REVIEW NOTES

OUTLINE IN CLINICAL CHEMISTRY SELECTED TOPICS

ANALYTICAL METHODS
A. COLORIMETRY
a. SPECTROPHOTOMETRY – measures the light intensity
b. FLAME EMISSION PHOTOMETRY – measures the light emitted by single
atom burned in a flame; excitation from lower to higher.
c. ATOMIC ABSORPTION SPECTROPHOTOMETRY – measures the light
absorbs by atoms dissociated by heat; unexcited, ground state
B. VOLUMETRIC (TITRIMETRIC)
- Unknown samples is tested with a known solution in the presence of an indicator.
C. TURBIDIMETRY
- Determines the amount of light blocked (reduction of light) by a particulate matter
in a turbid solution.
D. NEPHELOMETRY
- Determine the amount of scattered light
E. ELECTROPHORESIS – migration of charged particles in a field.
a. DENSITOMETRY – scans and quantitates electrophoretic pattern; like a
photocopying machine
b. ISOELECTRIC FOCUSING – separates molecules by migration through a pH
gradient. Ideal for separating of identical sizes but with different net charge
F. CHROMATOGRAPHY – involves separation of soluble components in a solution by
specific differences in physical and chemical characteristics
2 FORMS:
A. PLANAR
a. PAPER CHROMATOGRAPHY – sorbent (stationary phase)-
Whatman Paper
b. THIN LAYER CHROMATOGRAPHY – semi-quantitative drug
screening test
B. COLUMN
a. GAS CHROMATOGRAPHY – useful for compounds that are
naturally volatile or easily converted into volatile form
2 TYPES:
1. GAS SOLID CHROM – separation occurs based on
differences in absorption at the solid phase
2. GAS LIQUID CHROM – separation occurs by the
differences in soluble partitioning between the gaseous
mobile phase and liquid stationary phase

*MASS SPECTROSCOPY – based on the fragmentation and

ionization of molecules using a suitable source of energy; independent
method, detects the drug present whereas Gas Chromatography separates
*GAS CHROMATOGRAPHY-MASS SPECTROSCOPY (GC-
MC) – Gold Standard for drug testing

BY:
PRINCESS ALEN I. AGUILAR,RMT
 *TANDEM MS (MS/MS) – can determine 20 inborn errors of
metabolism from a single blood spot.
b. LIQUID CHROMATOGRAPHY – based on the distribution of
solutes between liquid mobile phase and a stationary phase.
1. HPLC – uses pressure for fast separations, controlled
temperature, in line detectors and gradient elution technique
2. LC-MS – determines non-volatile substances in body fluids

SEPARATION MECHANISMS:
a. GEL/GEL PERMEATION/GEL FILTARTION/ SIZE
EXCLUSION/MOLECULAR SIEVE CHROM – based
on their size and shape.
b. ION-EXCHANGE CHROM- exchanged of sample ions
with the charged group of the stationary phase
c. PARTITION CHROMATOGRAPHY (L-L CHROM) –
based on their partition between a liquid mobile phase and a
liquid stationary phase coated on a solid support.
d. AFFINITY CHROMATOGRAPHY – uses immobilized
biochem ligands as the stationary phase to separate a few
solutes from other unrestrained solutes.
e. ADSORPTION CHROMATOGRAPHY (L-S CHROM)
– based on the difference competition between the adsorption
and desorption of solutes at a surface of a solid particle.

G. FLUOROMETRY / MOLECULAR LUMINESCENCE

SPECTROPHOTOMETRY
- Measures the amount of light intensity present over a zero background.
- Determines the amount of light emitted by a molecule after excitation by
electromagnetic radiation; uses a 2 monochromators; affected by Quenching
effect.
H. CHEMILUMINESCENCE
- Emission of light is created from a chemical or electrochem reaction of the
excited compounds as it returns to its ground state.
I. OSMOMETRY
- Based on measuring charges in the colligative properties of solutions that occur
owing to variations in [particles]
- COLLIGATIVE PROPERTIES:
 Osmotic Pressure
 Boiling Point
 Freezing Point
 Vapor Pressure
*FREEZING PT. DEPRESSION OSMOMETRY - most commonly used
method for measuresing the charges in colligative properties of a solution.
J. ELECTROCHEMISTRY TECHNIQUES
a. POTENTIOMETRY – measuring of difference in voltage (potential) at a
constant current. Follows NERNST EQUATION; Uses: pH and pCO2.

BY:
PRINCESS ALEN I. AGUILAR,RMT
 ION SELCTIVE ELECTRODE (ISE) – it is very sensitive and selective for ion
measures; electrochem transducer capable of responding to one given ion.
1. DIRECT ISE – without serial dilution
2. INDIRECT ISE – with serial dilution
b. COULOMETRY - measures the amount of electricity (in coulumbs) at a fixed
potential. It follows the FARADAY’S LAW.
c. AMPEROMETRY – measures the current flow produced by an oxidation
reaction; Uses: pO2, glucose, Cl- and peroxidase determination
1. POLAROGRAPHY – measures differences in current at a constant
voltage. It follows ILKOVIC EQUATION.
d. VOLTAMETRY – measures current after which a potential is applied to an
electrochemical cell.

PIPET CALIBRATION
To Deliver  Distilled water
To Contain  Mercury
Verify pipette volume accuracy and precision  Gravimetric and Spectrometric Methods
Compare the reproducibility of brands of pipet tips  0.1% phenol red solution in distilled
water

ACID-BASE BALANCE
METABOLIC:
Acidosis – per mEq/L fall in HCO3, pCO2 drops 1-13mmHg (1:1)
Alkalosis – for every 10 mEq/L rise in HCO3, pCO2 rise by 6mmHg

RESPIRATORY
Acidosis - HCO3 rise 1mmHg for each 10mmHg rise in pCO2 (1:10)
Alkalosis - HCO3 falls 2mmHg for each 10mmHg fall in pCO2

ENZYME CLASSIFICATION
OXIDOREDUCTASE Dehydrogenases, Oxidases
TRANSFERASES Transferases and Kinases
Esterases (Phosphatases, CHS, LPS)
HYDROLASE Peptidases (Trypsin, Pepsin, LAP)
Glycosides (AMS, Galactosidase)
LYASES Decarboxylase, aldolase
ISOMERASES Isomerases
LIGASES Synthase

Enzyme Inhibited by
Regan ALP (Heat Stable) Phenylalaine reagent
Nagao ALP L-Leucine nd Phenylalanine
Bone ALP 3m-Urea, Levamisole
Liver ALP Levamisole rgt
Prostatic ACP 20mm L-tartrate ions
Red cell ACP 1mm cupric SO4 and 2% Formaldehyde ions

BY:
PRINCESS ALEN I. AGUILAR,RMT
Salivary AMS Endogenous ‘wheat germ”/ lectin
CK Urate and cystine

1˚ Test 2˚ Test
GGT – to confirm the source
of increase ALP
Obstructive Jaundice ALP
LAP
5’ Nucleotidase
Trypsin
AMS
Acute Pancreatitis Chymotrypsin
Lipase (Most specific)
Elastase 1

ROUTINELY
REFERENCE
ANALYTE USED OTHER METHODS
METHOD
METHOD
 Nickel Inhibition Test
5’ Nucleotidase  Campbell Method
 Belfield and Goldberg
Zimmerman
17-ketogenic steroid
Method
 Phenyl Hydrazine in
17-
Porter-Silber H2SO4 + Alcohol
hydroxycorticosteri
Method
od
Roy and Hillman
(Quanti-endpoint)
ACP a-napthyl PO4
(Thymolphthalein
MonoPO4)
Bromcresol
Green (Most
 HABA (dye binding
Bromcresol common)
Albumin @500nm)
Purple (specific) @500nm and
 Methyl Orange
630nm thus
>sensitivity
Bowers and
McComb (Szasz
ALP Modification- pH
of 10.15
@405nm)
Glutamate
Dehydrogenas
 Nesselerization
Ammonia e and
 Conway
Berthelot
Reaction
 Amyloclastic
Saccharogenic
Amylase  Chromogenic
(Somogyi units)
 Walleufels
BY:
PRINCESS ALEN I. AGUILAR,RMT
  Quanti: Polarographic;
Trinders
Reitman and
Frankel (color  Karmen
developer-2,4  Babson – uses MD and
AST/ALT
DNPH, color change in absorbance at
intensifier – 340nm
0.4N NaOH)
 Evelyn and Malloy
Jendrassic and
(acidic) (classical)
Bilirubin Groff
 Dicholorobenzenediazo
(alkaline)
nium Chloride
Enzymatic
 Colorimetric/Chemical
(indirect):
Blood Urea – Diacetyl Monoxide
IDMS GLD,
Nitrogen Method (inexpensive
Urease NH3
non-specific)
formation)
 Redox:
a. Clark Collip ppt
b. Ferro Chloranilic
Acid ppt
Calcium AAS ISE  Colorimetric: O-
cresolphthalein
complexone
 EDTA Titration
 EFP
 Mercurimetric (Schales
and Schales)
 Spectro:
a. Mercuric
Cholride ISE Thiocyanate
b. Ferric Perchlorate
 Coulometric
Amperometric Titration
 Cotlove (SWEAT)
 Libermann-Burchardt
Rxn
 Salkowski Rxn
Enzymatic:
Abell, Levy and  1step – Pearson, Stern
Cholesterol Cholesterol
Grodie  2step – Bloors
oxidase
 3step – Abell Kendall
 4step – Schoenheimer
Sperry
Oliver Rosalki/
 Tanzer-
CK Electrophoresis Reverse/
Gilbarg/Forward/Direct
Indirect

BY:
PRINCESS ALEN I. AGUILAR,RMT
  Enzymatic: Lim and
Moss
 Colorimetric:
Jaffe/Direct/Endpoint:
Enzymatic: a. Folin Wu
Creatinine b. Lloyds
Creatinine IDMS
Hydrogen c. Fullers earth
Peroxidase  Colorimetric Kinetic:
automated, rapid,
increase specificity
 Szasz
GGT
 Rosalki Tarlow
 Glucose Dehydrogenase
 Ferric Reduction
(Hagedorn Jensen)
 Condensation Method
Glucose (Dubowski Mtd)
Glucose Hexokinase Oxidase/  Copper reduction
Dextrostics Methods:
a. Folin Wu
b. Nelson Somogyi
c. Neocuprine
d. Benedict’s
Fiske
Inorganic Subbarow Mtd
Phosphorus (Ammonium
Molybdate)
Walker/
 Wrobleuski La Due –
LD Forward/
reverse/ indirect
Direct
Peroxidase  Tietz and Fiereck
Lipase Cherry-Krandal
coupling Templetion
 Electrophoresis
 Chromatography
Ultracentrifugatio Chemical
Lipoproteins  Immunochem
n precipitation
 Immunoassay
 Immunonephelometry
 Colorimetric:
a. Calmagite
Magnesium AAS b. Formazen Dye
c. Mg Thymol Blue
d. Dye Lake
Ellman Blood Gases Detection
pCO2 / pH
Potentiometry Technique (Gasometer)
*PCHe
(PCHe)  Natelson technique –
pO2 Amperometry Caprylic Alcohol (Anti-

BY:
PRINCESS ALEN I. AGUILAR,RMT
 foam) and Mercury
(production of vacuum)
 Yan Slyke
ISE
 EFP
Potassium AAS (Valinomycin
 Colorimetry (SWEAT)
Gel)
 Folin-Ciocalteu (Lowry)
 UV Absorption Mtd
Kjeldahl;
 SPE
Coomassie Biuret;
Protein  Turbidimetry
Brilliant Blue Ninhydrin
 Refractometry
sensitive (CSF)
 Nephelometry
 Salt Fractionation
ISE (Glass  Colorimetric (Albanese
Sodium AAS Aluminum Lein
Silicate)  EFP
Mod. Van Hadel Glycerol  Hantzsh Condensation
TAG (Neutral Fat)
and Zilversmith Kinase (Fluorometric method)
 Henry’s Classical Rxn
 Colorimetric endpt –
simple non-specific
 Enzymatic: UV (needs
special instrumentation
and optical cell) 230 nm
(Mercury lamp arc)
Uricase
 Enymatic: H2O2 –
Uric Acid IDMS (measures
interference by reducing
H2O2)
substance;
Phosphotungstic Acid
a. Trinders
b. Kabasakalian
c. Hans condensation
 Caraway

Chemiluminescen
Growth Hormone
ce IA
(Quanti) Pisano
Nor/Metanephrine
Method
Estrogen Kober Rxn

Hormonal Disorders Screening Test Confirmatory Test

Physical Activity
Growth Hormone Insulin Tolerance Test – Gold Standard
Test (Exercise
Deficiency 2nd: Arginine Stimulation Test
Test)

BY:
PRINCESS ALEN I. AGUILAR,RMT
Sertoli-Cellonly
Testicular Biopsy
Syndrome
Somatomedin-C
Glucose Suppression Test/ OGGT
Acromegaly or Insulin
(+) GH Fails to decrease
growth factor-1
24 hr Urine
Cushing’s / Inc.
Dexamethasone HPLC-MS
Cortisol
Suppression Test
Overnight Water Deprivation Test
- Neurogenic – (+) water conserve thru
Diabetes Insipidus
exogenous ADH
- Nephrogenic – (+) No effect
ACTH
Hypocorticolism Insulin Tolerance Test
Stimulation Test
Cystic Fibrosis Gibson and Cooke Pilocarpine Iontophoresis
Hashimoto’s Disease TPO (Thyroxine peroxidase)
1˚
PRA/PAC Ratio Saline Suppression Test
Hyperaldosteronism
Grave’s Disease TSH receptor Ab Test
Pheochromocytoma HPLC 24 hr urine/Glucagon Stimulation test
Euhtyroid TRH, inc. in rT3
Tumor Marker:
MTC Pentagastrin Stimulation test
Calcitonin
Drug-induced TSH
FT4
(inc)
2˚/3˚ Adrenal
Overnight Metyrapone Test
Insufficiency
Hypoglycemia WHIPPLES TRIAD

THERAPEUTIC DRUG MONITORING

Digoxin
Lidocaine (Xylocaine)
Metabolite: MEGX
Quinidine
Procainamide (Pronestyl)
CARDIOACTIVE
Metabolite: NAPA
Disopyramide
Propanolol
Amiodarone (Cordarone)
Verapamil
Aminoglycosides
ANTIBIOTICS Vancomycin (Red Man’s Syndrome)
Chloramphenicol

BY:
PRINCESS ALEN I. AGUILAR,RMT
 Phenobarbital
Inactive form: Primidone
Phentoin (Dilantin)
Injectable Proform: Fosphenytoin
Valproic Acid (Depakene)
for grand and petit mal seizure
ANTIEPILEPTICS
Carbamazepine (Tegretol)
grand mal with pain
Ethosuximide (Zarontin)
petit mal
Gabapentin (Neurontin)
not metabolized by human
Lithium
Manic Depressant treatment; Bipolar
PSYCHOACTIVE /
Tricyclic
ANTIDEPRESSANTS
Major Metabolite: Desipramine
Fluoxetine (Prozac)
BRONCHODILATOR Theophyline
Cyclosporine
Tacrolimus (Prograf/FK506)
IMMUNOSUPPRESSIVE Kapamycin (Sirolimus)
Leflunomide (LFM)
RA Treatment
Methotrexate
ANTINEOPLASTIC
Busulfan
Salicylates (Aspirin)
ANTI-INFLAMMATORY /
Acetaminophen (Tylenol)
ANALGESIC
Ibuprofen
Risperdal
Olonzapine (Zyprexa)
NEUROLEPTICS /
Quetiapine (Seroquel)
ANTIPSYCHOTIC MAJOR
Aripiprazole (Ability)
TRANQUILIZER
Phenothiazines (Chlorpromazine)
Butyrophenones (Haloperidol)

Protein Bound Drugs: >50%, expect the excretion is via hepatic way
- Digoxin – 25%
- Quinidine – 85%
- Procainamide – 20%
- Chloramphenicol – Antibiotic
- Phenobarbital – 50%
- Acetaminophen
- Phenytoin – 87-97%
- Valproic Acid – 93%
- Carbamazepine – 70-80%
- Cyclosporine and Tacrolimus
BY:
PRINCESS ALEN I. AGUILAR,RMT
 - TCA – 85-95%

NOTES:
 LITHIUM – inhibits iodine uptake = hypothyroidism
 DIGOXIN – patient with hyperthyroidism is resistant
 PROPANOLOL – treatment for thyrotoxicosis; suppress T4-T3 conversion
 AMIODARONE – iodine containing; can cause hyper/hypothyroidism

CARDIOACTIVE DRUGS CLASSES

CLASS I (Na+ Channel Blockers)
Quinidine
IA Procainamide
Disopyramide
Lidocaine
IB
Phenytoin

CLASS II (BETA-receptor Blocker)

Propanolol

CLASS III (K+ Channel Blocker)

Amiodarone

CLASS IV (Ca++ Channel Blocker)

Verapamil

CLASS V
Digoxin
Adenosine
MgSO4

TOXICOLOGY
TOXIC AGENTS
Ethanol (Grain)
“Hang-over Syndrome”
Methanol (Wood)
ALCOHOL “Blindness”
Isopropanol (Rubbing)
Ethylene Glycol (Anti-freezing agent)
“CaOx”
CARBON MONOXIDE Heme-binding
Fast acting / super toxic
CYANIDE
“Odor of bitter almonds”

BY:
PRINCESS ALEN I. AGUILAR,RMT
 Arsenic
“Odor of garlic”
Cadmium
“Electroplating and galvanizing”
Lead
METALS “Blocks D-ALA; wrist/foot drop”
Mercury
“Amalgite”
a. Elemental – Pink Disease (Acrodynia
and Erythism)
b. Alkyl – Congenital Minimata Disease

DRUGS OF ABUSE
AMPHETAMINES MDMA / Ecstasy / Designer Drug
(Treatment for Narcolepsy)
ANNABOLIC STEROIDS Improves Athletic Performance
Marijuana
CANNABINOIDS Metabolite: THC-COOH
Hashish
Chemically Synthesize:
 Hydromorphone
 Heroine
 Oxycodone
OPIATES
“Aneasthesia” (Lollipop / patches)
Naturally Synthesize:
 Opiates
 Morphine
 Codeine (Antitussive / cough)
Angel dust or hair
PHENYCYCLIDINE
“Isolation in Quiet Dark Room”
Commonly Abuse:
 Secobarbital
 Pentobarbital
 Phenobarbital
SEDATIVE HYPNOTICS  Thiopental
(CNS Depressants)
Commonly Used:
 Diazepam (Valium)
 Chlordiazepoxide (Librium)
 Lorazepam (Ativan)
(LSD, Lysergide)
LYSERGIC ACID DIET HYLAMIDE
“Bad Trip”
DMT “Businessman Lunch”
TRYPTAMINES
Psilocycin “Magic Mushroom”
METHAQUALONE Qualude

BY:
PRINCESS ALEN I. AGUILAR,RMT
 PIPERAZINES BZP/A2
“Pleasant feeling” MDMP
Same with Amphetamines TMPP or Molly

LEGALLY ACCEPTED /
ANALYTE REFERENCE METHOD
DEFINITIVE METHOD
Enzymatic (Alcohol
ETHANOL Gas Liquid-Chromatorgraphy
Dehydrogenase)
METHANOL GC-MS
ISOPROPANOL Gas Chromatography
ETHYLENE GLYCOL HPLC
Cooximetry (CO Hgb
CARBON MONOXIDE
measures)
CADMIUM (+) GGT in Urine
Zinc Protoporphyrin T;
LEAD
ALAD
Enzymatic Assay (Salicylate Colorimetric: Trinders
SALICYLATES
hydroxylase) HPLC, EMIT
ACETAMENOPHEN HPLC

TOXIC AGENTS ANTIDOTE

ETHANOL Diazepam
ARSENIC BAL rescue
CYANIDE Na+ Thiosulfate
OPIATES Naloxone (narcan)
COCAINE / TRYPTAMINES Benzediazepines
Cocaine  inhibitor of “Prozac”

BY:
PRINCESS ALEN I. AGUILAR,RMT