1. MUST TO KNOW IN CLINICAL CHEMISTRY
(From CC by Rodriguez)
Quality Control
Practicability Method is easily repeated
Reliability Maintain accuracy and precision
Intralab/Interlab QC Daily monitoring of accuracy and precision
Interlab/External QC Proficiency testing (Reference lab)
Long-term accuracy
Difference of >2: not in agreement w/ other lab
QC materials Available for a min. of 1 yr
Bovine control materials Preferred (Human: biohazard)
Not for immunochem, dye-binding and bilirubin
Matrix effect Improper product manufacturing
Unpurified analyte
Altered protein
Precision study First step in method evaluation
Nonlab. personnel 29% of errors (lab results)
SD Dispersion of values from the mean
CV Index of precision
Relative magnitude of variability (%)
Variance SD2
Measure of variability
Inferential statistics Compare means or SD of 2 groups of data
T-test Means of 2 groups of data
F-test SD of 2 groups of data
Cumulative Sum Graph V-mask
(CUSUM) Earliest indication of systematic errors (trend)
Youden/Twin Plot Compare results obtained from diff. lab
Shewhart Levey-Jennings Graphic representation of the acceptable limits of variation
Chart
Trend Gradual loss of reliability
Cause: Deterioration of reagents (Systematic error)
Shift Values: one side or either side of the mean
Cause: Improper calibration (Systematic error)
Outliers Values: far from the main set of values
Highly deviating values
Random or systematic errors
Kurtosis Degree of flatness or sharpness
Precision Random error
Accuracy Systematic error
Random error Causes:
(Imprecision; -Mislabeling
Indeterminate) -Pipetting error
-Improper mixing of sample and reagents
-Voltage/Temperature fluctuation
-Dirty optics
Parameters: SD and CV
Systematic error Causes:
(Inaccuracy/Determinate) -Improper calibration
-Deterioration of reagents
-Contaminated solution
-Sample instability/unstable reagent blanks
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2. -Diminishing lamp power
-Incorrect sample and reagent volume
Parameter: Mean
Multirule Shewhart Control rules + Control chart
procedure
Test method Westgard: at least 40 samples
Reference method Westgard: preferably 100 samples
Analytical Run Control and patient specimens assayed, evaluated, and report together
Physiologic Limit Referred to as absurd value
POCT Performed by nonlab personnel
Quality Assurance Tripod:
Program development
Assessment and monitoring
Quality improvement
Quality Patient Care Test request forms, clear instruction for patient prep., specimen handling…
Reference Range/ Interval At least 120 individuals should be tested in each age and sex category
Range/ Reference Values
Analytical Methods
Wavelength Distance bet 2 successive peaks (nm)
Lower frequency = Longer wavelength (Ex. Red)
Higher frequency = Shorter wavelength (Ex. Violet)
Spectrophotometric meas. Meas. light intensity in a narrower wavelength
Photometric measurement Meas. light intensity w/o consideration of wavelength
Multiple wavelength (uses filter only)
LASER Light Amplification by Stimulated Emission of Radiation
Light source for spectrophotometry
Visible region Tungsten light bulb
Mercury arc
UV Deuterium lamp
Mercury arc
Xenon lamp
Hydrogen lamp
IR Merst glower
Globar (Silicone carbide)
Stray light Wavelength outside the band
Most common cause of loss of linearity
Diffraction gratings Most commonly used monochromator
Cutting grooves
Prisms Rotatable
Nickel sulfate Prevents stray light
Cutoff filter Anti-stray light
Bandpass ½ peak transmittance
Alumina silica glass cuvet Most commonly used cuvet
Quartz/plastic cuvet UV
Borosilicate glass cuvet Strong bases
Photodetector Converts transmitted light into photoelectric energy
Barrier layer cell/ Simplest detector
photocell/ photovoltaic cell No external voltage
For filter photometers
Phototube Contains anode and cathode
Req external voltage
Photomultiplier tube Most common type
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3. Most sensitive
UV and visible region
Galvanometer/Ammeter Meter or read-out device
Absorbance A = abc (a = absorptivity; b = length of light (1cm); c = concentration)
A = 2 – log%T
Double beam spectro. Splits monochromatic light into two components:
One beam  sample
One beam  reference soln or blank (corrects for variation in light source
intensity)
Double-beam in space 2 photodetectors (sample beam and reference beam)
Double-beam in time 1 photodetector
Monochromatic light  sample cuvet and reference cuvet
Dydimium filter 600 nm
Holmium oxide filter 360 nm
Reagent blank Color of reagents
Sample blank Optical interference (Hgb)
FEP Meas. light emitted by a single atom burned in a flame
Principle: Excitation
Lt. source and cuvette: Flame
For excited ions (Na+, K+)
Cesium and Lithium Internal standards (FEP)
Correct variations in flame
Lithium Preferred internal std
Potent antidepressant
AAS Meas. light absorbed by atoms dissociated by heat
Principle: Dissociation (unionized, unexcited, ground state)
Lt. source: Hollow-cathode lamp
For unexcited trace metals (Ca++ and Mg++)
More sensitive than FEP
Atomizer (nebulizer) Convert ions  atoms
Chopper Modulate the light source
Lanthanum/Strontium Complex with phosphate
chloride Avoid calcium interference
Volumetric (Titrimetric) Unknown sample is made to react with a known solution in the presence of an
indicator
Turbidimetry Light blocked
Meas. abundant large particles (Proteins)
Depend on specimen concentration and particle size
Nephelometry Meas. amt of Ag-Ab complexes
Scattered light
Depends on wavelength and particle size
Electrophoresis Migration of charged particles in an electric field
Iontophoresis Migration of small charged ions
Zone electrophoresis Migration of charged macromolecules
Endosmosis Movement of buffer ions and solvent relative to the fixed support
Ex: gamma globulins
Cellulose acetate Molecular size
Agarose gel Electrical charge
Polyacrylamide gel Charge and molecular size
20 fractions (ex. isoenzymes)
Electrophoretic mobility Directly proportional to net charge
Inversely proportional to molecular size & viscosity of the supporting medium
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4. Isoelectric focusing Molecules migrate through a pH gradient
pH = pI
For isoenzymes: same size, different charge
Densitometry Scan & quantitate electrophoretic pattern
Capillary electrophoresis Electro-osmotic flow
Southern blot DNA
Northern blot RNA
Western blot Proteins
Chromatography Separation by specific differences in physical-chemical characteristics of the
different constituents
Paper chromatography Fractionation of sugar and amino acid
Sorbent: Whatman paper
TLC Screening: Drugs
Retention factor (Rf) value Relative distance of migration from the point of application
Rf = Distance leading edge of component moves
Total distance solvent front moves
Gas chromatography Separation of steroids, barbiturates, blood, alcohol, and lipids
Volatile compounds
Specimens  vaporized
Mobile phase: Inert gases
Gas Solid chromatography Differences in absorption at the solid phase surfaces
Gas Liquid chromatography Differences in solute partitioning between the gaseous mobile phase and the
liquid stationary phase
Mass Spectrometry Fragmentation and ionization
GC-MS Gold standard for drug testing
MS/MS Detect 20 inborn errors of metabolism from a single blood spot
HPLC Most widely used liquid chromatography
Fractionation of drugs, hormones, lipids, carbohydrates and proteins
Hydrophilic gel Gel filtration
Separation of enzymes, antibodies and proteins
Ex: Dextran and agarose
Hydrophobic gel Gel permeation
Separation of triglyceride and fatty acid
Ex: Sephadex
Ion exchange Separation depends on the sign and ionic charge density
chromatography
Partition chromatography Based on relative solubility in an organic solvent (nonpolar) and an aqueous
solvent (polar)
Affinity chromatography For lipoproteins, CHO and glycated hemoglobins
Adsorption Based on differences between the adsorption and desorption of solutes at the
chromatography surfaces of a solid particle
Fluorometry/Molecular Det. amt. of lt. emitted by a molecule after excitation by electromagnetic
Luminescence Spectro. radiation
Lt. sources: Mercury arc and Xenon lamp (UV)
Lt. detector: Photomultiplier tubes
2 monochromators:
Primary filter – selects wavelength absorbed by the solution to be measured
Secondary filter – prevents incident light from striking the photodetector
Sensitivity: 1000x than spectro
Quenching Major disadvantage of fluorometry
pH and temperature changes, chemical contaminants, UVL changes
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5. Instrumentation
Borosilicate glasswares For heating and sterilization
Ex: Pyrex and Kimax
Boron-free/Soft glasswares High resistance to alkali
Corex (Corning) Special alumina-silicate glass
Strengthened chemically than thermally
6x stronger than borosilicate
Vycor (Corning) For high thermal, drastic heat and shock
Can be heated to 900OC
Flint glass Soda-lime glass + Calcium, Silicon, Sodium oxides
Easy to melt
For making disposable glasswares
TD: To deliver Exact amount
TC: To contain Does not disperse the exact volume
Blowout w/ etched rings on top of pipet
Self-draining w/ o etched rings
Drain by gravity
Transfer pipet Volumetric: for non-viscous fluid; self-draining
Ostwald folin: for viscous fluid; w/ etched ring
Pasteur: w/o consideration of a specific volume
Automatic macro-/micropipets
Graduated or measuring Serological: w/ graduations to the tip (blowout)
pipet Mohr: w/o graduations to the tip (self-draining)
Bacteriologic
Ball, Kolmer and Kahn
Micropipettes: <1 mL
Micropipettes TC pipets:
Sahli-Hellige pipet
Lang-Levy pipet
RBC and WBC pipets
Kirk and Overflow pipets
Air displacement pipet Piston: suction
Disposable tip
Positive displacement pipet Piston  barrel (like a hypodermic syringe)
Dispenser/Dilutor pipet Liquid: common reservoir  dispense repeatedly
Distilled H2O Calibrating medium for TD pipettes
Mercury Calibrating medium for TC pipettes
Acid dichromate Cleaning solution for glasswares
(H2SO4 + K2Cr2O4)
Continuous flow analyzer Common reaction vessel
Air bubbles: separates and cleans
Glass coil: mix
Examples: “STS”
Simultaneous Multiple Analyzer (SMA)
Technicon Autoanalyzer II
SMAC
Centrifugal analyzer Acceleration and deceleration of the rotor
Advantage: Batch analysis
Examples: “RICC”
Cobas-Bio (Roche)
IL Monarch
CentrifiChem
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6. RotoChem
Discrete Analyzer Most popular
Req. vol: 2-6 μL
Uses positive-displacement pipets
Run multiple-tests-one-sample-at-a-time
Random access capability (STAT)
Examples:
Vitros
Dimension Dade
Beckman ASTRA System (4 & 8)
Hitachi
Bayer Advia
Roche Cobas Integra 800
Roche Analytics P Module
Automated Clinical Analyzer (ACA) Star (Dade)
Dupont ACA
Abbott ABA-100 Bichromatic Analyzer
ABA-200
VP Analyzer
American Monitor KDA
Olympus Demand
Thin-Film Analyzers 4 or 5 layers:
(Dry slide technology) -Spreading layer
-Scavenger layer - Ascorbate Oxidase
-Reagent layer
-Indicator layer
-Support layer
Colored reaction  Reflectance spectrophotometry
Examples: “KV2(75)”
Kodak Ektachem
Vitros 750XRC
Vitros 550XRC
Carry over Transport of quantity of analyte or rgt from one specimen rxn into another, and
contaminating a subsequent one
Batch testing All samples loaded at the same time
Single test is conducted on each sample
Parallel testing One specimen
More than one test is analyzed
Random access testing Any sample
Any test
Any sequence
STAT
Sequential testing Multiple tests analyzed one after another on a given specimen
Open reagent system System other than manufacturer’s reagents can be utilized for measurement
Closed reagent system The operator can only use the manufacturer’s reagents
Patient Preparation
Exercise Increased: GU2FT C2L3A5P2
GH
Urea
Urinary protein (Proteinuria)
Fatty acid
Testosterone
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7. CPK (muscle)
Creatinine (muscle)
Lactate
LH
LD (muscle)
ACP
Aldolase (muscle)
AST
ALT
Ammonia
Pyruvate
Prolactin
Decreased:
Glucose
Fist clenching Increased: “LPP”
Lactate
Potassium
Phosphate
Fasting 8-16 hours:
Glucose
Lipids
Lipoproteins
Increased:
Bilirubin (48 hours)
Triglyceride (72 hours)
Basal state collection Glucose
Cholesterol
Triglyceride
Electrolytes
Diet Increased: “GLUC2H”
Glucose
Lipids
Urea (High protein diet)
Caffeine: increases glucose
Catecholamines
5-HIAA (From Serotonin)
Turbidity/Lactescence Triglyceride >400mg/dL
Icterisia Bilirubin: 25.2 mg/dL
Icteric samples Interfere with: "TACGu”
Total Protein
Albumin
Cholesterol
Glucose
Upright/supine (lying) Preferred position
position Patient should be seated/supine at least 20 mins before blood collection to
prevent hemodilution or hemoconcentration
Supine  Sitting/Standing Vasoconstriction  Reduced plasma volume
Increased: “ECA”
Enzymes
Calcium
Albumin
Sitting  Supine Hemoconcentration
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8. Increased: “P(u)BLIC”
Proteins
BUN
Lipids
Iron
Calcium
Standing  Supine Hemodilution
Decreased: “TLC”
Triglycerides
Lipoproteins
Cholesterol
Prolonged standing Increased: K+ (muscles)
Prolonged bedrest Decreased: Albumin (Fluid retention)
Tourniquet Recommended: 1 minute application
Prolonged tourniquet app. Hemoconcentration
Anaerobiosis
Increased: “C2LEA2K”
Calcium
Cholesterol
Lactate
Enzymes
Ammonia
Albumin
K+
Tobacco smoking (Nicotine) Increased: “TUNG2C3”
Triglycerides
Urea
Nonesterified fatty acid
Glucose
GH
Catecholamines
Cortisol
Cholesterol
Alcohol ingestion Increased: “THUG”
Triglycerides
Hypoglycemia (chronic alcoholism)
Uric acid/Urates
GGT
Ammonia Increases by 100-200μg/L/cigar
Stress (anxiety) Increased: “LAGIC”
Lactate
Albumin
Glucose
Insulin
Cholesterol
Drugs Medications affecting plasma volume can affect protein, BUN, iron, calcium
Hepatotoxic drugs: increased liver function enzymes
Diuretics: decreased sodium and potassium
Diurnal variation "CA3PI2TG”
Cortisol
ACTH
ACP
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9. Aldosterone
Prolactin
Iron
Insulin
Thyroxine
GH
Specimen Collection and Handling
Sleeping patients Must be awakened before blood collection
Unconscious patients Ask nurse or relative
Identification bracelet
Venipuncture Median Cubital (1st)  Cephalic (2nd)  Basilic (3rd)
Tourniquet Velcro or Seraket type
3-4 inches above the site
Not exceed 1 minute
Needle Bevel up
15-30O angle
Length: 1 or 1.5 inch (Butterfly needle: ½ to ¾ inch)
After blood collection Cotton  site
Apply pressure for 3-5 minutes
BP cuff as tourniquet Inflate to 60 mmHg
Benzalkonium chloride Disinfectant for ethanol testing
(Zephiran) Dilution – 1:750
IV line on both arms Discontinue IV for 2 minutes
Collect sample below the IV site
Initial sample (5mL)  discard
IV fluid contamination Increased:
Glucose (10% contam. w/ 5% dextrose  increased bld glucose by 500 mg/dL)
Chloride
Potassium
Sodium
Decreased:
Urea
Creatinine
Renin blood level Collected after a 3-day diet, from a peripheral vein
Basal state collection Early morning blood collection
12 hours after the last ingestion of food
Lancet 1.75mm: preferred length to avoid penetrating the bone
Incision (Skin puncture) <2.0mm (infants and children)
2-3mm (adults)
1.5-2.4mm Distance from the skin surface to bone or cartilage (middle finger)
Arterialized capillary blood Earlobe: Preferred site
Lateral plantar heel surface: most commonly used site
Flea Minute metal filling which may be inserted into the capillary tube before
collecting blood to help mix the specimen while the blood is entering the tube
Indwelling umbilical artery Best site for blood gas analysis (newborns)
1000-3000 RCF for 10 mins Centrifugation requirement
Hemolysis Increased:
“KLA6MP ITC2”
-K+
-LDH (150x)
-ACP
-ALP
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10. -Aldolase
-ALT
-AST
-Albumin
-Mg2+
-Phosphorus
-Iron
-Total protein
Affects bilirubin levels
Inhibits lipase
Refrigeration/Chilling Required for: “ABCGLRP2”
(Low temp) Ammonia
Blood gases
Catecholamines
Gastrin
Lactic acid
Renin
PTH
Pyruvate
Decreased:
LD 4 and 5
Increased:
ALP
Photosensitive analytes Bilirubin
Beta-carotene
Folate
Porphyrins
Vitamins A and B6
Oxalate Insoluble salt
1-2 mg/mL blood
Citrate Non-ionized form
3.2-3.8 g/dL (1:9 ratio)
EDTA Chelation
1-2 mg/mL blood
Versene: disodium salt
Sequestrene: Dipotassium salt
Fluoride Weakly dissociated calcium component
2 mg/mL blood: anti-glycolytic
10 mg/mL blood: anticoagulant
Heparin A.k.a. Mucoitin polysulfuric acid
Universal anticoagulant
Antithrombin
0.2 mg/mL blood
Lithium heparin For glucose, BUN, ionized calcium, electrolyte studies (K+: best) and creatinine
Orange top tube Additive: Thrombin
Royal blue top tube Additives:
None;
Na2EDTA
Sodium heparin
Brown top tube Lead testing
Tan top tube Lead testing
Black top tube Additive: Buffered sodium citrate
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11. For ESR
Respinning gel tubes Increases potassium
Thixotropic gel Gel separator (SG: 1.04)
Serum: (SG: 1.03)
RBC: (SG: 1.05)
Laboratory Mathematics
% w/v Grams of solute = % solution desired x total volume desired
100
% v/v mL of solute = % solution desired x total volume desired
100
% w/w Grams of solute = % solution desired x grams of the total solution
100
Molarity M= _grams of solute_______
GMW x volume of solution
Moles Mol = weight (grams)
GMW
To prepare a molar solution Grams of solute = Molarity x GMW of the solute x Volume (L) desired
To convert % w/v to M = % w/v  10
Molarity GMW
Normality N = _Grams of solute_
EW x volume (L)
Equivalent weight (EW) EW = __MW___
valence
To prepare a normal Grams of solute = Normality x EW x Volume (L)
solution of solids
To convert % w/v to N = w/v  10
Normality EW
Normality N = Molarity x Valence
Molarity M = Normality
valence
Molality m = Grams of solute__
MW x kg of solvent
Milliequivalents mEq/L = mg/dL  10  valence
MW
Millimoles mmol/L = mg/dL  10
MW
Ratio Ratio = _Volume of solute_
Volume of solvent
Dilution Dilution = __Volume of solute__
Volume of solution
0.179 Conversion factor for iron (mg/dL  μmol/L)
0.01 Conversion factor for phospholipid (g/dL to g/L)
2.27 Conversion factor for folate
Analytical reagent (AR) For qualitative and quantitative analyses
grade For accuracy
Established by American Chemical Society (ACS)
Uses: Trace metal analysis and preparation of standard solutions
Ultrapure reagents Additional purification steps
Ex: Spectrograde, nanograde, HPLC grade
Uses: Chromatography, atomic absorption, immunoassays
Chemically Pure (CP) or Indicates that the impurity limitations are not stated
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12. Pure Grade Purity is delivered by meas. of melting point or boiling point
Technical/Commercial In manufacturing
grade Never used in clin. lab. testing
United States For human consumption
Pharmacopoeia (USP) and Not applicable for lab. analysis
National Formulery (NF) Purpose: For drug manufacturing
Preparation of reagent Filtration (1st)  Distillation, Ion exchange, Reverse Osmosis
grade water
Type I Rgt Water Min. interference
Max. water purity
Used immediately
For ultramicrochemical analyses, measurements of nanogram or subnanogram
concentrations, tissue or cell methods (microscopy) and preparation of
standard solutions
Uses: FEP, AAS, blood gases and pH, enzyme studies, electrolyte testing, HPLC,
trace metal and iron studies
Type II Rgt Water For clinical laboratory use (hematology, microbiology, immunology, chemistry)
For prep. of rgts and QC materials
Type III For washing glasswares
For urinalysis, parasitology and histology
Distilled water Purified to remove almost all organic materials
Deionized water Free from mineral salts; removed by ion exchange processes
Organic material may still be present
Occupational Safety and Req. manuf. to indicate lot no., physical or biological health hazard of the chem..
Health Act (OSHA) rgts, and precautions for safe use and storage
College of American Recommends that a lab. document culture growth, pH and specific water
Pathologists (CAP) resistance on reagent grade water
Tests for water purity Microbiological content
pH
Resistivity
Chemical oxygen demand
Ammonia
Ions
Metals
Detergent-contaminated Alkaline pH
water
Hard water Contains calcium, iron and other dissolved elements
NCCLS Now: Clinical and Laboratory Standards Institute (CLSI)
Dilute solution Relatively little solute
Concentrated solution Large quantity of solute in solution
Saturated solution Excess of undissolved solute particles
Super saturated solution Greater concentration of undissolved solute particles than does a saturated
solution of the same substance
Primary standard Highly purified
(IUPAC) Measured directly to produce a substance of exact known concentration
Secondary standard Low purity
Concentration is determined by comparison w/ a primary standard
Laboratory Safety
National Fire Protection Association (NFPA) Classification of Fires
Class A fire Ordinary combustibles: paper, cloth, rubbish, plastics, wood
Extinguisher: Water (A), Dry chemical (ABC), loaded steam
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13. Class B fire Flammable liquids: grease, gasoline, paints, oil
Extinguisher: Dry chemical (ABC), carbon dioxide (BC), halon foam (BC)
Class C fire Electrical equipment and motor switches
Extinguisher: Dry chemical (ABC), Carbon dioxide (BC), halon (BC)
Class D fire Flammable metals: mercury, magnesium, sodium, lithium
Extinguisher: Metal X
Fought be fire fighters only
Class E fire Detonation (Arsenal fire)
Allowed to burn out and nearby materials protected
Standard Hazards Identification System (Diamond-shaped color coded symbol)
Blue quadrant Health hazard
Red quadrant Flammable hazard
Yellow quadrant Reactivity/Stability hazard
White quadrant Other special information
Chemical spills 1st step: assist/evacuate personnel
1:10 dilution of chlorine To disinfect and clean bench tops
bleach (10%) In contact with the area for at least 20 minutes
HBV: 10 minutes
HIV: 2 minutes
Poisonous vapors Chloroform
Methanol
Carbon tetrachloride
Bromide
Ammonia
Formaldehyde
Mercury
Flammable and Acetone
combustible solvents Ethanol
Toluene
Methanol
Xylene
Benzene
Isopropanol
Heptane
Flammable liquids Flash point below 37.8OC
Combustible liquids Flash point at or above 37.8OC
Strong acids or bases Neutralized before disposal
Water should NEVER be added to concentrated acid
Ether Deteriorate over time  hazardous
Forms explosive peroxides
Benzidine Known carcinogen
Fumehoods Ventilation: velocity of 100-120 ft/min
Safety showers Deliver 30-50 gal/min of H2O at 20-50 psi
Carbohydrates
Glycol aldehyde The simplest carbohydrate
Sucrose Most common nunreducing sugar
Pancreas Exocrine: Enzymes (AMS, LPS)
Endocrine: Hormones (Insulin, glucagon, somatostatin)
Hyperglycemic Hormones “GAG CHET”
Glucagon
ACTH
GH
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14. Cortisol
Human Placental Lactogen
Epinephrine
Thyroxine
Hyperglycemia Electrolyte Imbalance:
(≥126 mg/dL) Decreased: Sodium, Bicarbonate
Increased: Potassium
Hypoglycemia 50-55 mg/dL = Symptoms
≤50 mg/dL = Diagnostic
Whipple’s triad Low blood glucose concentration
(Hypoglycemia) Typical symptoms
Symptoms alleviated by glucose administration
6:1 Ratio of BHA to AA in severe DM
(Normal = 1:1)
Type 1 DM IDDM
Juvenile Onset
Brittle
Ketosis-prone
80-90% reduction of beta-cells  Symptomatic Type 1 DM
HLA-DR3 and DR4
(+) Glutamic acid decarboxylase (GAD65)
(+) Insulin autoantibodies
(+) Microalbuminuria: 50-200 mg/24 hours = Diabetic nephropathy
(-) C-peptide
Complications of Type I DM Microvascular disorders:
Nephropathy
Neuropathy
Retinopathy
Type 2 NIDDM
Adult type/Maturity Onset
Stable
Ketosis-resistant
Receptor-deficient
Insulin resistance: relative insulin deficiency
Strong genetic predisposition
Geneticist’s nightmare
If untreated  glucose: >500 mg/dL  nonketotic hyperosmolar coma
Gestational DM Screening: 1hr GCT (50g) – bet. 24 and 28 weeks of gestation
Confirmatory: 3-hr GTT (100g)
Infants: at risk for respiratory distress syndrome, hypocalcemia,
hyperbilirubinemia
After giving birth, evaluate 6-12 weeks postpartum
Converts to DM w/in 10 years in 30-40% of cases
OGTT (GDM) FBS = ≥95 mg/dL
1-Hr = ≥ 180 mg/dL
2-Hr = ≥ 155 mg/dL
3-Hr = ≥ 140 mg/dL
GDM = 2 plasma values of the above glucose levels are exceeded
Impaired fasting glucose FBS = 100-125 mg/dL
(Pre-diabetes)
Impaired glucose tolerance FBS = <126 mg/dL
2-Hr OGTT = 140-199 mg/dL
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15. FBS WB = 15% lower than in serum or plasma
VB = 7 mg/dL lower than capillary and arterial blood
CSF glucose 60-70% of the plasma glucose
Peritoneal fluid glucose Same with plasma glucose
Plasma glucose increases Fasting: 2 mg/dL/decade
w/ age Postprandial: 4 mg/dL/decade
Glucose challenge: 8-13 mg/dL/decade
w/in 1 hour Separate serum/plasma from the cells
(Preferably w/in 30 mins)
5-7%/hr Glycolysis at room temperature
1-2 mg%/hr Glycolysis at refrigerated temperature
Copper reduction methods Cupric  Cuprous  Cuprous oxide
Folin Wu Cuprous ions + phosphomolybdate  phosphomolybdenum blue
Nelson-Somogyi Cuprous ions + arsenomolybdate  arsenomolybdenum blue
Neocuproine method Cuprous ions + neocuproine  Cuprous-neocuproine complex (yellow)
Benedict’s method Reducing substances in blood and urine
Alkaline Ferric Reduction Ferricyanide ---(Glucose)--> Ferrocyanide
method (Hagedorn-Jensen) (Yellow) (Colorless)
Ortho-toluidine Schiff’s base
(Dubowski method)
Glucose oxidase Measures beta-D-glucose (65%)
Mutarotase Converts alpha-D-glucose (35%) to beta-D-glucose (65%)
NADH/NADPH Absorbance at 340nm
Polarographic glucose Consumption of oxygen on an oxygen-sensing electrode
oxidase O2 consumption α glucose concentration
Hexokinase method Most specific method
Reference method
Uses G-6-PD
G-6-PD Most specific enzyme rgt for glucose testing
Interfering substances False-decreased
(Glucose oxidase) Bilirubin
Uric acid
Ascorbate
Hemolysis (>0.5 g/dL Hgb) Major interfering substance in hexokinase method (false-decreased)
Dextrostics Cellular strip
Strip w/ glucose oxidase, peroxidase and chromogen
OGTT Janney-Isaacson method (Single dose) = most common
Exton Rose (Double dose)
Drink the glucose load within 5 mins
IVGTT For patients with gastrointestinal disorders (malabsorption)
Glucose: 0.5 g/kg body weight
Given w/in 3 mins
1st blood collection: after 5 mins of IV glucose
Requirements for OGTT Ambulatory
Fasting: 8-14 hours
Unrestricted diet of 150g CHO/day for 3 days
Do not smoke or drink alcohol
Glucose load 75 g = adult (WHO std)
100 g = pregnant
1.75 g glucose/kg BW = children
HbA1c 2-3 months
Glucose = beta-chain of HbA1
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16. 1% increase in HbA1c = 35 mg/dL increase in plasma glucose
18-20% = prolonged hyperglycemia
7% = cutoff
Specimen: EDTA whole blood
Test: Affinity chromatography (preferred)
IDA and older RBCs High HbA1c
RBC lifespan disorders Low HbA1c
Fructosamine 2-3 weeks
(Glycosylated albumin/ Useful for patients w/ hemolytic anemias and Hgb variants
plasma protein ketoamine) Not used in cases of low albumin
Specimen: Serum
Galactosemia Congenital deficiency of 1 of 3 enzymes in galactose metabolism
Galactose-1-phosphate uridyl transferase (most common)
Galactokinase
Uridine diphosphate galactose-4-epimerase
Essential fructosuria Autosomal recessive
Fructokinase deficiency
Hereditary fructose Defective fructose-1,6-biphosphate aldolase B activity
intolerance
Fructose-1,6-biphosphate Failure of hepatic glucose generation by gluconeogenic precursors such as
deficiency lactate and glycerol
Glycogen Storage Disease Autosomal recessive
Defective glycogen metabolism
Test: IVGTT (Type I GSD)
Ia = Von Gierke Glucose-6-Phosphatase deficiency (most common worldwide)
II = Pompe Alpha-1,4-glucosidase deficiency (most common in the Philippines)
III = Cori Forbes Debrancher enzyme deficiency
IV = Andersen Brancher enzyme deficiency
V = McArdle Muscle phosphorylase deficiency
VI = Hers Liver phosphorylase deficiency
VII = Tarui Phosphofructokinase deficiency
XII = Fanconi-Bickel Glucose transporter 2 deficiency
CSF glucose Collect blood glucose at least 60 mins (to 2 hrs) before the lumbar puncture
(Because of the lag in CSF glucose equilibrium time)
< 0.5 Normal CSF : serum glucose ratio
C-peptide Formed during conversion of pro-insulin to insulin
5:1 to 15:1 Normal C-peptide : insulin ratio
D-xylose absorption test Differentiate pancreatic insufficiency from malabsorption (low blood or urine
xylose)
Gerhardt’s ferric chloride Acetoacetate
test
Nitroprusside test 10x more sensitive to acetoacetate than to acetone
Acetest tablets Acetoacetate and acetone
Ketostix Detects acetoacetate better than acetone
KetoSite assay Detects beta-hydroxybutyrate but not widely used
Normal Values RBS = <140 mg/dL
(Carbohydrates) FBS = 70-100 mg/dL
HbA1c = 3-6%
Fructosamine = 205-285 μmol/L
2-Hr PPBS = <140 mg/dL
GTT:
30 mins = 30-60 mg/dL above fasting
lec.mt 04 |Page | 16

17. 1-Hr = 20-50 mg/dL above fasting
2-Hr = 5-15 mg/dL above fasting
3-Hr = fasting level or below
Lipids
Phospholipids Most abundant lipid
Amphipathic: polar (hydrophilic head) and nonpolar (hydrophobic side chain)
Sphingomyelin Reference material during 3rd trimester of pregnancy
Concentration is constant as opposed to lecithin
Not derived from glycerol but from sphingosine (amino alcohol)
Forms of phospholipids 70% Lecithin/Phosphatidyl choline
20% Sphingomyelin
10% Cephalin
TLC + Densitometric Method for L/S ratio
quantitation
Microviscosity Measured by fluorescence polarization
Cholesterol Not a source of fuel
Not affected by fasting
70% Cholesterol ester (plasma/serum)
30% Free cholesterol (plasma/serum and RBC)
LCAT Esterification of cholesterol
Apo A-1 Activator of LCAT
Cholesterol increases after 2 mg/dL/year between 50 and 60 years old
the age of 50
Liebermann Burchardt Cholestadienyl Monosulfonic acid
Green end color
Salkowski Cholestadienyl Disulfonic acid
Red end color
Color developer mixture Glacial acetic acid
(Cholesterol) Acetic anhydride
Conc. H2SO4
One-step method Colorimetry (Pearson, Stern and Mac Gavack)
Two-step method Color. + Extraction (Bloor’s)
Three-step method Color. + Extract. + Saponification (Abell-Kendall)
Four-step method Color. +Extract. + Sapon. + Precipitation
(Schaenheimer Sperry, Parekh and Jung)
Abell, Levy and Brodie mtd CDC reference method for cholesterol:
(Chemical method) -Hydrolysis/saponification (Alc. KOH)
-Hexane extraction
-Colorimetry (Liebermann-Burchardt)
Triglycerides Most insoluble lipid
Main storage lipid in man (adipose tissue) – 95%
Fasting: 12 hours
Triglyceride increases after 2 mg/dL/year between 50 and 60 years old
the age of 50
Van Handel & Zilversmith Chromotropic acid
(Colorimetric) (+) Blue color compound
Hantzsch Condensation Diacetyl acetone
(Fluorometric) (+) Diacetyl lutidine compound
Modified Van Handel and CDC reference method for triglycerides:
Zilversmith -Alkaline hydrolysis
(Chemical method) -Chloroform extraction  extract treated w/ silicic acid
-Color reaction w/ chromotropic acid – meas. HCHO
lec.mt 04 |Page | 17

18. (+) Pink colored
Fatty acids Short chain = 4-6 C atoms
Medium chain = 8-12 C atoms
Long chain = >12 C atoms
Saturated = w/o double bonds
Unsaturated = w/ double bonds
Substrate for gluconeogenesis
Most is bound to albumin
Palmitic acid 16:0
Stearic acid 18:0
Oleic acid 18:1
Linoleic acid 18:2
Arachidonic acid 20:4
Lipoprotein lipase Hydrolyzes TAG in lipoproteins, releasing fatty acid and glycerol
(Lipemia clearing factor)
Hepatic lipase Hydrolyzes TAG and phospholipids from HDL
Hydrolyzes lipids on VLDL and IDL
Endothelial lipase Hydrolyzes phospholipids and TAG in HDL
Apolipoprotein Protein component of lipoprotein
Amphipathic helix – ability of proteins to bind to lipids
Chylomicrons Largest and least dense
Produced by the intestine
SG: <0.95
80-95% TAG (exogenous)
Apo B-48 (Major)
EP: Origin
VLDL Secreted by the liver
SG: 0.95-1.006
65% TAG (endogenous)
Apo B-100 (Major)
EP: pre-beta
LDL Synthesized by the liver
SG: 1.006-1.063
50% CE
Apo B-100 (Major)
EP: beta
Cholesterol transport: LiverTissues
Target of cholesterol lowering therapy
Better marker for CHD risk
HDL Smallest but dense
SG: 1.063-1.21
45-55% protein
26-32% phospholipid
Apo A-1 (Major)
EP: alpha
Produced by the liver and intestine
Reverse cholesterol transport: TissueLiver
IDL Product of VLDL catabolism
Seen in Type 3 hyperlipoproteinemia (Apo E-III def.; beta-VLDL)
SG: 1.006-1.019
Lp(a) Sinking pre-beta lipoprotein
SG: 1.045-1.080
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19. Apo B-100
EP: pre-beta (VLDL)
UC: like LDL
Independent risk factor for atherosclerosis
LpX Found in obstructive jaundice (cholestasis) and LCAT deficiency
90% FC and PL
Apo C and albumin
Beta-VLDL Floating beta-lipoprotein
SG: <1.006
EP: beta (LDL)
UC: like VLDL
Found in type 3 hyperlipoproteinemia (Apo E-III def; IDL)
Rich in cholesterol content than VLDL
Lipoprotein methodologies Specimen: sample from serum separator tubes (preferred)
EDTA plasma: choice for research studies of LPP fractions
Fasting state: TAG  VLDL
Nonfasting state: TAG  CM
Ultracentrifugation Reference method for LPP quantitation
Reagent: Potassium bromide (SG: 1.063)
Ultracentrifugation of plasma for 24 hours
Expressed in Svedberg units
Electrophoresis Electrophoretic pattern:
(+) HDL VLDL  LDL  CM (Origin) (-)
Agarose gel: sensitive medium
VLDL: migrates w/ alpha2-globulin (pre-beta)
Chemical precipitation Uses polyanions (heparin and divalent cations) and polyethylene glycol
Dextran sulfate-Mg2+
Heparin-Mn2+
3-step procedure: CDC Reference method for HDL
Ultracentrifugation
Precipitation
Abell-Kendall assay
Beta quantification + Method for LDL
Ultracentrifugation + Sample: EDTA plasma
Chemical precipitation
Immunoturbidimetric assay Measures Lipoprotein (a)
LDL Cholesterol Total Cholesterol – HDL – VLDL
Friedewald method Most commonly used
VLDL = TAG/2.175 (mmol/L)
VLDL = TAG/5 (mg/dL)
Not applicable if TAG is >400 mg/dL
De Long method VLDL = TAG/2.825 (mmol/L)
VDL = TAG/6.5 (mg/dL)
Apo A-1 Activates LCAT
Apo B-100 LDL  LDL receptor
Apo B-48 CM (major)
Not recognized by LDL receptor
Apo C-II Activates LPL
Apo D Activates LCAT
Apo E Apo E-4: associated w/ high LDL, higher risk of CHD and Alzheimer’s disease
Apo(a) Lp(a)
Homologous to plasminogen
lec.mt 04 |Page | 19

20. Abetalipoproteinemia Autosomal recessive
(Basses-Kornzweig syn.) Defective apo B synthesis
Deficient fat soluble vitamins
Niemann-Pick disease Sphingomyelinase deficiency
Tangier’s disease Deficiency of HDL (1-2 mg/dL)
Defects in the gene for the ABCA1 transporter
LPL deficiency TAG = 10,000 mg/dL
(Chylomicronemia) Do not develop premature coronary disease (CM are not atherogenic)
Abdominal pain and pancreatitis
LCAT deficiency Fish-eye disease
Low HDL
Tay-Sachs disease Hexosaminidase A deficiency
Fredrickson Classification
Type 1 LPL deficiency (Chylomicronemia)
Increased: CM (TAG)
Type 2a Familial hypercholesterolemia
Increased: LDL (cholesterol)
Type 2b Combined hyperlipidemia (most common primary hyperlipidemia)
Increased: LDL (cholesterol), VLDL (TAG)
Type 3 Dysbetalipoproteinemia
Increased: IDL, (+) beta-VLDL
(+) Apo E-II
(+) Eruptive and palmar xanthomas
Type 4 Hypertriglyceridemia
Increased: VLDL (TAG)
Type 5 Increased: VLDL (Endo.TAG), CM (Exo.TAG)
Normal Values Cholesterol:
(Lipids) Desirable = <200 mg/dL
Borderline high = 200-239 mg/dL
High = >240 mg/dL
Triglycerides:
Desirable = <150 mg/dL
Borderline high = 150-199 mg/dL
High = 200-499
Very high = >500 mg/dL
HDL:
Low = <40 mg/dL (Cutoff)
High = >60 mg/dL
LDL:
Optimal = <100 mg/dL
Near/above optimal = 100-129 mg/dL
Borderline high = 130-159 mg/dL
High = 160-189 mg/dL
Very high = >190 mg/dL
Proteins
Proteis First rank of importance
Proteins Amphoteric: positive and negative charges
Effective blood buffers
Synthesized by the liver except immunoglobulins (plasma cells)
Provide 12-20% of total daily body energy requirement
Composed of 50-70% of the cell’s dry weight
lec.mt 04 |Page | 20

21. Primary structure Amino acid sequence
Det. the identity of protein, molecular structure, function binding capacity,
recognition ability
Secondary structure Winding of polypeptide chain
Specific 3-D conformations: alpha-helix, beta-pleated sheath, bend form
Tertiary structure Actual 3-D configuration
Folding pattern
Physical and chemical properties of proteins
Quarternary structure Association of 2 or more polypeptide chains  protein
Albumin No quarternary structure
Glucogenic amino acids Alanine (pyruvate)
Arginine (alpha-ketoglutarate)
Aspartate (oxaloacetate)
Ketogenic amino acids Degraded to acetyl-CoA
Leucine
Lysine
Simple proteins Hydrolysis  Amino acids
Fibrous: fibrinogen, troponins, collagen
Globular: hemoglobin, plasma proteins, enzymes, peptide hormones
Conjugated proteins Protein (apoprotein) + nonprotein moiety (prosthetic group)
Metalloproteins: ferritin, ceruloplasmin, hemoglobin, flavoproteins
Lipoproteins: VLDL, HDL, LDL, CM
Glycoproteins: haptoglobin, alpha1-antitrypsin (10-40% CHO)
Mucoproteins or proteoglycans: Mucin (CHO > CHON)
Nucleoproteins: Chromatin (combined w/ nucleic acids)
Nitrogen balance Balance bet. anabolism and catabolism
Negative nitrogen balance Catabolism > anabolism
Excessive tissue destruction
Positive nitrogen balance Anabolism > catabolism
Growth and repair processes
Prealbumin (Transthyretin) Transports thyroxine and retinol (Vit. A)
Landmark to confirm that the specimen is really CSF
Albumin Maintains osmotic pressure
Negative acute phase reactant
Alpha1-antitrypsin Acute phase reactant
Major inhibitor of protease activity
90% of alpha1-globulin band
Alpha1-fetoprotein Gestational marker
Tumor marker: hepatic and gonodal cancers
Screening test for fetal conditions (Spx: maternal serum)
Amniotic fluid: confirmatory test
Increased: Hepatoma, spina bifida, neural tube defects
Decreased: Down Syndrome (Trisomy 21)
Alpha1-acid glycoprotein/ Low pI (2.7)
orosomucoid Negatively charged even in acid solution
Alpha1-antichymotrypsin Acute phase reactant
Binds and inactivates PSA
Increased: Alzheimer’s disease, AMI, infection, malignancy, burns
Haptoglobin (alpha2) Acute phase reactant
Binds free hemoglobin (alpha chain)
Ceruloplasmin (alpha2) Copper binding (6-8 atoms of copper are attached to it)
Has enzymatic activities
lec.mt 04 |Page | 21

22. Decreased: Wilson’s disease (copper  skin, liver, brain, cornea [Kayser-
Fleisher rings])
Alpha2-macroglobulin Larges major nonimmunoglobulin protein
Increased: Nephrotic syndrome (10x)
Forms a complex w/ PSA
Group-specific component Affinity w/ vitamin D and actin
(Gc)-globulin (bet. alpha1
and alpha2)
Hemopexin (beta) Binds free heme
Beta2-microglobulin HLA
Filtered by glomeruli but reabsorbed
Transferrin/Siderophilin Negative acute phase reactant
(beta) Major component of beta2-globulin fraction
Pseudoparaproteinemia in severe IDA
Increased: Hemochromatosis (bronze-skin), IDA
Complement (beta) C3: major
Fibrinogen (bet. beta and Acute phase reactant
gamma) Between beta and gamma globulins
CRP (gamma) General scavenger molecule
Undetectable in healthy individuals
hsCRP: warning test to persons at risk of CAD
Immunoglobulins (gamma) Synthesized by the plasma cells
IgG>IgA>IgM>IgD>IgE
Myoglobin Marker: Ischemic muscle cells, chest pain (angina), AMI
Troponins Most important marker for AMI
TnT (Tropomyosin-binding Specific for heart muscle
subunit) Det. unstable angina (angina at rest)
TnI (Inhibitory subunit or Only found in the myocardium
Actin-binding unit) Greater cardiac specificity than TnT
Highly specific for AMI
13x more abundant in the myocardium than CK-MB
Very sensitive indicator of even minor amount of cardiac necrosis
TnC Binds calcium ions and regulate muscle contractions
Glomerular proteinuria Most common and serious type
Often called albuminuria
Tubular proteinuria Defective reabsorption
Slightly increased albumin excretion
Overload proteinuria Hemoglobinuria
Myoglobinuria
Bence-Jones proteinuria
Postrenal proteinuria Urinary tract infection, bleeding, malignancy
Microalbuminuria Type 1 DM
Albumin excretion ≥30 mg/g creatinine (cutoff: DM) but ≤300 mg/g creatinine
Microalbuminuria: 2 out of 3 specimens submitted are w/ abnormal findings
(w/in 6 months)
CSF Oligoclonal banding 2 or more IgG bands in the gamma region:
Multiple sclerosis
Encephalitis
Neurosyphilis
Guillain-Barre syndrome
Neoplastic disorders
lec.mt 04 |Page | 22

23. Serum Oligoclonal banding Leukemia
Lymphoma
Viral infections
Alkaptonuria Ochronosis (tissue pigmentation)
Homocystinuria Impaired activity of cystathione beta-synthetase
Elevated homocysteine and methionine in blood and urine
Screen: Modified Guthrie test (Antagonist: L-methionine sulfoximine)
MSUD Markedly reduced or absence of alpha-ketoacid decarboxylase
4 mg/dL of leucine is indicative of MSUD
Screen: Modified Guthrie test (Antagonist: 4-azaleucine)
Diagnostic: Amino acid analysis (HPLC)
PKU Deficiency of tetrahydrobiopterin (BH4)  elevated blood phenylalanine
Normal Values Total protein = 6.5-8.3 g/dL
(Proteins) Albumin = 3.5-5.0 g/dL
Globulin = 2.3-3.5 g/dL
Kidney Function Tests
Tests for GFR Clearance:
-Inulin clearance
-Creatinine clearance
-Urea clearance
Phenolsulfonphthalein dye test
Cystatin C
Tests for Renal Blood Flow BUN
Creatinine
Uric acid
Tests Measuring Tubular Excretion:
Function -Para-amino hippurate test (Diodrast test)
-Phenolsulfonphthalein dye test
Concentration:
-Specific gravity
-Osmolality
GFR Decreases by 1.0 mL/min/year after age 20-30 years
150 L of glomerular filtrate is produced daily
Inulin clearance Reference method
Creatinine clearance Best alternative method
Measure of the completeness of a 24-hour urine collection
Excretion: 1.2-1.5 g creatinine/day
Urea clearance Demonstrate progression of renal disease or response to therapy
Cystatin C Low MW protease inhibitor
FilteredNot secretedCompletely reabsorbed (PCT)
Indirect estimates of GFR
Its presence in urine denotes damage to PCT
BUN Synthesized from Ornithine or Kreb’s Henseleit cycle
First metabolite to elevate in kidney diseases
Better indicator of nitrogen intake and state of hydration
2.14 BUN  Urea (mg/dL)
Fluoride or citrate Inhibit urease
Thiosemicarbazide Enhance color development (BUN mtd)
Ferric ions
Diacetyl monoxime method Yellow diazine derivative
Urease method Routinely used
Urease: prepared from jack beans
lec.mt 04 |Page | 23

24. Urea ---(Urease)--> NH4 + Berthelot reagent (Measure ammonia)
Coupled urease Glutamate dehydrogenase method
UV enzymatic method
Isotope dilution mass Reference method
spectrometry For research purposes
NPN 45% Urea
20% Amino acid
20% Uric acid
5% Creatinine
1-2% Creatine
0.2% Ammonia
Creatinine Derived from alpha-methyl guanidoacetic acid (creatine)
Produced by 3 amino acids (methionine, arginine, lysine)
Most commonly used to monitor renal function
Enzymatic methods Creatinine Aminohydrolase – CK method
(Creatinine) Creatinase-Hydrogen Peroxide method – benzoquinonemine dye (red)
Creatininase (a.k.a. creatinine aminohydrolase)
Direct Jaffe method Formation of red tautomer of creatinine picrate
Interferences (Direct Jaffe) Falsely increased:
Ascorbate
Glucose
Uric acid
Alpha-keto acids
Folin Wu Method (+) Red orange tautomer
Lloyd’s or Fuller’s Earth True measure of creatinine
method Sensitive and specific
Uses adsorbent to remove interferences (UA, Hgb, Bili)
Lloyd’s reagent Sodium aluminum silicate
Fuller’s earth reagent Aluminum magnesium silicate
Jaffe reagent (Alk. picrate) Satd. picric acid + 10% NaOH
Kinetic Jaffe method Popular, inexpensive, rapid and easy to perform
Requires automated equipment
Azotemia Elevated urea and creatinine in blood
Pre-renal azotemia Decreased GFR but normal renal function
Dehydration, shock, CHF
Increased: BUN
Normal: Creatinine
Renal azotemia True renal disease
Decreased GFR
Striking BUN level but slowly rising creatinine value
BUN = >100 mg/dL
Creatinine = >20 mg/dL
Uric acid = >12 mg/dL
Post renal azotemia Urinary tract obstruction
Decreased GFR
Nephrolithiasis, cancer or tumors of GUT
Creatinine = normal or slightly increased
Uremia Marked elevation of urea, accompanied by acidemia and electrolyte imbalance
(K+ elevation) of renal failure
Normocytic, normochromic anemia
Uremic frost (dirty skin)
Edema
lec.mt 04 |Page | 24

25. Foul breath
Urine-like sweat
Uric acid From purine (adenine and guanine) catabolism
Excretion: 1g/day
Hyperuricemia -Gout
-Increased nuclear metabolism (leukemia, lymphoma, MM, polycythemia,
hemolytic and megaloblastic anemia) – Tx: Allopurinol
-Chronic renal disease
-Lesch-Nyhan syndrome (HGPRT deficiency)
Hypouricemia Fanconi’s syndrome
Wilson’s disease
Hodgkin’s disease
Methods (Uric acid) Stable for 3 days
Potassium oxalate cannot be used
Major interferences: Ascorbate and bilirubin
Phosphotungstic acid mtd Uric acid + Phosphotungstic acid ---(NaCN/NaCO3)--> Tungsten blue + Allantoin
NaCN Folin
Newton
Brown
Benedict
NaCO3 Archibald
Henry
Caraway
Lagphase Incubation period after the addition of an alkali to inactivate non-uric acid
reactants
Uricase method Simplest and most specific method
Candidate reference method
Uric acid (Absorbance at 293nm) ---[Uricase]--> Allantoin (No absorbance)
Decrease in absorbance α uric acid concentration
Para-amino hippurate test Measures renal plasma flow
Reference method for tubular function
Phenolsulfonphthalein dye Measures excretion of dye proportional to renal tubular mass
test 6 mg of PSP is administered IV
Concentration tests Collecting tubules and loops of Henle
Specimen: 1st morning urine
Specific gravity Affected by solute number and mass
SG >1.050: X-ray dye and mannitol
1.010 = SG of ultrafiltrate in Bowman’s space
Osmolality Total number solute particles present/kg of solvent (moles/kg solvent)
Affectted only by number of solutes present
Urine osmolality = due to urea
Serum osmolality = due to sodium and chloride
Det. by Colligative properties:
Freezing point (incr. osm. = decr. FP)
Vapor pressure (incr. osm. = decr. VP)
Osmotic pressure (incr. osm. = incr. OP)
Boiling point (incr. osm. = incr. BP)
Direct methods Freezing point osmometry = popular method
(Osmolality) Vapor pressure osmometry (Seebeck effect)
Incr. plasma osmolality Incr. vasopressin (H2O reabsorption)  decr. plasma osmolality
Tubular failure Increased: BUN, creatinine, calcium
Decreased: Phosphate
lec.mt 04 |Page | 25

26. Osmolal gap Difference between measured and calculated osmolality
Sensitive indicator of alcohol or drug overdose
Osmolal gap: >12 mOsm/kg DKA
Drug overdose
Renal failure
Normal Values Creatinine Clearance:
(Kidney Function Tests) Male = 85-125 mL/min
Female = 75-112 mL/min
BUN = 8-23 mg/dL
Creatinine = 0.5-1.5 mg/dL
Uric acid:
Male = 3.5-7.2 mg/dL
Female = 2.6-6.0 mg/dL
Renal plasma flow (PAH) = 600-700 mL/min
Renal blood flow (PSP) = 1200 mL/min
SG = 1.005-1.030
Osmolality:
Serum = 275-295 mOsm/kg
Urine (24-hr) = 300-900 mOsm/kg
[<290 mOsm/kg = kidney damage]
Urine osmolality: Serum osmolality = 1:1 to 3:1
[>1:1 = Glomerular disease]
[1.2:1 = loss of renal concentrating ability]
[<1:1 = Diabetes Insipidus]
Liver Function Tests
Liver Receives 15 mL of blood per minute
Lobule: anatomic unit
Synthetic function Proteins, CHO, lipids, LPP, clotting factors, ketone bodies, enzymes
Albumin: 12g/day
Conjugation function Bilirubin metabolism
Bilirubin: 200mg/day
Detoxification and Drug Drugs
metabolism Ammonia  Urea  Excreted
Excretory and Secretory Bile acids: cholic acid and chenodeoxycholic acid
functions Bile salts: bile acids + amino acids (glycine and taurine)
Storage function Vitamins
Glycogen
Test measuring the Hepatic Total Protein Determination:
Synthetic Ability -Kjeldahl method
-Biuret method
-Folin-Ciocalteu (Lowry) method
-UV absorption method
-Electrophoresis
-Refractometry
-Turbidimetric and Nephelometric methods
-Salt fractionation
Prothrombin Time (Vitamin K Response Test)
Test measuring Bilirubin Assay:
Conjugation/Excretion -Evelyn and Malloy method
Function -Jendrassik and Grof
Bromsulfonphthalein (BSP) Dye Excretion test
lec.mt 04 |Page | 26

27. Test for Detoxification Enzyme tests: ALP, AST, ALT, 5’NT, GGT, OCT, LAP, LDH
Function Ammonia:
-Kjeldahl (Digestion) method
-Nesslerization reaction
-Berthelot reaction
Plasma protein 0.2-0.4 g/dL higher than serum due to fibrinogen
Kjeldahl (Digestion) mtd Standard reference method
Measurement of nitrogen content
Serum + Tungstic acid  PFF
1g N2 = 6.54g protein
15.1-16.8% = N2 content of proteins
Rgt: H2SO4
End product: NH3
Biuret method Most widely used method (IFCC recommended)
Req. at least 2 peptide bonds and an alkaline medium
Rgts:
Alkaline CuSO4
Rochelle salt (NaK Tartrate)
NaOH
KI
End product: Violet color (545nm)
Folin-Ciocalteu (Lowry) Highest analytical sensitivity
method Oxidation of phenolic compounds (tyrosine, tryptophan, histidine)
Rgts:
Phenol (or phosphotungstic-molybdic acid)
Biuret (color enhancer)
End product: Blue color
Electrophoresis MI: elevated APRs (AAT, HPG, a1-x)
Gamma-spike Monoclonal gammopathy (multiple myeloma)
Beta-gamma bridging In serum: Hepatic cirrhosis (IgA)
In plasma: normal (fibrinogen)
Alpha2-globulin band spike Nephrotic syndrome
Alpha1-globulin flat curve Juvenile cirrhosis (AAT deficiency)
Alpha1, alpha2, beta- Inflammation
globulin band spikes
Polyclonal gammopathy Chronic inflammation (RA, malignancy)
Small spikes in beta region IDA (transferrin)
Free hemoglobin “Blip” in the late alpha2 or early beta region
Refractometry Refractive index
Turbidimetric and SSA
nephelometric methods TCA
Salt fractionation Salt: Sodium sulfate
Albumin Soluble:
Water
Moderately concentrated salt solution
Concentrated salt solution
Insoluble:
Hydrocarbon solvents
Highly concentrated salt solution
Saturated salt solution
lec.mt 04 |Page | 27

28. Globulin Soluble:
Hydrocarbon solvents
Weak salt solution
Insoluble:
Water
Saturated salt solution
Concentrated salt solution
Prothrombin time Differentiates intrahepatic disorder (prolonged PT) from extrahepatic
obstructive liver disease (normal PT)
Albumin Inversely proportional to the severity of the liver disease
Hepatic cirrhosis Low total protein + low albumin
Bromcresol green Most commonly used dye for albumin
Bromcresol purple Most specific dye for albumin
Other dyes for albumin Hydroxyazobenzene benzoic acid (HABA)
Methyl orange (MO)
Nephrotic syndrome Albumin excretion: 20-30 g/day
Analbuminemia (-) albumin
Bisalbuminemia EP: 2 albumin bands
Therapeutic drugs in serum
Inverted A/G ratio Hepatic cirrhosis (IgA)
Multiple Myeloma (IgG)
Waldenström’s macroglobulinemia (IgM)
Chronic inflammation
Bilirubin Derived from hemoglobin myoglobin, catalase and cytochrome oxidase
Heme oxygenase Protoporphyrin  Biliverdin
Biliverdin reductase Biliverdin B1
Urobilinogen Deconjugated bilirubin
Bilirubin 1 Non-polar bilirubin
Free/Slow bilirubin
Bilirubin 2 Polar bilirubin
One-minute/prompt bilirubin
Regurgitative bilirubin
Delta bilirubin Bilirubin tightly bound to albumin
Delta bilirubin = TB-DB+IB
Jaundice Bilirubin >2 or 3 mg/dL
Pre-hepatic jaundice Hemolytic
B1 = increased
B2 = normal
UG = increased
UB = negative
Hepatic jaundice Hepatocellular
B1 = increased
B2 = increased
UG = increased
UB = positive
ALT = increased
AST = increased
Post-hepatic jaundice Obstructive
B1 = normal
B2 = increased
UG = decreased/negative
UB = positive
lec.mt 04 |Page | 28

29. ALP = increased
GGT = increased
Cholesterol = increased
Gilbert’s syndrome Bilirubin transport deficit (uptake)
B1 = increased
B2 = decreased
Crigler-Najjar syndrome Conjugation deficit
Type I = total UDPGT deficiency
Type II = partial UDPGT deficiency
B1 = increased
B2 = decreased
Danger: Kernicterus
Bile is colorless
Dubin-Johnson syndrome & Bilirubin excretion deficit
Rotor syndrome Blockade of excretion into the canaliculi
TB = increased
B2 = increased
Lucey-Driscoll syndrome Circulating inhibitor of bilirubin conjugation
B1 = increased
Methods (Bilirubin) Free from hemolysis and lipemia
Store in the dark
Measured ASAP or w/in 2-3 hours
Van den Berg reaction Diazotization of bilirubin
Evelyn and Malloy method Accelerator: Methanol
Diazo rgts:
Diazo A (0.1% Sulfanilic acid + HCl)
Diazo B (0.5% Sodium nitrite)
Diazo blank (1.5% HCl)
(+) pink to purple azobilirubin
Affected by hemolysis
Jendrassik and Grof Candidate reference method
Accelerator: Caffeine sodium benzoate
Buffer: Sodium acetate
Ascorbic acid: terminates the initial reaction and destroys the excess diazo rgt
Not falsely elevated by hemolysis
Total bilirubin is measured 15 minutes after adding methanol or caffeine soln
Bilirubin Absorbs light maximally at 450nm
Rosenthal White method Double collection method
Collection:
-After 5 mins (50% dye retention)
-After 30 mins (0% dye retention)
Mac Donald method Single collection method
Collection:
-After 45 mins (+/- 5% dye retention)
Ammonia From deamination of amino acids
Elevated levels are neurotoxic and often associated w/ encephalopathy and
acetaminophen poisoning
Diagnosis of hepatic failure and Reye’s syndrome
In severe liver disorder: NH3  circulation  brain (conv. to glutamine) 
increases pH  compromise the Kreb’s cycle  Coma due to lack of ATP for the
brain
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30. Methods (Ammonia) Specimen: Heparin or EDTA plasma
Fasting is required
Avoid smoking
Prolonged standing of specimen: increased NH3 due to deamination
Place on iced water immediately
Avoid hemolysis
Kjeldahl (Digestion) Specimen  PFF
method N2 ----------(hot conc. H2SO4 + CuSO4 + Hg + Selenium)----------> NH3
Nesslerization of ammonia NH3 + K2Hg2I2 ----------(Gum Ghatti)----------> NH2Hg2I2
End color:
Yellow (low to moderate N2)
Orange brown (high N2)
Berthelot reaction NH3 + Phenol + Hypochlorite -----(Na Nitroprusside)-----> Indophenol blue
Normal Values Total protein = 6.5-8.3 g/dL
(Liver Function Tests) Albumin = 3.5-5.0 g/dL
Globulin = 2.3-3.5 g/dL
α1-globulin = 0.1-0.3 g/dL
α2-globulin = 0.6-1.0 g/dL
β-globulin = 0.7-1.1 g/dL
γ-globulin = 0.8-1.6 g/dL
Total bilirubin = 0.2-1.0 mg/dL
Indirect bilirubin = 0.2-0.8 mg/dL
Direct bilirubin = 0-0.2 mg/dL
Urobilinogen:
Urine = 0.1-1.0 Ehrlich units/2hrs (or 0.54 Ehrlich units/day)
Stool = 75-275 Ehrlich units/100g feces (or 75-400 Ehrlich units/24hrs)
Ammonia = 19-60 μg/dL
Enzymes
Enzyme concentration Serum
 Enzyme concentration =  reaction rate
Substrate concentration Reagent
If enzyme > substrate,  substrate =  reaction rate
Saturation kinetics When substrate concentration reaches a maximal value, higher concentration
of substrate no longer results in increased rate of reaction
Cofactors Nonprotein entities
Coenzymes Organic compound
Ex. NADP
 Coenzyme =  Velocity
Activators Inorganic ions
Alters spatial configuration of the enzyme for proper substrate binding
Ex. Ca2+ (#1 activator), Zn2+ (LDH), Cl- (AMS), Mg2+ (CK, ALP)
Metalloenzymes Inorganic ion attached to a molecule
Ex. Catalase, cytochrome oxidase
Inhibitors Interferes with the enzymatic reactions
Competitive inhibitor Binds to the active site of an enzyme
Reversible (Substrate > Inhibitor)
Noncompetitive inhibitor Binds to the allosteric site (cofactor site)
Irreversible
Uncompetitive inhibitor Binds to the enzyme-substrate complex
 Substrate = ES = Inhibition
Isoenzymes Same catalytic reactions but slightly different molecular structures
Fractionation of isoenzymes
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31. Temperature 37’C = optimum temperature for enzyme activity
Temperature = Reaction rate (movement of molecules)
40-50’C Denaturation of enzymes
60-65’C Inactivation of enzymes
Temperature coefficient For every 10OC increase in temperature, there will be a two-fold increase in
(Q10) enzyme activity
pH Most physiologic reactions occur in the pH range of 7-8
Storage Enzymes: -20’C = for longer period of time
Substrate and Coenzymes: 2-8’C
LDH (LD4 & 5): Room temperature
Hemolysis Mostly increases enzyme concentration
Lactescence or milky Decreases enzyme concentration
specimen
Enzyme nomenclature 1st digit: classification
2nd and 3rd digits: subclass
4th digit(s): serial number
Enzyme classification “OTHLIL”
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Oxidoreductases Redox reaction
Dehydrogenases:
-Cytochrome oxidase
-LDH
-MDH
-Isocitrate dehydrogenase
-G-6-PD
Transferases Transfer of a chemical group other than hydrogen from 1 substrate to another
Kinases, Transaminases, Aminotransferases:
-CK
-GGT
-AST
-ALT
-OCT
Hydrolases Hydrolysis/splitting by addition of water
Esterases:
-ACP
-ALP
-CHS
-LPS
Peptidases:
-Trypsin
-Pepsin
-LAP
Glycosidases:
-AMS
-Galactosidases
Lyases Removal of groups w/o hydrolysis (product contains double bonds)
Aldolase
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32. Decarboxylases:
-Glutamate decarboxylase
-Pyruvate decarboxylase
-Tryptophan decarboxylase
Isomerases Intramolecular arrangements
Glucose phosphate isomerase
Ribose phosphate isomerase
Ligases Joining of 2 substrate molecules
Synthases
Active site Water-free cavity
Where the substrate interacts
Allosteric site Cavity other than the active site
May bind regulatory molecules
Prosthetic group Coenzyme that is bound tightly to the enzyme
Holoenzyme Apoenzyme + Prosthetic group
Zymogen/proenzyme Inactive form of enzyme
Emil Fisher’s/Lock and Key Shape of the key (substrate) must fit into the lock (enzyme)
theory
Kochland’s/Induced fit Based on the substrate binding to the active site of the enzyme
theory Acceptable theory
Enzyme kinetics Enzymes catalyze reactions by lowering the activation energy level that the
substrate must reach for the reaction to occur
Absolute specificity Enzyme combines w/ only 1 substrate and catalyzes only 1 reaction
Group specificity Enzymes combine w/ all the substrates in a chemical group
Bond specificity Enzymes reacting w/ specific chemical bonds
Zero-order reaction Reaction rate depends only on enzyme concentration
Independent on substrate concentration
First-order reaction Reaction rate is directly proportional to substrate concentration
Independent on enzyme concentration
Measurement of enzyme Change in substrate concentration
activity Change in product concentration
Change in coenzyme concentration
International Unit 1 micromole of substrate/minute
Katal Unit 1 mole of substrate/second
Nonkinetic assay Absorbance is made at 10-second intervals for 100 seconds
Alkaline Phosphatase pH = 10.5
405nm
Electrophoresis:
(+) Liver  Bone (Regan)  Placenta  Intestine (-)
Heat fractionation:
(Δ Stable) Regan  Placenta  Intestine  Liver  Bone (Δ Labile)
Phenylalanine Inhibits Regan, placental and intestinal ALP
L-leucine Inhibits Nagao ALP
Levamisole Inhibits liver and bone ALP
3M urea Inhibits bone ALP
Methods (ALP) Low temperature = Increased ALP
1. Bowers and McComb (PNPP) – IFCC recommended
2. Bessy, Lowry and Brock (PNPP)
3. Bodansky, Shinowara, Jones, Reinhart = BGP (beta glycerophosphate)
4. King and Armstrong = PP (phenylphosphate)
5. Klein, Babson & Read = Buffered PPP (phenolphthalein phosphate)
6. Huggins and Talalay = PPDP (phenolphthalein diphosphate)
lec.mt 04 |Page | 32

33. 7. Moss = ANP (alpha naphthol phosphate)
Increased ALP Sprue
Hyperparathyroidism
Rickets (children) and osteomalacia (adults)
Acid Phosphatase pH = 5.5
405nm
Sources: Prostate (major), RBC, platelets, bone
Prostatic ACP Inhibited by L-tartrate ions
RBC ACP Inhibited by cupric and formaldehyde ions
Methods (ACP) Room temperature (1-2 hrs) = decreased ACP
Thymolphthalein monophosphate = specific substrate, substrate of choice
(endpoint)
Alpha-naphthyl phosphate = preferred for continuous monitoring methods
1. Gutman and Gutman = PP
2. Shinowara = PNPP
3. Babsonm Read and Phillips = ANP (continuous monitoring)
4. Roy and Hillman = Thymolphthalein monophosphate (endpoint)
Aspartate Aminotransferase pH 7.5
(AST/SGOT) 340nm
Sources: Cardiac tissue > Liver > Skeletal muscle > Kidney, pancreas, RBCs
Alanine Aminotransferase pH 7.5
(ALT/SGPT) 340nm
Major Source: Liver
Methods (AST and ALT) 1. Karmen method = Kinetic
2. Reitman and Frankel = Endpoint
-Color developer: DNPH
-Color intensifier: 0.4N NaOH
Increased Transaminases DeRitis ratio (ALT:AST) >1.0 = Acute hepatitis (Highest)
20x = viral or toxic hepatitis
Moderate elevation = chronic hepatitis, hepatic cancer, IM
Slight elevation = Hepatic cirrhosis, alcoholic hepatitis, obstructive jaundice
Amylase Smallest enzyme (appears in urine)
Earliest pancreatic marker
P3: most predominant pancreatic AMS isoenzyme in AP
Isoenzymes:
S-type (ptyalin): anodal
P-type (amylopsin): cathodal
Methods (AMS) Samples w/ high activity of AMS should be diluted w/ NaCl to prev. inactivation
Salivary AMS = inhibited by wheat germ lectin
Substrate: Starch
Saccharogenic Reducing sugars produced
Classic reference method (SU)
Amyloclastic Degradation of starch
Chromogenic Increase in color intensity
Coupled-enzyme Continuous-monitoring technique
Lipase Late marker (AP)
Most specific pancreatic marker
Methods (LPS) Substrate: Olive oil/Triolein
1. Cherry Crandal (Reference method)
2. Tietz and Fiereck
3. Peroxidase coupling (most commonly used method)
Lactate dehydrogenase Lacks specificity
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