ephedrine, pseudoephedrine and and physical defects
phenylpropanolamine
 Signs of acute intoxication: SEDATIVE HYPNOTICS
hyperpyrexia
 Have therapeutic roles and they are
 Acute psychotic syndrome: auditory
CNS depressants
and visual hallucinations, suicidal
 Used also to potentiate the effects of
tendency and paranoia
heroine
 Toxic effects: palpitation, hypertension,
o EXAMPLES: Barbiturates and
cardiac arrhythmias, convulsion,
benzodiazepine
pancytopenia, mental impairment and
 Commonly abused barbiturates:
teeth grinding
secobarbital, phentobarbital,
phenobarbital and theopental
  Commonly abused benzodiazepine: (Phecyclidine), most powerful is
diazepam (Valium), chlordiazepoxide LSD (Lysergic acid diethylamide)
(Librium), and Lorazepam (Ativan) 1. PHENCYCLIDINE- PCP, angel dust, hog,
 Toxicity: initiated by ethanol killer weed, Love boat
 Major metabolite: secobarbital 2. LYSERGIC ACID DIETHYLAMIDE- LSD,
(barbiturates) Acid, Green or Red dragon, White
 Barbiturate chemoadsorbent: activated lightning, Blue heavens
charcoal 3. MESCALINE AND PEYOTE- Mesc,
 Toxic effects: Cheyne-Stokes Buttons, Cactus
respiration, depression, cyanosis, 4. PSILOCYBIN- Magic mushrooms,
areflexia, stupor, coma mushrooms (classified also as
 Barbiturates are condensation products tryptamines are derivatives of serotonin
of urea and malonic acid and components of some components
 Benx=ziodiazepines are used for the in plants)
treatment of cocaine addictions  MESCALINE- the most active
 Diazepam has a clinical use for rapid component of cactus peyote
control of acute seizure activity; a minor (Loporophora Williamsii) grows wild in
tranquilizer Mexico, Southern and Western US.
 Phenobarbital structurally resembles Favorite ritualistic hallucinogen.
phenytoin (Dilantin)  DOM- effects heightened awareness of
sensory input. The phenomenon as a
METHAQUALONE (QUALUDE) result to use of other hallucinogen:
Psilon or Psilocybin from Psilocybin
 A 2,3 – distubstituted quinazoline with
Mexicana
anesthetic, antihistamine and
 PIPTADENIE PERIGRINA (found in
antitussive properties
Orinoco region of Amazonia) SOURCE
 It has also sedative-hypnotic properties;
OF TRYPTEMINES: a. DMT
a hallucinogen
(Dimethyltryptamine) b. DET
 Has a similar symptoms of toxicity to
(Diethyltryptamine)
barbiturates, as well as pyramidal signs
 EFFECTS: finds difficulty in
(hypertonicity, hyperreflexia and
distinguishing between fantasy and
myoclonus)
reality, one object to another, even
 CHEMOADSORBENT: activated charcoal
oneself to surroundings
HALLUCINOGEN  PCP- KILLER WEED or Angel Dust: is
usually smoked with tobacco can
 Are chemically diverse group which provoke serious toxic psychosis,
produce mental changes like euphoria, schizophrenic in character, violent
anxiety, sensory distortion, vivid visual aggressive or self-destructive behavior
and auditory hallucination  LSD- users sits quietly in a dream-like
o Example: DOM (dimethoxy-4- state. Altered senses of sight, hearing
methylamphetamine) PCP and touch as well as images. Drug
 blocks the pain receptor of the brain LYSERGIC ACID DIETHYLAMIDE
and may cause self-inflicted injuries. (LSD/LYSERGIDE)
Large doses may produce convulsions,
coma and death.  Semi-synthetic indolalkalylamine
 A hallucinogen
TRYPTAMINES  One of the most potent pharmacologic
materials known
 Derivatives of serotonin; some
 Produces effects at low doses: 20 ug (IV
compounds are present in palnts
or ingestion)
 Examples: N,N-dimethyltrypthamine
 Most common adverse reaction: panic
(DMT), psilocin
reaction- “bad trip”
 DMT is a short-term hallucinogen and
 Toxic effects: blurred or undulating
taken by smoking; businessman’s lunch
vision and synthesis
 Psilocyn is a component of “magic
mushrooms” (psilocybe); a hallucinogen NARCOTICS
 Monoamine oxidase inhibitor (b-
carboline) enhances the hallucinogenic  HEROIN- Smoke. Horse, brown sugar,
effect of tryptamines black Tar
 Ayahuasca- a tea which contains  METHADINE- Dolly, Dolophine,
tryptamines Methadose, Amidone
 Antagonist: benzodiazepine  MORPHINE- Pectoral syrup, sweet
 Toxic effects: tachycardia, amorphous
hypertension, dystonia, seizures,  CODEINE- Empirine compound with
rhabdomyolysis and paralysis codeine, codeine in cough medicines
 MEPERIDINE- Pethidine, Demerol
PHENCYCLIDINE (ANGEL DUST/ANGEL HAIR)
Mepergan
 A depressant, stimulant, has  OPIUM- Paregoric, Dakers powder,
hallucinogen and anaesthetic properties parapectolin
 Can be ingested or inhaled by smoking  OTHER NARCOTICS- Fentanyl, Darvon,
 About 10-15% is unchanged when Valium, Lomotil
excreted in urine – acidification of the  Narcotics also classified as Opiates
urine helps in immediate excretion  HEROIN- POPPY PLANT (Papaver
 Physiologic effects: analgesia and somniferum L.) – coagulated juice from
anaesthesia the unripe capsule. Originated in the
 Major metabolite: Phencyclidine HCI Mediterrenean and spreads to Persia,
 Mode of treatment: Isolation (kept in Egypt, China and Europe
quiet and dark room)  PHILIPPUS PARACELSUS, the 16th
 Toxic effects: tachycardia, seizure and century alchemist and physician who
coma (eventually death) started use and preparation of Opium
for treatment of various illness.
 MORPHINE- main active principle of
opium. Contains 10% morphine.
 Extracted wither from opium or directly  Fentanyl “lollipops” or “patches” are
from poppy straw. Still the classical more potent analgesics than morphine
effective analgesic for the relief of  Commonly tested opiates: morphine
severe pain; and codeine
 CODEINE (METHYLMORPHINE) – used  Major cause of drug related death:
extensively as an effective cough darvon overdose combine with alcohol
suppressant (anti-tussive). And a mild  Major metabolites of heroin: N-acetyl
analgesic with a low addictive potential. morphine (heroin) and morphine
 SS OF NARCOTICS ABUSER: (Heroin,  Withdrawal symptoms of heroin: cold
Morphine, Opium) feeling of euphoria, seweats, nightmares and hypothermia
followed by drowsiness, nausea and  Antagonist for opiate overdose:
vomiting, constricted pupils water and naloxone (narcan)
itchy eyes.  Toxic effects: respiratory acidosis,
 Overdose: may produce slow and myoglobinuria, cardiopulmonary failure
shallow breathing grayish color of skin, and pupillary constriction (“pin-point
convulsion], coma, and death, multiple pupils”)
scar marks in arm and due to
contaminated needles may acquire MORPHINE AND HEROIN
AIDS and other infectious diseases
 Morphine is a metabolite of heroin; a
 Addiction to pregnant women may
powerful analgesic; used in treatment
result to premature delivery, still birth
of acute congestive heart failure
and addicted infants who experience
 Morphine binds to mu-receptors in the
severe withdrawal symptoms
limbic system( CNS) producing analgesic
OPIATES effect.
 Morphine and meperidine increase liver
 Capable of analgesia, sedation and and pancreatic enzymes.
anesthesia  Heroin is highly addictive (true physical
 Derived chemically from opium poppy dependence)
 Naturally occurring opiates: opium,  Heroin crosses the blood-brain barrier-
morphine and codeine elevated levels in the CNS
 Common modified opiates: heroin,  Heroin is taken by IV administration
hydromerphone and oxycodone
(Percodan) ANABOLIC STEROIDS
 COMMON SYNTHETIC OPIATES:
 Chemically associated to male
MERPERIDINE (Demerol), methadone
hormone, testosterone
(Dolophine), Propoxyphene (Darvon),
(dihydotestosterone and testosterone.)
pentazocine (Talwin) and fentanyl
 Improves athletic performance by
(Sublimaze)
increasing muscle mass.
 Codeine is an antitussive drug
 Methadone is a nonbicyclic drug that
binds with morphine in the brain
  Toxic effects: chronic hepatitis, WAYS OF TAMPERING SPECIMEN
atherosclerosis, abnormal platelet
aggregation and cardiomegaly  Adulteration- specimen containing a
substance a not normal constituent for
SPECIMEN CONSIIDERATIONS: that type of spx/ containing
endogenous substance that is not
 Alcohol in blood samples may be normal physiological concentration;
analysed even after a moment of delay EXAMPLES: bleach, salt, vinegar
provided the samples in tubes remain  Dilution (internal or external)- with <
sealed normal physiological contents; internal-
 Urine temperature is a vital factor to drinking plenty of water EXAMPLE:
assure it is freshly voided (abused diuretics; external- addition of water to
drugs) the specimen.
 Aspiration of gastric contents or  Substitution
vomitus may reveal tablets or capsules
from which the ingested drug can be METHODOLOGY
determined
 SCREENING METHOD:
SAMPLRE REQUIREMENTS: o FDA-DOH registered kits using
immunoassay technique
 Urine, serum, hair, nails, whole blood or o Instrumented screening
plasma (alcohol), sweat, saliva and method: ELISA, FPIA
exhales breath (alcohol intoxication) (immunoassay), TLC, HPLC
 Urine specimen collected → (chromatographic)
concentration and extraction  CONFIRMATORY METHOD
procedures; least expensive; within the o GC-MS- gold standard
week  ENZYMATIC TEST
 Drugs deposited in hair are generally o Alcohol is measured from blood
present in relatively low levels using alcohol dehydrogenase as
 Sweating testing- parent drug is reagent
increased than metabolites o Quantitates the sum of all
 Saliva testing- drug concentration in it alcohols present in a sample
reflects the free or active fraction o Does not distinguish alcohols
 2 basic techniques for identification of from its metabolites during
drugs: immunochemical and quantitation
chromatography  CAPILLARY ELECTROPHORESIS
 Hair specimen twice more sensitive, o Different analyte selectivity is
requires 1.5x1.5 based on different
 Urine 60 ml single container physochemical principle of
separation without changes in
CHECK AUBF BOOK FOR MORE INFO
instrument hardware, a distinct
STRASINGER 6TH ED.
advantage of this technique.
 oRecent variant of TLC that oMay be used as an alternative
includes the advantages of to GC-MS in definitive
HPLC identification of drugs
 HOMOGENOUS IMMUNOASSAY  Gas Chromotography (GLC, GC-MS)
o Done in one solution without o Gas Liquid Chromotography
separation (GLC) - Legally accepted in
o EMIT- method uses enzyme ethanol testing
labeled drug that competes o GC With Infrared Spectroscopy
with the drug in the sample; the – detection of amphetamines
active site of the enzyme is o Gas Chromatography-Mass
blocked with the anti-drug Spectroscopy (GC-MS)- gold
antibosy, resulting to decreased standard for confirmation of
enzymatic activity; free drug screening methods such as TLC
(analyte in the serum or urine) and EMIT; allows for detection
competes with the antibody- of low levels of drugs like
drug-enzyme complex, cocaine and drug metabolites
 CHROMATOGRAPHIC METHODS
FACTORS REESULTING TO INNOCENT DRUG
 TLC
TESTING RESULTS
o Uses serum, urine, or gastric
fluid for analysis  Presence to detergents will be result to
o Extraction of drugs is pH alkaline pH
dependent – acidic drugs at pH  Use of sodium chloride (table salt)
4.5 (barbiturates) and alkaline  Low specific gravity (diluted urine)
drugs (opiates, amphetamines)
 High pH (alkaline urine)
at pH 9.0
 Blood in urine (hematuria)
 LC-MS
o Used to confirm positive test URINE IS THE PREFERRED SAMPLE ESPECIALLY
results from a screening assay IN DRUG TESTING BECAUSE OF THE
(non-volatile compounds) FOLLOWING REASONS:
o Used for detection of poisons in
acute or chronic intoxication, 1. Drugs that their metabolites are present
therapeutic drug identification in higher concentrations in urine than in
and quantitation, blood
pharmacokinetics and drug 2. Larger sample volumes are easily
metabolism studies collected
 HPLC 3. There is no pain or discomfort when
o Allows quantitative collecting the sample
measurement of drugs as well 4. The process of obtaining the sample is
as separation of these same noninvasive
drugs, especially tricyclic
antidepressants including its
active and inactive metabolites
DRUG TEST KIT (DKT)