1. must to know in clinical chemistry 001

MUST TO KNOW IN CLINICAL CHEMISTRY(From CC by Rodriguez)

Quality ControlPracticability Method is easily repeatedReliability Maintain accuracy and precisionIntralab/Interlab QC Daily monitoring of accuracy and precisionInterlab/External QC Proficiency testing (Reference lab)

Long-term accuracyDifference of >2: not in agreement w/ other lab

QC materials Available for a min. of 1 yrBovine control materials Preferred (Human: biohazard)

Not for immunochem, dye-binding and bilirubinMatrix effect Improper product manufacturing

Unpurified analyteAltered protein

Precision study First step in method evaluationNonlab. personnel 29% of errors (lab results)SD Dispersion of values from the meanCV Index of precision

Relative magnitude of variability (%)Variance SD2

Measure of variabilityInferential statistics Compare means or SD of 2 groups of dataT-test Means of 2 groups of dataF-test SD of 2 groups of dataCumulative Sum Graph (CUSUM)

V-maskEarliest indication of systematic errors (trend)

Youden/Twin Plot Compare results obtained from diff. labShewhart Levey-Jennings Chart

Graphic representation of the acceptable limits of variation

Trend Gradual loss of reliabilityCause: Deterioration of reagents (Systematic error)

Shift Values: one side or either side of the meanCause: Improper calibration (Systematic error)

Outliers Values: far from the main set of valuesHighly deviating valuesRandom or systematic errors

Kurtosis Degree of flatness or sharpnessPrecision Random errorAccuracy Systematic errorRandom error(Imprecision; Indeterminate)

Causes:-Mislabeling-Pipetting error-Improper mixing of sample and reagents-Voltage/Temperature fluctuation-Dirty opticsParameters: SD and CV

Systematic error (Inaccuracy/Determinate)

Causes:-Improper calibration-Deterioration of reagents-Contaminated solution-Sample instability/unstable reagent blanks

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-Diminishing lamp power-Incorrect sample and reagent volumeParameter: Mean

Multirule Shewhart procedure

Control rules + Control chart

Test method Westgard: at least 40 samplesReference method Westgard: preferably 100 samplesAnalytical Run Control and patient specimens assayed, evaluated, and report togetherPhysiologic Limit Referred to as absurd valuePOCT Performed by nonlab personnelQuality Assurance Tripod:

Program developmentAssessment and monitoringQuality improvement

Quality Patient Care Test request forms, clear instruction for patient prep., specimen handling…Reference Range/ Interval Range/ Reference Values

At least 120 individuals should be tested in each age and sex category

Analytical MethodsWavelength 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 wavelengthPhotometric 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 spectrophotometryVisible region Tungsten light bulb

Mercury arcUV Deuterium lamp

Mercury arcXenon lampHydrogen lamp

IR Merst glowerGlobar (Silicone carbide)

Stray light Wavelength outside the bandMost common cause of loss of linearity

Diffraction gratings Most commonly used monochromatorCutting grooves

Prisms RotatableNickel sulfate Prevents stray lightCutoff filter Anti-stray lightBandpass ½ peak transmittanceAlumina silica glass cuvet Most commonly used cuvetQuartz/plastic cuvet UVBorosilicate glass cuvet Strong basesPhotodetector Converts transmitted light into photoelectric energyBarrier layer cell/ photocell/ photovoltaic cell

Simplest detectorNo external voltageFor filter photometers

Phototube Contains anode and cathodeReq external voltage

Photomultiplier tube Most common typelec.mt 04 |Page | 2

Most sensitiveUV and visible region

Galvanometer/Ammeter Meter or read-out deviceAbsorbance A = abc (a = absorptivity; b = length of light (1cm); c = concentration)

A = 2 – log%TDouble beam spectro. Splits monochromatic light into two components:

One beam sampleOne 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 cuvetDydimium filter 600 nmHolmium oxide filter 360 nmReagent blank Color of reagentsSample blank Optical interference (Hgb)FEP Meas. light emitted by a single atom burned in a flame

Principle: ExcitationLt. source and cuvette: FlameFor excited ions (Na+, K+)

Cesium and Lithium Internal standards (FEP)Correct variations in flame

Lithium Preferred internal stdPotent antidepressant

AAS Meas. light absorbed by atoms dissociated by heatPrinciple: Dissociation (unionized, unexcited, ground state)Lt. source: Hollow-cathode lampFor unexcited trace metals (Ca++ and Mg++)More sensitive than FEP

Atomizer (nebulizer) Convert ions atomsChopper Modulate the light sourceLanthanum/Strontium chloride

Complex with phosphateAvoid calcium interference

Volumetric (Titrimetric) Unknown sample is made to react with a known solution in the presence of an indicator

Turbidimetry Light blockedMeas. abundant large particles (Proteins)Depend on specimen concentration and particle size

Nephelometry Meas. amt of Ag-Ab complexesScattered lightDepends on wavelength and particle size

Electrophoresis Migration of charged particles in an electric fieldIontophoresis Migration of small charged ionsZone electrophoresis Migration of charged macromoleculesEndosmosis Movement of buffer ions and solvent relative to the fixed support

Ex: gamma globulinsCellulose acetate Molecular sizeAgarose gel Electrical chargePolyacrylamide 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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Isoelectric focusing Molecules migrate through a pH gradientpH = pIFor isoenzymes: same size, different charge

Densitometry Scan & quantitate electrophoretic patternCapillary electrophoresis Electro-osmotic flowSouthern blot DNANorthern blot RNAWestern blot ProteinsChromatography Separation by specific differences in physical-chemical characteristics of the

different constituentsPaper chromatography Fractionation of sugar and amino acid

Sorbent: Whatman paperTLC Screening: DrugsRetention 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 lipidsVolatile compoundsSpecimens vaporizedMobile phase: Inert gases

Gas Solid chromatography Differences in absorption at the solid phase surfacesGas Liquid chromatography Differences in solute partitioning between the gaseous mobile phase and the

liquid stationary phaseMass Spectrometry Fragmentation and ionizationGC-MS Gold standard for drug testingMS/MS Detect 20 inborn errors of metabolism from a single blood spotHPLC Most widely used liquid chromatography

Fractionation of drugs, hormones, lipids, carbohydrates and proteinsHydrophilic gel Gel filtration

Separation of enzymes, antibodies and proteinsEx: Dextran and agarose

Hydrophobic gel Gel permeationSeparation of triglyceride and fatty acidEx: Sephadex

Ion exchange chromatography

Separation depends on the sign and ionic charge density

Partition chromatography Based on relative solubility in an organic solvent (nonpolar) and an aqueous solvent (polar)

Affinity chromatography For lipoproteins, CHO and glycated hemoglobinsAdsorption chromatography

Based on differences between the adsorption and desorption of solutes at the surfaces of a solid particle

Fluorometry/Molecular Luminescence Spectro.

Det. amt. of lt. emitted by a molecule after excitation by electromagnetic radiationLt. sources: Mercury arc and Xenon lamp (UV)Lt. detector: Photomultiplier tubes2 monochromators:Primary filter – selects wavelength absorbed by the solution to be measuredSecondary filter – prevents incident light from striking the photodetectorSensitivity: 1000x than spectro

Quenching Major disadvantage of fluorometrypH and temperature changes, chemical contaminants, UVL changes

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InstrumentationBorosilicate glasswares For heating and sterilization

Ex: Pyrex and KimaxBoron-free/Soft glasswares High resistance to alkaliCorex (Corning) Special alumina-silicate glass

Strengthened chemically than thermally6x stronger than borosilicate

Vycor (Corning) For high thermal, drastic heat and shockCan be heated to 900OC

Flint glass Soda-lime glass + Calcium, Silicon, Sodium oxidesEasy to meltFor making disposable glasswares

TD: To deliver Exact amountTC: To contain Does not disperse the exact volumeBlowout w/ etched rings on top of pipetSelf-draining w/ o etched rings

Drain by gravityTransfer pipet Volumetric: for non-viscous fluid; self-draining

Ostwald folin: for viscous fluid; w/ etched ringPasteur: w/o consideration of a specific volumeAutomatic macro-/micropipets

Graduated or measuring pipet

Serological: w/ graduations to the tip (blowout)Mohr: w/o graduations to the tip (self-draining)BacteriologicBall, Kolmer and KahnMicropipettes: <1 mL

Micropipettes TC pipets:Sahli-Hellige pipetLang-Levy pipetRBC and WBC pipetsKirk and Overflow pipets

Air displacement pipet Piston: suctionDisposable tip

Positive displacement pipet Piston barrel (like a hypodermic syringe)Dispenser/Dilutor pipet Liquid: common reservoir dispense repeatedlyDistilled H2O Calibrating medium for TD pipettesMercury Calibrating medium for TC pipettesAcid dichromate(H2SO4 + K2Cr2O4)

Cleaning solution for glasswares

Continuous flow analyzer Common reaction vesselAir bubbles: separates and cleansGlass coil: mixExamples: “STS”Simultaneous Multiple Analyzer (SMA)Technicon Autoanalyzer IISMAC

Centrifugal analyzer Acceleration and deceleration of the rotorAdvantage: Batch analysisExamples: “RICC”Cobas-Bio (Roche)IL MonarchCentrifiChem

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RotoChemDiscrete Analyzer Most popular

Req. vol: 2-6 LμUses positive-displacement pipetsRun multiple-tests-one-sample-at-a-timeRandom access capability (STAT)Examples:VitrosDimension DadeBeckman ASTRA System (4 & 8)HitachiBayer AdviaRoche Cobas Integra 800Roche Analytics P ModuleAutomated Clinical Analyzer (ACA) Star (Dade)Dupont ACAAbbott ABA-100 Bichromatic AnalyzerABA-200VP AnalyzerAmerican Monitor KDAOlympus Demand

Thin-Film Analyzers(Dry slide technology)

4 or 5 layers:-Spreading layer-Scavenger layer - Ascorbate Oxidase-Reagent layer-Indicator layer-Support layerColored reaction Reflectance spectrophotometryExamples: “KV2(75)”Kodak EktachemVitros 750XRCVitros 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 timeSingle test is conducted on each sample

Parallel testing One specimenMore than one test is analyzed

Random access testing Any sampleAny testAny sequenceSTAT

Sequential testing Multiple tests analyzed one after another on a given specimenOpen reagent system System other than manufacturer’s reagents can be utilized for measurementClosed reagent system The operator can only use the manufacturer’s reagents

Patient PreparationExercise Increased: GU2FT C2L3A5P2

GHUreaUrinary protein (Proteinuria)Fatty acidTestosterone

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CPK (muscle)Creatinine (muscle)LactateLHLD (muscle)ACPAldolase (muscle)ASTALTAmmoniaPyruvateProlactinDecreased:Glucose

Fist clenching Increased: “LPP”LactatePotassiumPhosphate

Fasting 8-16 hours:GlucoseLipidsLipoproteinsIncreased:Bilirubin (48 hours)Triglyceride (72 hours)

Basal state collection GlucoseCholesterolTriglycerideElectrolytes

Diet Increased: “GLUC2H”GlucoseLipidsUrea (High protein diet)Caffeine: increases glucoseCatecholamines5-HIAA (From Serotonin)

Turbidity/Lactescence Triglyceride >400mg/dLIcterisia Bilirubin: 25.2 mg/dLIcteric samples Interfere with: "TACGu”

Total ProteinAlbuminCholesterolGlucose

Upright/supine (lying) position

Preferred positionPatient should be seated/supine at least 20 mins before blood collection to prevent hemodilution or hemoconcentration

Supine Sitting/Standing Vasoconstriction Reduced plasma volumeIncreased: “ECA”EnzymesCalciumAlbumin

Sitting Supine Hemoconcentration

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Increased: “P(u)BLIC”ProteinsBUNLipidsIronCalcium

Standing Supine HemodilutionDecreased: “TLC”TriglyceridesLipoproteinsCholesterol

Prolonged standing Increased: K+ (muscles)Prolonged bedrest Decreased: Albumin (Fluid retention)Tourniquet Recommended: 1 minute applicationProlonged tourniquet app. Hemoconcentration

AnaerobiosisIncreased: “C2LEA2K”CalciumCholesterolLactateEnzymesAmmoniaAlbuminK+

Tobacco smoking (Nicotine) Increased: “TUNG2C3”TriglyceridesUreaNonesterified fatty acidGlucoseGHCatecholaminesCortisolCholesterol

Alcohol ingestion Increased: “THUG”TriglyceridesHypoglycemia (chronic alcoholism)Uric acid/UratesGGT

Ammonia Increases by 100-200 g/L/cigarμStress (anxiety) Increased: “LAGIC”

LactateAlbuminGlucoseInsulinCholesterol

Drugs Medications affecting plasma volume can affect protein, BUN, iron, calciumHepatotoxic drugs: increased liver function enzymesDiuretics: decreased sodium and potassium

Diurnal variation "CA3PI2TG”CortisolACTHACP

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AldosteroneProlactinIronInsulinThyroxineGH

Specimen Collection and HandlingSleeping patients Must be awakened before blood collectionUnconscious patients Ask nurse or relative

Identification braceletVenipuncture Median Cubital (1st) Cephalic (2nd) Basilic (3rd)Tourniquet Velcro or Seraket type

3-4 inches above the siteNot exceed 1 minute

Needle Bevel up15-30O angleLength: 1 or 1.5 inch (Butterfly needle: ½ to ¾ inch)

After blood collection Cotton siteApply pressure for 3-5 minutes

BP cuff as tourniquet Inflate to 60 mmHgBenzalkonium chloride (Zephiran)

Disinfectant for ethanol testingDilution – 1:750

IV line on both arms Discontinue IV for 2 minutesCollect sample below the IV siteInitial sample (5mL) discard

IV fluid contamination Increased:Glucose (10% contam. w/ 5% dextrose increased bld glucose by 500 mg/dL)ChloridePotassiumSodiumDecreased:UreaCreatinine

Renin blood level Collected after a 3-day diet, from a peripheral veinBasal state collection Early morning blood collection

12 hours after the last ingestion of foodLancet 1.75mm: preferred length to avoid penetrating the boneIncision (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 siteFlea 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 tubeIndwelling umbilical artery Best site for blood gas analysis (newborns)1000-3000 RCF for 10 mins Centrifugation requirementHemolysis Increased:

“KLA6MP ITC2”-K+

-LDH (150x)-ACP-ALP

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-Aldolase-ALT-AST-Albumin-Mg2+

-Phosphorus-Iron-Total proteinAffects bilirubin levelsInhibits lipase

Refrigeration/Chilling(Low temp)

Required for: “ABCGLRP2”AmmoniaBlood gasesCatecholaminesGastrinLactic acidReninPTHPyruvateDecreased:LD 4 and 5Increased:ALP

Photosensitive analytes BilirubinBeta-caroteneFolatePorphyrinsVitamins A and B6

Oxalate Insoluble salt1-2 mg/mL blood

Citrate Non-ionized form3.2-3.8 g/dL (1:9 ratio)

EDTA Chelation1-2 mg/mL bloodVersene: disodium saltSequestrene: Dipotassium salt

Fluoride Weakly dissociated calcium component2 mg/mL blood: anti-glycolytic10 mg/mL blood: anticoagulant

Heparin A.k.a. Mucoitin polysulfuric acidUniversal anticoagulantAntithrombin0.2 mg/mL blood

Lithium heparin For glucose, BUN, ionized calcium, electrolyte studies (K+: best) and creatinineOrange top tube Additive: ThrombinRoyal blue top tube Additives:

None;Na2EDTASodium heparin

Brown top tube Lead testingTan top tube Lead testingBlack top tube Additive: Buffered sodium citrate

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For ESRRespinning gel tubes Increases potassiumThixotropic 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

100Molarity M = _grams of solute_______

GMW x volume of solutionMoles Mol = weight (grams)

GMWTo prepare a molar solution Grams of solute = Molarity x GMW of the solute x Volume (L) desiredTo convert % w/v to Molarity

M = % w/v 10 GMW

Normality N = _Grams of solute_ EW x volume (L)

Equivalent weight (EW) EW = __MW___ valence

To prepare a normal solution of solids

Grams of solute = Normality x EW x Volume (L)

To convert % w/v to Normality

N = w/v 10 EW

Normality N = Molarity x ValenceMolarity M = Normality

valenceMolality m = Grams of solute__

MW x kg of solventMilliequivalents mEq/L = mg/dL 10 valence

MWMillimoles mmol/L = mg/dL 10

MWRatio Ratio = _Volume of solute_

Volume of solventDilution Dilution = __Volume of solute__

Volume of solution0.179 Conversion factor for iron (mg/dL mol/L)μ0.01 Conversion factor for phospholipid (g/dL to g/L)2.27 Conversion factor for folateAnalytical reagent (AR) grade

For qualitative and quantitative analysesFor accuracyEstablished by American Chemical Society (ACS)Uses: Trace metal analysis and preparation of standard solutions

Ultrapure reagents Additional purification stepsEx: Spectrograde, nanograde, HPLC gradeUses: Chromatography, atomic absorption, immunoassays

Chemically Pure (CP) or Indicates that the impurity limitations are not stated


Pure Grade Purity is delivered by meas. of melting point or boiling pointTechnical/Commercial grade

In manufacturingNever used in clin. lab. testing

United States Pharmacopoeia (USP) and National Formulery (NF)

For human consumptionNot applicable for lab. analysisPurpose: For drug manufacturing

Preparation of reagent grade water

Filtration (1st) Distillation, Ion exchange, Reverse Osmosis

Type I Rgt Water Min. interferenceMax. water purityUsed immediatelyFor ultramicrochemical analyses, measurements of nanogram or subnanogram concentrations, tissue or cell methods (microscopy) and preparation of standard solutionsUses: 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 glasswaresFor urinalysis, parasitology and histology

Distilled water Purified to remove almost all organic materialsDeionized water Free from mineral salts; removed by ion exchange processes

Organic material may still be presentOccupational Safety and Health Act (OSHA)

Req. manuf. to indicate lot no., physical or biological health hazard of the chem.. rgts, and precautions for safe use and storage

College of American Pathologists (CAP)

Recommends that a lab. document culture growth, pH and specific water resistance on reagent grade water

Tests for water purity Microbiological contentpHResistivityChemical oxygen demandAmmoniaIonsMetals

Detergent-contaminated water

Alkaline pH

Hard water Contains calcium, iron and other dissolved elementsNCCLS Now: Clinical and Laboratory Standards Institute (CLSI)Dilute solution Relatively little soluteConcentrated solution Large quantity of solute in solutionSaturated solution Excess of undissolved solute particlesSuper saturated solution Greater concentration of undissolved solute particles than does a saturated

solution of the same substancePrimary standard(IUPAC)

Highly purifiedMeasured directly to produce a substance of exact known concentration

Secondary standard Low purityConcentration is determined by comparison w/ a primary standard

Laboratory SafetyNational Fire Protection Association (NFPA) Classification of FiresClass A fire Ordinary combustibles: paper, cloth, rubbish, plastics, wood

Extinguisher: Water (A), Dry chemical (ABC), loaded steam


Class B fire Flammable liquids: grease, gasoline, paints, oilExtinguisher: Dry chemical (ABC), carbon dioxide (BC), halon foam (BC)

Class C fire Electrical equipment and motor switchesExtinguisher: Dry chemical (ABC), Carbon dioxide (BC), halon (BC)

Class D fire Flammable metals: mercury, magnesium, sodium, lithiumExtinguisher: Metal XFought 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 hazardRed quadrant Flammable hazardYellow quadrant Reactivity/Stability hazardWhite quadrant Other special informationChemical spills 1st step: assist/evacuate personnel1:10 dilution of chlorine bleach (10%)

To disinfect and clean bench topsIn contact with the area for at least 20 minutesHBV: 10 minutesHIV: 2 minutes

Poisonous vapors ChloroformMethanolCarbon tetrachlorideBromideAmmoniaFormaldehydeMercury

Flammable and combustible solvents

AcetoneEthanolTolueneMethanolXyleneBenzeneIsopropanolHeptane

Flammable liquids Flash point below 37.8OCCombustible liquids Flash point at or above 37.8OCStrong acids or bases Neutralized before disposal

Water should NEVER be added to concentrated acidEther Deteriorate over time hazardous

Forms explosive peroxidesBenzidine Known carcinogenFumehoods Ventilation: velocity of 100-120 ft/minSafety showers Deliver 30-50 gal/min of H2O at 20-50 psi

CarbohydratesGlycol aldehyde The simplest carbohydrateSucrose Most common nunreducing sugarPancreas Exocrine: Enzymes (AMS, LPS)

Endocrine: Hormones (Insulin, glucagon, somatostatin)Hyperglycemic Hormones “GAG CHET”

GlucagonACTHGH


CortisolHuman Placental LactogenEpinephrineThyroxine

Hyperglycemia(≥126 mg/dL)

Electrolyte Imbalance:Decreased: Sodium, BicarbonateIncreased: Potassium

Hypoglycemia 50-55 mg/dL = Symptoms≤50 mg/dL = Diagnostic

Whipple’s triad(Hypoglycemia)

Low blood glucose concentrationTypical symptomsSymptoms alleviated by glucose administration

6:1 Ratio of BHA to AA in severe DM(Normal = 1:1)

Type 1 DM IDDMJuvenile OnsetBrittleKetosis-prone80-90% reduction of beta-cells Symptomatic Type 1 DMHLA-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:NephropathyNeuropathyRetinopathy

Type 2 NIDDMAdult type/Maturity OnsetStableKetosis-resistantReceptor-deficientInsulin resistance: relative insulin deficiencyStrong genetic predispositionGeneticist’s nightmareIf untreated glucose: >500 mg/dL nonketotic hyperosmolar coma

Gestational DM Screening: 1hr GCT (50g) – bet. 24 and 28 weeks of gestationConfirmatory: 3-hr GTT (100g)Infants: at risk for respiratory distress syndrome, hypocalcemia, hyperbilirubinemiaAfter giving birth, evaluate 6-12 weeks postpartumConverts to DM w/in 10 years in 30-40% of cases

OGTT (GDM) FBS = ≥95 mg/dL1-Hr = ≥ 180 mg/dL2-Hr = ≥ 155 mg/dL3-Hr = ≥ 140 mg/dLGDM = 2 plasma values of the above glucose levels are exceeded

Impaired fasting glucose (Pre-diabetes)

FBS = 100-125 mg/dL

Impaired glucose tolerance FBS = <126 mg/dL2-Hr OGTT = 140-199 mg/dL


FBS WB = 15% lower than in serum or plasmaVB = 7 mg/dL lower than capillary and arterial blood

CSF glucose 60-70% of the plasma glucosePeritoneal fluid glucose Same with plasma glucosePlasma glucose increases w/ age

Fasting: 2 mg/dL/decadePostprandial: 4 mg/dL/decadeGlucose challenge: 8-13 mg/dL/decade

w/in 1 hour(Preferably w/in 30 mins)

Separate serum/plasma from the cells

5-7%/hr Glycolysis at room temperature1-2 mg%/hr Glycolysis at refrigerated temperatureCopper reduction methods Cupric Cuprous Cuprous oxideFolin Wu Cuprous ions + phosphomolybdate phosphomolybdenum blueNelson-Somogyi Cuprous ions + arsenomolybdate arsenomolybdenum blueNeocuproine method Cuprous ions + neocuproine Cuprous-neocuproine complex (yellow)Benedict’s method Reducing substances in blood and urineAlkaline Ferric Reduction method (Hagedorn-Jensen)

Ferricyanide ---(Glucose)--> Ferrocyanide (Yellow) (Colorless)

Ortho-toluidine(Dubowski method)

Schiff’s base

Glucose oxidase Measures beta-D-glucose (65%)Mutarotase Converts alpha-D-glucose (35%) to beta-D-glucose (65%)NADH/NADPH Absorbance at 340nmPolarographic glucose oxidase

Consumption of oxygen on an oxygen-sensing electrodeO2 consumption α glucose concentration

Hexokinase method Most specific methodReference methodUses G-6-PD

G-6-PD Most specific enzyme rgt for glucose testingInterfering substances(Glucose oxidase)

False-decreasedBilirubinUric acidAscorbate

Hemolysis (>0.5 g/dL Hgb) Major interfering substance in hexokinase method (false-decreased)Dextrostics Cellular strip

Strip w/ glucose oxidase, peroxidase and chromogenOGTT 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 weightGiven w/in 3 mins1st blood collection: after 5 mins of IV glucose

Requirements for OGTT AmbulatoryFasting: 8-14 hoursUnrestricted diet of 150g CHO/day for 3 daysDo not smoke or drink alcohol

Glucose load 75 g = adult (WHO std)100 g = pregnant1.75 g glucose/kg BW = children

HbA1c 2-3 monthsGlucose = beta-chain of HbA1


1% increase in HbA1c = 35 mg/dL increase in plasma glucose18-20% = prolonged hyperglycemia7% = cutoffSpecimen: EDTA whole bloodTest: Affinity chromatography (preferred)

IDA and older RBCs High HbA1c

RBC lifespan disorders Low HbA1c

Fructosamine(Glycosylated albumin/ plasma protein ketoamine)

2-3 weeksUseful for patients w/ hemolytic anemias and Hgb variantsNot used in cases of low albuminSpecimen: Serum

Galactosemia Congenital deficiency of 1 of 3 enzymes in galactose metabolismGalactose-1-phosphate uridyl transferase (most common)GalactokinaseUridine diphosphate galactose-4-epimerase

Essential fructosuria Autosomal recessiveFructokinase deficiency

Hereditary fructose intolerance

Defective fructose-1,6-biphosphate aldolase B activity

Fructose-1,6-biphosphate deficiency

Failure of hepatic glucose generation by gluconeogenic precursors such as lactate and glycerol

Glycogen Storage Disease Autosomal recessiveDefective glycogen metabolismTest: 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 deficiencyIV = Andersen Brancher enzyme deficiencyV = McArdle Muscle phosphorylase deficiencyVI = Hers Liver phosphorylase deficiencyVII = Tarui Phosphofructokinase deficiencyXII = Fanconi-Bickel Glucose transporter 2 deficiencyCSF 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 ratioC-peptide Formed during conversion of pro-insulin to insulin5:1 to 15:1 Normal C-peptide : insulin ratioD-xylose absorption test Differentiate pancreatic insufficiency from malabsorption (low blood or urine

xylose)Gerhardt’s ferric chloride test

Acetoacetate

Nitroprusside test 10x more sensitive to acetoacetate than to acetoneAcetest tablets Acetoacetate and acetoneKetostix Detects acetoacetate better than acetoneKetoSite assay Detects beta-hydroxybutyrate but not widely usedNormal Values(Carbohydrates)

RBS = <140 mg/dLFBS = 70-100 mg/dLHbA1c = 3-6%Fructosamine = 205-285 mol/Lμ2-Hr PPBS = <140 mg/dLGTT:30 mins = 30-60 mg/dL above fasting


1-Hr = 20-50 mg/dL above fasting2-Hr = 5-15 mg/dL above fasting3-Hr = fasting level or below

LipidsPhospholipids 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 lecithinNot derived from glycerol but from sphingosine (amino alcohol)

Forms of phospholipids 70% Lecithin/Phosphatidyl choline20% Sphingomyelin10% Cephalin

TLC + Densitometric quantitation

Method for L/S ratio

Microviscosity Measured by fluorescence polarizationCholesterol Not a source of fuel

Not affected by fasting70% Cholesterol ester (plasma/serum)30% Free cholesterol (plasma/serum and RBC)

LCAT Esterification of cholesterolApo A-1 Activator of LCATCholesterol increases after the age of 50

2 mg/dL/year between 50 and 60 years old

Liebermann Burchardt Cholestadienyl Monosulfonic acidGreen end color

Salkowski Cholestadienyl Disulfonic acidRed end color

Color developer mixture (Cholesterol)

Glacial acetic acidAcetic anhydrideConc. 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(Chemical method)

CDC reference method for cholesterol:-Hydrolysis/saponification (Alc. KOH)-Hexane extraction-Colorimetry (Liebermann-Burchardt)

Triglycerides Most insoluble lipidMain storage lipid in man (adipose tissue) – 95%Fasting: 12 hours

Triglyceride increases after the age of 50

2 mg/dL/year between 50 and 60 years old

Van Handel & Zilversmith(Colorimetric)

Chromotropic acid(+) Blue color compound

Hantzsch Condensation(Fluorometric)

Diacetyl acetone(+) Diacetyl lutidine compound

Modified Van Handel and Zilversmith(Chemical method)

CDC reference method for triglycerides:-Alkaline hydrolysis-Chloroform extraction extract treated w/ silicic acid-Color reaction w/ chromotropic acid – meas. HCHO


(+) Pink coloredFatty acids Short chain = 4-6 C atoms

Medium chain = 8-12 C atomsLong chain = >12 C atomsSaturated = w/o double bondsUnsaturated = w/ double bondsSubstrate for gluconeogenesisMost is bound to albumin

Palmitic acid 16:0Stearic acid 18:0Oleic acid 18:1Linoleic acid 18:2Arachidonic acid 20:4Lipoprotein lipase(Lipemia clearing factor)

Hydrolyzes TAG in lipoproteins, releasing fatty acid and glycerol

Hepatic lipase Hydrolyzes TAG and phospholipids from HDLHydrolyzes lipids on VLDL and IDL

Endothelial lipase Hydrolyzes phospholipids and TAG in HDLApolipoprotein Protein component of lipoprotein

Amphipathic helix – ability of proteins to bind to lipidsChylomicrons Largest and least dense

Produced by the intestineSG: <0.9580-95% TAG (exogenous)Apo B-48 (Major)EP: Origin

VLDL Secreted by the liverSG: 0.95-1.00665% TAG (endogenous)Apo B-100 (Major)EP: pre-beta

LDL Synthesized by the liverSG: 1.006-1.06350% CEApo B-100 (Major)EP: betaCholesterol transport: LiverTissuesTarget of cholesterol lowering therapyBetter marker for CHD risk

HDL Smallest but denseSG: 1.063-1.2145-55% protein26-32% phospholipidApo A-1 (Major)EP: alphaProduced by the liver and intestineReverse cholesterol transport: TissueLiver

IDL Product of VLDL catabolismSeen in Type 3 hyperlipoproteinemia (Apo E-III def.; beta-VLDL)SG: 1.006-1.019

Lp(a) Sinking pre-beta lipoproteinSG: 1.045-1.080


Apo B-100EP: pre-beta (VLDL)UC: like LDLIndependent risk factor for atherosclerosis

LpX Found in obstructive jaundice (cholestasis) and LCAT deficiency90% FC and PLApo C and albumin

Beta-VLDL Floating beta-lipoproteinSG: <1.006EP: beta (LDL)UC: like VLDLFound 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 fractionsFasting state: TAG VLDLNonfasting state: TAG CM

Ultracentrifugation Reference method for LPP quantitationReagent: Potassium bromide (SG: 1.063)Ultracentrifugation of plasma for 24 hoursExpressed in Svedberg units

Electrophoresis Electrophoretic pattern:(+) HDL VLDL LDL CM (Origin) (-)Agarose gel: sensitive mediumVLDL: migrates w/ alpha2-globulin (pre-beta)

Chemical precipitation Uses polyanions (heparin and divalent cations) and polyethylene glycolDextran sulfate-Mg2+

Heparin-Mn2+

3-step procedure:UltracentrifugationPrecipitationAbell-Kendall assay

CDC Reference method for HDL

Beta quantification + Ultracentrifugation + Chemical precipitation

Method for LDLSample: EDTA plasma

Immunoturbidimetric assay Measures Lipoprotein (a)LDL Cholesterol Total Cholesterol – HDL – VLDLFriedewald 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 LCATApo B-100 LDL LDL receptorApo B-48 CM (major)

Not recognized by LDL receptorApo C-II Activates LPLApo D Activates LCATApo E Apo E-4: associated w/ high LDL, higher risk of CHD and Alzheimer’s diseaseApo(a) Lp(a)

Homologous to plasminogen


Abetalipoproteinemia(Basses-Kornzweig syn.)

Autosomal recessiveDefective apo B synthesisDeficient fat soluble vitamins

Niemann-Pick disease Sphingomyelinase deficiencyTangier’s disease Deficiency of HDL (1-2 mg/dL)

Defects in the gene for the ABCA1 transporterLPL deficiency(Chylomicronemia)

TAG = 10,000 mg/dLDo not develop premature coronary disease (CM are not atherogenic)Abdominal pain and pancreatitis

LCAT deficiency Fish-eye diseaseLow HDL

Tay-Sachs disease Hexosaminidase A deficiencyFredrickson ClassificationType 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 HypertriglyceridemiaIncreased: VLDL (TAG)

Type 5 Increased: VLDL (Endo.TAG), CM (Exo.TAG)Normal Values(Lipids)

Cholesterol:Desirable = <200 mg/dLBorderline high = 200-239 mg/dLHigh = >240 mg/dLTriglycerides:Desirable = <150 mg/dLBorderline high = 150-199 mg/dLHigh = 200-499Very high = >500 mg/dLHDL:Low = <40 mg/dL (Cutoff)High = >60 mg/dLLDL:Optimal = <100 mg/dLNear/above optimal = 100-129 mg/dLBorderline high = 130-159 mg/dLHigh = 160-189 mg/dLVery high = >190 mg/dL

ProteinsProteis First rank of importanceProteins Amphoteric: positive and negative charges

Effective blood buffersSynthesized by the liver except immunoglobulins (plasma cells)Provide 12-20% of total daily body energy requirementComposed of 50-70% of the cell’s dry weight


Primary structure Amino acid sequenceDet. the identity of protein, molecular structure, function binding capacity, recognition ability

Secondary structure Winding of polypeptide chainSpecific 3-D conformations: alpha-helix, beta-pleated sheath, bend form

Tertiary structure Actual 3-D configurationFolding patternPhysical and chemical properties of proteins

Quarternary structure Association of 2 or more polypeptide chains proteinAlbumin No quarternary structureGlucogenic amino acids Alanine (pyruvate)

Arginine (alpha-ketoglutarate)Aspartate (oxaloacetate)

Ketogenic amino acids Degraded to acetyl-CoALeucineLysine

Simple proteins Hydrolysis Amino acidsFibrous: fibrinogen, troponins, collagenGlobular: hemoglobin, plasma proteins, enzymes, peptide hormones

Conjugated proteins Protein (apoprotein) + nonprotein moiety (prosthetic group)Metalloproteins: ferritin, ceruloplasmin, hemoglobin, flavoproteinsLipoproteins: VLDL, HDL, LDL, CMGlycoproteins: haptoglobin, alpha1-antitrypsin (10-40% CHO)Mucoproteins or proteoglycans: Mucin (CHO > CHON)Nucleoproteins: Chromatin (combined w/ nucleic acids)

Nitrogen balance Balance bet. anabolism and catabolismNegative nitrogen balance Catabolism > anabolism

Excessive tissue destructionPositive nitrogen balance Anabolism > catabolism

Growth and repair processesPrealbumin (Transthyretin) Transports thyroxine and retinol (Vit. A)

Landmark to confirm that the specimen is really CSFAlbumin Maintains osmotic pressure

Negative acute phase reactantAlpha1-antitrypsin Acute phase reactant

Major inhibitor of protease activity90% of alpha1-globulin band

Alpha1-fetoprotein Gestational markerTumor marker: hepatic and gonodal cancersScreening test for fetal conditions (Spx: maternal serum)Amniotic fluid: confirmatory testIncreased: Hepatoma, spina bifida, neural tube defectsDecreased: Down Syndrome (Trisomy 21)

Alpha1-acid glycoprotein/ orosomucoid

Low pI (2.7)Negatively charged even in acid solution

Alpha1-antichymotrypsin Acute phase reactantBinds and inactivates PSAIncreased: Alzheimer’s disease, AMI, infection, malignancy, burns

Haptoglobin (alpha2) Acute phase reactantBinds free hemoglobin (alpha chain)

Ceruloplasmin (alpha2) Copper binding (6-8 atoms of copper are attached to it)Has enzymatic activities


Decreased: Wilson’s disease (copper skin, liver, brain, cornea [Kayser-Fleisher rings])

Alpha2-macroglobulin Larges major nonimmunoglobulin proteinIncreased: Nephrotic syndrome (10x)Forms a complex w/ PSA

Group-specific component (Gc)-globulin (bet. alpha1 and alpha2)

Affinity w/ vitamin D and actin

Hemopexin (beta) Binds free hemeBeta2-microglobulin HLA

Filtered by glomeruli but reabsorbedTransferrin/Siderophilin (beta)

Negative acute phase reactantMajor component of beta2-globulin fractionPseudoparaproteinemia in severe IDAIncreased: Hemochromatosis (bronze-skin), IDA

Complement (beta) C3: majorFibrinogen (bet. beta and gamma)

Acute phase reactantBetween beta and gamma globulins

CRP (gamma) General scavenger moleculeUndetectable in healthy individualshsCRP: warning test to persons at risk of CAD

Immunoglobulins (gamma) Synthesized by the plasma cellsIgG>IgA>IgM>IgD>IgE

Myoglobin Marker: Ischemic muscle cells, chest pain (angina), AMITroponins Most important marker for AMITnT (Tropomyosin-binding subunit)

Specific for heart muscleDet. unstable angina (angina at rest)

TnI (Inhibitory subunit or Actin-binding unit)

Only found in the myocardiumGreater cardiac specificity than TnTHighly specific for AMI13x more abundant in the myocardium than CK-MBVery sensitive indicator of even minor amount of cardiac necrosis

TnC Binds calcium ions and regulate muscle contractionsGlomerular proteinuria Most common and serious type

Often called albuminuriaTubular proteinuria Defective reabsorption

Slightly increased albumin excretionOverload proteinuria Hemoglobinuria

MyoglobinuriaBence-Jones proteinuria

Postrenal proteinuria Urinary tract infection, bleeding, malignancyMicroalbuminuria Type 1 DM

Albumin excretion ≥30 mg/g creatinine (cutoff: DM) but ≤300 mg/g creatinineMicroalbuminuria: 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 sclerosisEncephalitisNeurosyphilisGuillain-Barre syndromeNeoplastic disorders


Serum Oligoclonal banding LeukemiaLymphomaViral infections

Alkaptonuria Ochronosis (tissue pigmentation)Homocystinuria Impaired activity of cystathione beta-synthetase

Elevated homocysteine and methionine in blood and urineScreen: Modified Guthrie test (Antagonist: L-methionine sulfoximine)

MSUD Markedly reduced or absence of alpha-ketoacid decarboxylase4 mg/dL of leucine is indicative of MSUDScreen: Modified Guthrie test (Antagonist: 4-azaleucine)Diagnostic: Amino acid analysis (HPLC)

PKU Deficiency of tetrahydrobiopterin (BH4) elevated blood phenylalanineNormal Values(Proteins)

Total protein = 6.5-8.3 g/dLAlbumin = 3.5-5.0 g/dLGlobulin = 2.3-3.5 g/dL

Kidney Function TestsTests for GFR Clearance:

-Inulin clearance-Creatinine clearance-Urea clearancePhenolsulfonphthalein dye testCystatin C

Tests for Renal Blood Flow BUNCreatinineUric acid

Tests Measuring Tubular Function

Excretion:-Para-amino hippurate test (Diodrast test)-Phenolsulfonphthalein dye testConcentration:-Specific gravity-Osmolality

GFR Decreases by 1.0 mL/min/year after age 20-30 years150 L of glomerular filtrate is produced daily

Inulin clearance Reference methodCreatinine clearance Best alternative method

Measure of the completeness of a 24-hour urine collectionExcretion: 1.2-1.5 g creatinine/day

Urea clearance Demonstrate progression of renal disease or response to therapyCystatin C Low MW protease inhibitor

FilteredNot secretedCompletely reabsorbed (PCT)Indirect estimates of GFRIts presence in urine denotes damage to PCT

BUN Synthesized from Ornithine or Kreb’s Henseleit cycleFirst metabolite to elevate in kidney diseasesBetter indicator of nitrogen intake and state of hydration

2.14 BUN Urea (mg/dL)Fluoride or citrate Inhibit ureaseThiosemicarbazideFerric ions

Enhance color development (BUN mtd)

Diacetyl monoxime method Yellow diazine derivativeUrease method Routinely used

Urease: prepared from jack beans


Urea ---(Urease)--> NH4 + Berthelot reagent (Measure ammonia)Coupled urease Glutamate dehydrogenase method

UV enzymatic methodIsotope dilution mass spectrometry

Reference methodFor research purposes

NPN 45% Urea20% Amino acid20% Uric acid5% Creatinine1-2% Creatine0.2% 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)

Creatinine Aminohydrolase – CK methodCreatinase-Hydrogen Peroxide method – benzoquinonemine dye (red)Creatininase (a.k.a. creatinine aminohydrolase)

Direct Jaffe method Formation of red tautomer of creatinine picrateInterferences (Direct Jaffe) Falsely increased:

AscorbateGlucoseUric acidAlpha-keto acids

Folin Wu Method (+) Red orange tautomerLloyd’s or Fuller’s Earth method

True measure of creatinineSensitive and specificUses adsorbent to remove interferences (UA, Hgb, Bili)

Lloyd’s reagent Sodium aluminum silicateFuller’s earth reagent Aluminum magnesium silicateJaffe reagent (Alk. picrate) Satd. picric acid + 10% NaOHKinetic Jaffe method Popular, inexpensive, rapid and easy to perform

Requires automated equipmentAzotemia Elevated urea and creatinine in bloodPre-renal azotemia Decreased GFR but normal renal function

Dehydration, shock, CHFIncreased: BUNNormal: Creatinine

Renal azotemia True renal diseaseDecreased GFRStriking BUN level but slowly rising creatinine valueBUN = >100 mg/dLCreatinine = >20 mg/dLUric acid = >12 mg/dL

Post renal azotemia Urinary tract obstructionDecreased GFRNephrolithiasis, cancer or tumors of GUTCreatinine = normal or slightly increased

Uremia Marked elevation of urea, accompanied by acidemia and electrolyte imbalance (K+ elevation) of renal failureNormocytic, normochromic anemiaUremic frost (dirty skin)Edema


Foul breathUrine-like sweat

Uric acid From purine (adenine and guanine) catabolismExcretion: 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 syndromeWilson’s diseaseHodgkin’s disease

Methods (Uric acid) Stable for 3 daysPotassium oxalate cannot be usedMajor interferences: Ascorbate and bilirubin

Phosphotungstic acid mtd Uric acid + Phosphotungstic acid ---(NaCN/NaCO3)--> Tungsten blue + AllantoinNaCN Folin

NewtonBrownBenedict

NaCO3 ArchibaldHenryCaraway

Lagphase Incubation period after the addition of an alkali to inactivate non-uric acid reactants

Uricase method Simplest and most specific methodCandidate reference methodUric acid (Absorbance at 293nm) ---[Uricase]--> Allantoin (No absorbance)Decrease in absorbance α uric acid concentration

Para-amino hippurate test Measures renal plasma flowReference method for tubular function

Phenolsulfonphthalein dye test

Measures excretion of dye proportional to renal tubular mass6 mg of PSP is administered IV

Concentration tests Collecting tubules and loops of HenleSpecimen: 1st morning urine

Specific gravity Affected by solute number and massSG >1.050: X-ray dye and mannitol1.010 = 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 presentUrine osmolality = due to ureaSerum osmolality = due to sodium and chlorideDet. 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 (Osmolality)

Freezing point osmometry = popular methodVapor pressure osmometry (Seebeck effect)

Incr. plasma osmolality Incr. vasopressin (H2O reabsorption) decr. plasma osmolalityTubular failure Increased: BUN, creatinine, calcium

Decreased: Phosphate


Osmolal gap Difference between measured and calculated osmolalitySensitive indicator of alcohol or drug overdose

Osmolal gap: >12 mOsm/kg DKADrug overdoseRenal failure

Normal Values(Kidney Function Tests)

Creatinine Clearance:Male = 85-125 mL/minFemale = 75-112 mL/minBUN = 8-23 mg/dLCreatinine = 0.5-1.5 mg/dLUric acid:Male = 3.5-7.2 mg/dLFemale = 2.6-6.0 mg/dLRenal plasma flow (PAH) = 600-700 mL/minRenal blood flow (PSP) = 1200 mL/minSG = 1.005-1.030Osmolality:Serum = 275-295 mOsm/kgUrine (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 TestsLiver Receives 15 mL of blood per minute

Lobule: anatomic unitSynthetic function Proteins, CHO, lipids, LPP, clotting factors, ketone bodies, enzymes

Albumin: 12g/dayConjugation function Bilirubin metabolism

Bilirubin: 200mg/dayDetoxification and Drug metabolism

DrugsAmmonia Urea Excreted

Excretory and Secretory functions

Bile acids: cholic acid and chenodeoxycholic acidBile salts: bile acids + amino acids (glycine and taurine)

Storage function VitaminsGlycogen

Test measuring the Hepatic Synthetic Ability

Total Protein Determination:-Kjeldahl method-Biuret method-Folin-Ciocalteu (Lowry) method-UV absorption method-Electrophoresis-Refractometry-Turbidimetric and Nephelometric methods-Salt fractionationProthrombin Time (Vitamin K Response Test)

Test measuring Conjugation/Excretion Function

Bilirubin Assay:-Evelyn and Malloy method-Jendrassik and GrofBromsulfonphthalein (BSP) Dye Excretion test


Test for Detoxification Function

Enzyme tests: ALP, AST, ALT, 5’NT, GGT, OCT, LAP, LDHAmmonia:-Kjeldahl (Digestion) method-Nesslerization reaction-Berthelot reaction

Plasma protein 0.2-0.4 g/dL higher than serum due to fibrinogenKjeldahl (Digestion) mtd Standard reference method

Measurement of nitrogen contentSerum + Tungstic acid PFF1g N2 = 6.54g protein15.1-16.8% = N2 content of proteinsRgt: H2SO4

End product: NH3

Biuret method Most widely used method (IFCC recommended)Req. at least 2 peptide bonds and an alkaline mediumRgts:Alkaline CuSO4

Rochelle salt (NaK Tartrate)NaOHKIEnd product: Violet color (545nm)

Folin-Ciocalteu (Lowry) method

Highest analytical sensitivityOxidation 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 syndromeAlpha1-globulin flat curve Juvenile cirrhosis (AAT deficiency)Alpha1, alpha2, beta-globulin band spikes

Inflammation

Polyclonal gammopathy Chronic inflammation (RA, malignancy)Small spikes in beta region IDA (transferrin)Free hemoglobin “Blip” in the late alpha2 or early beta regionRefractometry Refractive indexTurbidimetric and nephelometric methods

SSATCA

Salt fractionation Salt: Sodium sulfateAlbumin Soluble:

WaterModerately concentrated salt solutionConcentrated salt solutionInsoluble:Hydrocarbon solventsHighly concentrated salt solutionSaturated salt solution


Globulin Soluble:Hydrocarbon solventsWeak salt solutionInsoluble:WaterSaturated salt solutionConcentrated 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 diseaseHepatic cirrhosis Low total protein + low albuminBromcresol green Most commonly used dye for albuminBromcresol purple Most specific dye for albuminOther dyes for albumin Hydroxyazobenzene benzoic acid (HABA)

Methyl orange (MO)Nephrotic syndrome Albumin excretion: 20-30 g/dayAnalbuminemia (-) albuminBisalbuminemia EP: 2 albumin bands

Therapeutic drugs in serumInverted 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 BiliverdinBiliverdin reductase Biliverdin B1 Urobilinogen Deconjugated bilirubinBilirubin 1 Non-polar bilirubin

Free/Slow bilirubinBilirubin 2 Polar bilirubin

One-minute/prompt bilirubinRegurgitative bilirubin

Delta bilirubin Bilirubin tightly bound to albuminDelta bilirubin = TB-DB+IB

Jaundice Bilirubin >2 or 3 mg/dLPre-hepatic jaundice Hemolytic

B1 = increasedB2 = normalUG = increasedUB = negative

Hepatic jaundice HepatocellularB1 = increasedB2 = increasedUG = increasedUB = positiveALT = increasedAST = increased

Post-hepatic jaundice ObstructiveB1 = normalB2 = increasedUG = decreased/negativeUB = positive


ALP = increasedGGT = increasedCholesterol = increased

Gilbert’s syndrome Bilirubin transport deficit (uptake)B1 = increasedB2 = decreased

Crigler-Najjar syndrome Conjugation deficitType I = total UDPGT deficiencyType II = partial UDPGT deficiencyB1 = increasedB2 = decreasedDanger: KernicterusBile is colorless

Dubin-Johnson syndrome & Rotor syndrome

Bilirubin excretion deficitBlockade of excretion into the canaliculiTB = increasedB2 = increased

Lucey-Driscoll syndrome Circulating inhibitor of bilirubin conjugationB1 = increased

Methods (Bilirubin) Free from hemolysis and lipemiaStore in the darkMeasured ASAP or w/in 2-3 hours

Van den Berg reaction Diazotization of bilirubinEvelyn 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 azobilirubinAffected by hemolysis

Jendrassik and Grof Candidate reference methodAccelerator: Caffeine sodium benzoateBuffer: Sodium acetateAscorbic acid: terminates the initial reaction and destroys the excess diazo rgtNot falsely elevated by hemolysisTotal bilirubin is measured 15 minutes after adding methanol or caffeine soln

Bilirubin Absorbs light maximally at 450nmRosenthal White method Double collection method

Collection:-After 5 mins (50% dye retention)-After 30 mins (0% dye retention)

Mac Donald method Single collection methodCollection:-After 45 mins (+/- 5% dye retention)

Ammonia From deamination of amino acidsElevated levels are neurotoxic and often associated w/ encephalopathy and acetaminophen poisoningDiagnosis of hepatic failure and Reye’s syndromeIn 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


Methods (Ammonia) Specimen: Heparin or EDTA plasmaFasting is requiredAvoid smokingProlonged standing of specimen: increased NH3 due to deaminationPlace on iced water immediatelyAvoid hemolysis

Kjeldahl (Digestion) method

Specimen PFFN2 ----------(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 blueNormal Values(Liver Function Tests)

Total protein = 6.5-8.3 g/dLAlbumin = 3.5-5.0 g/dLGlobulin = 2.3-3.5 g/dLα1-globulin = 0.1-0.3 g/dLα2-globulin = 0.6-1.0 g/dL

-globulin = 0.7-1.1 g/dLβ-globulin = 0.8-1.6 g/dLγ

Total bilirubin = 0.2-1.0 mg/dLIndirect bilirubin = 0.2-0.8 mg/dLDirect bilirubin = 0-0.2 mg/dLUrobilinogen: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 g/dLμ

EnzymesEnzyme concentration Serum

Enzyme concentration = reaction rateSubstrate concentration Reagent

If enzyme > substrate, substrate = reaction rateSaturation kinetics When substrate concentration reaches a maximal value, higher concentration

of substrate no longer results in increased rate of reactionCofactors Nonprotein entitiesCoenzymes Organic compound

Ex. NADP Coenzyme = Velocity

Activators Inorganic ionsAlters spatial configuration of the enzyme for proper substrate bindingEx. Ca2+ (#1 activator), Zn2+ (LDH), Cl- (AMS), Mg2+ (CK, ALP)

Metalloenzymes Inorganic ion attached to a moleculeEx. Catalase, cytochrome oxidase

Inhibitors Interferes with the enzymatic reactionsCompetitive inhibitor Binds to the active site of an enzyme

Reversible (Substrate > Inhibitor)Noncompetitive inhibitor Binds to the allosteric site (cofactor site)

IrreversibleUncompetitive inhibitor Binds to the enzyme-substrate complex

Substrate = ES = InhibitionIsoenzymes Same catalytic reactions but slightly different molecular structures

Fractionation of isoenzymes


Temperature 37’C = optimum temperature for enzyme activityTemperature = Reaction rate (movement of molecules)

40-50’C Denaturation of enzymes60-65’C Inactivation of enzymesTemperature coefficient (Q10)

For every 10 O C increase in temperature, there will be a two-fold increase in enzyme activity

pH Most physiologic reactions occur in the pH range of 7-8Storage Enzymes: -20’C = for longer period of time

Substrate and Coenzymes: 2-8’CLDH (LD4 & 5): Room temperature

Hemolysis Mostly increases enzyme concentrationLactescence or milky specimen

Decreases enzyme concentration

Enzyme nomenclature 1st digit: classification2nd and 3rd digits: subclass4th digit(s): serial number

Enzyme classification “OTHLIL”OxidoreductasesTransferasesHydrolasesLyasesIsomerasesLigases

Oxidoreductases Redox reactionDehydrogenases:-Cytochrome oxidase-LDH-MDH-Isocitrate dehydrogenase-G-6-PD

Transferases Transfer of a chemical group other than hydrogen from 1 substrate to anotherKinases, Transaminases, Aminotransferases:-CK-GGT-AST-ALT-OCT

Hydrolases Hydrolysis/splitting by addition of waterEsterases:-ACP-ALP-CHS-LPSPeptidases:-Trypsin-Pepsin-LAPGlycosidases:-AMS-Galactosidases

Lyases Removal of groups w/o hydrolysis (product contains double bonds)Aldolase


Decarboxylases:-Glutamate decarboxylase-Pyruvate decarboxylase-Tryptophan decarboxylase

Isomerases Intramolecular arrangementsGlucose phosphate isomeraseRibose phosphate isomerase

Ligases Joining of 2 substrate moleculesSynthases

Active site Water-free cavityWhere the substrate interacts

Allosteric site Cavity other than the active siteMay bind regulatory molecules

Prosthetic group Coenzyme that is bound tightly to the enzymeHoloenzyme Apoenzyme + Prosthetic groupZymogen/proenzyme Inactive form of enzymeEmil Fisher’s/Lock and Key theory

Shape of the key (substrate) must fit into the lock (enzyme)

Kochland’s/Induced fit theory

Based on the substrate binding to the active site of the enzymeAcceptable 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 reactionGroup specificity Enzymes combine w/ all the substrates in a chemical groupBond specificity Enzymes reacting w/ specific chemical bondsZero-order reaction Reaction rate depends only on enzyme concentration

Independent on substrate concentrationFirst-order reaction Reaction rate is directly proportional to substrate concentration

Independent on enzyme concentrationMeasurement of enzyme activity

Change in substrate concentrationChange in product concentrationChange in coenzyme concentration

International Unit 1 micromole of substrate/minuteKatal Unit 1 mole of substrate/secondNonkinetic assay Absorbance is made at 10-second intervals for 100 secondsAlkaline Phosphatase pH = 10.5

405nmElectrophoresis:(+) Liver Bone (Regan) Placenta Intestine (-)Heat fractionation:( Stable) Regan Δ Placenta Intestine Liver Bone ( Labile)Δ

Phenylalanine Inhibits Regan, placental and intestinal ALPL-leucine Inhibits Nagao ALPLevamisole Inhibits liver and bone ALP3M urea Inhibits bone ALPMethods (ALP) Low temperature = Increased ALP

1. Bowers and McComb (PNPP) – IFCC recommended2. 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)


7. Moss = ANP (alpha naphthol phosphate)Increased ALP Sprue

HyperparathyroidismRickets (children) and osteomalacia (adults)

Acid Phosphatase pH = 5.5405nmSources: Prostate (major), RBC, platelets, bone

Prostatic ACP Inhibited by L-tartrate ionsRBC ACP Inhibited by cupric and formaldehyde ionsMethods (ACP) Room temperature (1-2 hrs) = decreased ACP

Thymolphthalein monophosphate = specific substrate, substrate of choice (endpoint)Alpha-naphthyl phosphate = preferred for continuous monitoring methods1. Gutman and Gutman = PP2. Shinowara = PNPP3. Babsonm Read and Phillips = ANP (continuous monitoring)4. Roy and Hillman = Thymolphthalein monophosphate (endpoint)

Aspartate Aminotransferase (AST/SGOT)

pH 7.5340nmSources: Cardiac tissue > Liver > Skeletal muscle > Kidney, pancreas, RBCs

Alanine Aminotransferase (ALT/SGPT)

pH 7.5340nmMajor Source: Liver

Methods (AST and ALT) 1. Karmen method = Kinetic2. 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 hepatitisModerate elevation = chronic hepatitis, hepatic cancer, IMSlight elevation = Hepatic cirrhosis, alcoholic hepatitis, obstructive jaundice

Amylase Smallest enzyme (appears in urine)Earliest pancreatic markerP3: most predominant pancreatic AMS isoenzyme in APIsoenzymes:S-type (ptyalin): anodalP-type (amylopsin): cathodal

Methods (AMS) Samples w/ high activity of AMS should be diluted w/ NaCl to prev. inactivationSalivary AMS = inhibited by wheat germ lectinSubstrate: Starch

Saccharogenic Reducing sugars producedClassic reference method (SU)

Amyloclastic Degradation of starchChromogenic Increase in color intensityCoupled-enzyme Continuous-monitoring techniqueLipase Late marker (AP)

Most specific pancreatic markerMethods (LPS) Substrate: Olive oil/Triolein

1. Cherry Crandal (Reference method)2. Tietz and Fiereck3. Peroxidase coupling (most commonly used method)

Lactate dehydrogenase Lacks specificity


RBC: 150x LDH than in serumSources:LD1 (α-HBD) and LD2 = Heart, RBC, KidneysLD3 = pancreas, lungs, spleenLD4 an LD5 = liver and muscleLD6 = alcohol dehydrogenase

Methods (LDH) 1. Wacker method (forward/direct) = pH 8.8, 340 nm, most commonly used2. Wrobleuski LaDue (reverse/indirect) = pH 7.2, 2x faster3. Wrobleuski Cabaud4. Berger Broida

10-fold increase (LDH) Hepatic carcinoma and toxic hepatitis2-3x URL Viral hepatitis and cirrhosisCreatine Kinase Isoenzymes:

CK-BB = most anodal, brainCK-MB = myocardium (20%)CK-MM = least anodal, skeletal and smooth muscles (Major, 94-100%)

Duchenne’s muscular dystrophy

Total CK: 50x URL (highest)

CK-MB Most specific indicator of myocardial damage (AMI)Not elevated in angina

Methods (CK) 1. Tanzer-Gilbarg (forward/direct) = pH 9.0, 340nm2. Oliver-Rosalki/ Rosalki & Hess (reverse/indirect) = most commonly used method, faster reaction; pH 6.8, 340nm

Adenylate kinase Inside RBCsInterferes w/ CK assayInhibited by adenosine monophosphate

N-acetylcysteine Activate CKLiver cells and RBC Do not contain CKCleland’s reagent and glutathione

Partially restore lost activity of CK

Electrophoresis Reference method for CKCK relative index (CKI) CKI (%) = CK-MB/Total CK x 100Aldolase Isoenzymes:

Aldolase A = Skeletal musclesAldolase B = WBC, liver, kidneyAldolase C = brain tissue

5’ Nucleotidase Marker for hepatobiliary diseases and infiltrative lesions of the liverMethods:1. Dixon and Purdon2. Campbell, Belfield and Goldberg

GGT Located in the canaliculi of the hepatic cellsDifferentates the source of an elevated ALP levelSensitive indicator of occult alcoholismIncreased:Obstructive jaundiceAlcoholic hepatitis (most sensitive)

Methods (GGT) Substrate: gamma-glutamyl-p-nitroanilide1. Szass2. Rosalki and Tarrow3. Orlowski

Cholinesterase/ Monitor effects of relaxants (succinylcholine) after surgery


Pseudocholinesterase Marker for organophosphate poisoning (Low CHS)Methods:1. Ellman technic2. Potentiometric

Angiotensin-Converting Enzyme

A.k.a. peptidyldipeptidase A or Kininase IIConverts angiotensin I angiotensin II (lungs)Indicator of neuronal dysfunction (Alzheimer’s disease – CSF)

Ceruloplasmin Ferrooxidase enzymeOrnithine carbamoyl transferase

For hepatobiliary diseases

G-6-PD Drug induced hemolytic anemia (primaquine, antimalarial drug)Normal Values (Enzymes) ALP = 30-90 U/L

ACP:Total ACP (male) = 2.5-11.7 U/LProstatic ACP = 0-3.5 ng/mLAST = 5-37 U/LALT = 6-37 U/LAMS = 60-180 SU/dL (95-290 U/L)LPS = 0-1.0 U/mLLDH:Forward = 100-225 U/LReverse = 80-280 U/L

Acute Myocardial Infarction MarkersMyoglobin Troponin T Troponin I CK-MB AST LD

Rise 1-3 h 3-4 h 3-6 h 4-8 h 6-8 h 12-24 hPeak 5-12 h 10-24 h 12-18 h 12-24 h 24 h 48-72 hNormalize 18-30 h 7 d (10-14 d) 5-10 d 48-72 h 5 d 10-14 d

Acute Pancreatitis MarkersAmylase Lipase

Rise 2-12 h 6 hPeak 24 h 24 hNormalize 3-5 d 7 d

ElectrolytesElectroneutrality Equal no. of cations and anions

Balance of charges40-75% Average water content of the human bodyECF 1/3 of total body waterICF 2/3 of total body waterNormal plasma 93% water (Plasma: 13% > Whole blood)

7% solutes: (Increased in dehydration)-Proteins-Glucose-NPN-Lipids-Ions

Vasopressin deficiency Excretion of 10-20L H2O everydayVolume and Osmotic regulation

SodiumPotassiumChloride

Electrolytes EC = Na+ > Cl- > HCO3- > Ca2+(5th) > iPO4

IC = K+ > Mg2+(4th)Myocardial rhythm and Potassium


contractilityNeuromuscular excitability

CalciumMagnesium

Cofactors (enzyme) CalciumMagnesium (CK)ZincChloride (AMS)Potassium

ATPase ion pump MagnesiumProduction and use of ATP from glucose

MagnesiumPhosphate

Acid-base balance BicarbonateReplication of DNA and translation of mRNA

Magnesium

Sodium Major contributor of osmolality (92%, together w/ Chloride and Bicarbonate)100 mg/dL glucose = 1.6 mmol/L sodium

Aldosterone SodiumPotassium = Magnesium

Atrial natriuretic factor SodiumHypernatremia Excess water loss

Decreased water intakeHyperaldosteronism (Conn’s disease)Hypothalamic disease (Chronic hypernatremia)

Hyponatremia Renal failureSIADH (increased water retention)Marked hemolysis (dilutional effect)<125 mmol/L = severe neuropsychiatric symptoms

Thirst Major defense against hyperosmolality and hypernatremia1-2% water deficit = severe thirst150-160 mEq/L Na+ = Moderate deficit of water>165 mEq/L Na+ = Severe water deficit

Pseudohyponatremia (artifactual)

Hyperlipidemia (turbidity)Hyperproteinemia

Methods (Na+) 1. FEP2. AAS3. ISE = Glass aluminum silicate4. Colorimetry = Albanese Lein

Potassium Concentration in RBC is 105 mmol/LReciprocal relationship with H+

Specimen Considerations (K+)

0.5% hemolysis = 0.5 mmol/LGross hemolysis = 30%Serum K+ > Plasma K+ by 0.1-0.7 mmol/L because of platelets (clot)10-20% in muscle activity0.3-1.2 mmol/L = mild to moderate exercise2-3 mmol/L = vigorous exercise; fist clenching

Hyperkalemia Decreased resting membrane potential incr. contractility lack of muscle excitabilityDecreased renal excretion (Dehydration, renal failure, Addison’s disease)Acidosis (DM)Muscle injurySpironolactone

Hypokalemia Increased resting membrane potential arrhythmia


Leads to hypomagnesemiaVomitingDiureticsCushing’s syndromeAlkalosisInsulin overdose

pH and K+ pH by 0.1 = K+ by 0.2-1.7 mmol/LMethods (K+) Lithium heparin plasma = preferred

1. FEP2. AAS3. ISE = Valinomycin gel4. Colorimetry = Lockhead and Purcell

Chloride Chief counter ion of sodium in ECFSpecimen Considerations (Cl-)

Chloride methods measure bromide and iodideCl- = HCO3

-

Methods (Cl-) 1. Schales and Schales:-Mercurimetric titration-Diphenylcarbazone-Excess Hg++

-(+) Blue violet2. Whiterhorn Titration method-Mercuric thiocyanate-Reddish complex3. Ferric perchlorate4. Cotlove chloridometer-Coulometric amperometric titration-Excess Ag++

5. ISE-Ion exchange membrane-Tri-n-octylpropylammonium chloride decanol

Hyperchloremia Renal tubular acidosisMetabolic acidosisDiabetes insipidus (Dehydration)Prolonged diarrhea

Hypochloremia Prolonged vomiting (HCl)Aldosterone deficiency (Na+ = Cl- = K+)Metabolic alkalosis (HCO3

- = Cl-)Marked hemolysis (dilutional effect)

Calcium 99% Bones1% ECFAbsorbed in the duodenumAbsorption is favored at an acidic pH

3 Forms of Calcium 50% = Free/Ionized/Unbound/Active Calcium40% = Protein-bound (Albumin)10% = Complexed with anions

Vitamin D3 Ca2+ = absorption (intestine) and reabsorption (kidney)PTH Ca2+ = resorption (bone) and reabsorption (kidney)Calcitonin Ca2+ = urinary excretion (major net loss of calcium)Practical considerations (Ca2+)

Serum = specimen of choice Albumin (1g/dL) = Ca2+ (0.8 mg/dL)

Hypercalcemia Acidosis (Ca2+: from Bones Blood)


CancerHyperthyroidismMilk-alkali syndrome

Hypocalcemia TetanyAlkalosis (Ca2+: from Blood Bones)Acute pancreatitis (Ca2+: binds to damage pancreatic tissues)

Primary hypocalcemia Low PTHParathyroid gland disease

Secondary hypocalcemia High PTHRenal failure ( excretion)

Methods (Ca2+) 1. Clark Collip precipitation method-(+) Oxalic acid-Renal calculi2. Ferro Ham Chloranilic acid precipitation method-(+)Chloranilic acid3. Colorimetric = Ortho-Cresolphthalein complexone dyes-Dye: Arzeno III-8-hydroxyquinoline = chelates (inhibits) Mg2+

4. EDTA titration method (Bachra, Dawer and Sobel)5. AAS = Reference method6. ISE = Liquid membrane7. FEP

Inorganic Phosphorus 85% Bones15% ECF (iPO4)Maximally absorbed in the jejunum (Ca2+: duodenum)Trancellular shift: Once absorbed inside cells, it no longer comes out used for energy productionDirunal variation: late morning, eveningOrganic phosphate = principal anion within cellsInorganic phosphate = part of the blood buffer (Measured in the clin.lab.)

3 Forms of Inorganic Phosphorus

55% = Free35% = Complexed with ions10% = Protein-bound

PTH PO4 = Ca2+

Calcitonin PO4 = Ca2+

Growth hormone PO4 (renal reabsorption)Practical considerations Fasting is required (Nonfasting: PO4)Hyperphosphatemia Hypoparathyroidism

Renal failureHypervitaminosis D

Hypophosphatemia Alcohol abuse = most common causePrimary hyperparathyroidismAvitaminosis D (Rickets, Osteomalacia)

Methods (iPO4) Most accurate: unreduced phosphomolybdate formation (340nm)1. Fiske Subbarow Method (Ammonium molybdate method)-Reducing agents: Pictol, Elon, Senidine, Ascorbic acid-(+) Phosphomolybdenum blue

Magnesium 53% Bones46% Muscles and soft tissues1% Serum and RBCVasodilator

3 Forms of Magnesium 55% = Free/Ionized/Physiologically active


30% = Protein-bound10% = Complexed with ions

PTH Mg2+ = Ca2+ = PO4

Aldosterone (& Thyroxine) Mg2+ = K+ = Na+

Hypermagnesemia Addison’s diseaseChronic renal failure

Hypomagnesemia Acute renal failureChronic alcoholism

Methods (Mg2+) 1. Calmagite-(+) Reddish-violet complex2. Formazen dye method-(+) Colored complex3. Magnesium Thymol blue method-(+) Colored complex4. AAS = reference method5. Dye-lake Method-Titan Yellow dye (Clayton Yellow or Thiazole yellow)

Bicarbonate 90% of the total CO2

Chloride shift HCO3- diffuses out of the cell in exchange for Cl- to maintain ionic charge

neutrality w/in the cellAnion Gap Difference between unmeasured anions and unmeasured cations

QC for ISEIncreased AG Uremia/renal failure

KetoacidosisLactic acidosisMethanol poisoningEthanol poisoningEthylene glycol poisoningSalicylate poisoning

Decreased AG HypoalbuminemiaHypercalcemiaHyperlipidemiaMultiple myeloma

Cystic Fibrosis (Mucoviscidosis)

Defective gene: Cystic fibrosis transmembranous conductance regulator (Chromosome 7)Miconeum ileus (Infants)Foul-smelling stoolURT infection Na+ and Cl-

Pilocarpine Sweat inducerGibson & Cooke pilocarpine iontophoresis

Reference method (Sweat sodium and chloride)

Iron Prooxidant3-5g = Total body ironFerrous = HgbFerric = Transferrin and Ferritin

Methods (Iron) 1. Colorimetric = HCl and Ferrozine-(+) Blue color2. Anodic stripping voltammetry

Increased iron Hemochromatosis


Viral hepatitisNon-IDA

Decreased iron IDAMalnutritionChronic infection

TIBC UIBC + Serum IronIncreased: IDA, hepatitis, iron-supplemented pregnancyDecreased: Non-IDA, nephrosis

UIBC TIBC – Serum ironMeasure of reserve iron binding capacity of transferrin

% Transferrin Saturation Index of iron storageIncreased: Iron overdose, hemochromatosis, sideroblastic anemiaDecreased: IDA (lowest), malignancy, chronic infection

Transferrin TIBC ( g/dL) x 0.70 = mg/dLμNote Sodium 1/α Potassium

Potassium 1/α Hydrogen ionPotassium α MagnesiumMagnesium α CalciumCalcium 1/α Inorganic phosphateChloride 1/α Bicarbonate

Normal Values (Electrolytes)

Sodium:Serum = 135-145 mmol/L[Critical: 160 mmol/L and 120 mmol/L]CSF = 136-150 mmol/LPotassium:Serum = 3.5-5.2 mmol/L[Critical: 6.5 mmol/L and 2.5 mmol/L]Chloride:Serum = 98-107 mmol/LSweat = 5-40 mmol/L [Critical: >65 mmol/L]Calcium:Total = 8.6-10 mg/dL (adult) and 8.8-10.8 mg/dL (child)Ionized = 4.6-5.3 mg/dL (adult) and 4.8-5.5 mg/dL (child)[Critical: <7.5 mg/dL]Inorganic Phosphate:Adult = 2.7-4.5 mg/dLChild = 4.5-5.5 mg/dLMagnesium:Serum = 1.2-2.1 mEq/LAnion Gap:w/ K+ = 10-20 mmol/Lw/o K+ = 7-16 mmol/LIron:Male = 50-160 g/dLμFemale = 45-150 g/dLμTIBC:Adult = 245-425 g/dLμ>40 y.o. = 10-250 g/dLμNB and Child = 100-200 g/dLμ% Transferrin Saturation = 20-50%


Blood Gases and pHRegulation of Acid-Base balance

Lungs and KidneysCO2 + H2O <--(Carbonic anhydrase)--> H2CO3

H2CO3 <-------(Carbonic anhydrase)--> H+ + HCO3-

20:1 HCO3-: H2CO3 ratio

4:1 HPO4: H2PO4 ratioExpanded Henderson-Hasselbalch equation

pH = 6.1 + log [Total CO2 – (pCO2 x 0.03)] pCO2 x 0.03

Chloride-isohydric shift Buffering effect of hemoglobinpCO2 Index of efficiency of gas exchange

Increased: Barbiturates, morphine, alcohol, heparin (12-15%)pO2 Reflects the availability of the gas in blood but not its content

Excessive O2 supply acidosisMetabolic Acidosis Causes:

-Bicarbonate deficiency-DKA (normochloremic acidosis)-Renal failure-Diarrhea (HCO3

-)Compensation: HyperventilationCompensated: HCO3

- + pCO2 + pH <7.4

Metabolic Alkalosis Causes:-Bicarbonate excess-Vomiting (Cl-)-Hypochloremia-HypokalemiaCompensation: HypoventilationCompensated: HCO3

- + pCO2 + pH >7.4Respiratory Acidosis Causes:

-CO2 excess (Hypoventilation)-COPD-Drug overdose (morphine, barbiturates, opiates)Compensation: Bicarbonate retentionCompensated: HCO3

- + pCO2 + pH <7.4Respiratory Alkalosis Causes

-CO2 loss (Hyperventilation)Compensation: Bicarbonate excretionCompensated: HCO3

- + pCO2 + pH >7.4Full compensation pH normal rangePartial compensation pH near normalBuffer base All forms of base that will titrate hydrogen ionsMethods for Blood Gases and pH

Specimen: Arterial bloodBlood gas analyzers: meas. pH, pCO2, pO2

Factors affecting Blood gases & pH measurements

For every 1 O C above 37 O C : pH by 0.015 pO2 by 7% pCO2 by 3%Bacterial contamination: consume O2 (pO2)Excess heparin (acid MPS) = pHAir exposure (bubbles):pO2 = 4 mmHg/2minspCO2 = 4 mmHg/2mins


Methods(Blood gases & pH)

1. Gasometera. Van Slykeb. Natelson-Mercury: produce vacuum-Caprylic alcohol: anti-foam reagent-Lactic acid-NaOH-NaHSO3

2. Electrodesa. pH = potentiometry-Silver-silver chloride electrode (Reference electrode)-Calomel electrode [Hg2Cl2] (Reference electrode)b. pCO2 = Severinghaus electrode (potentiometry)c. pO2 = Clark electrode (polarography-amperometry)

Whole blood total CO2 Dissolved CO2 + H2CO3 + HCO3-

Transcutaneous electrodes Continuous monitoring of pO2

Directly placed on the skinBlood gas QC Min. requirement:

-1 sample every 8 hours-3 levels of control (acidosis, normal, alkalosis) every 24 hours

Normal Values(Blood gases and pH)

pH = 7.35-7.45pCO2 = 35-45 mmHgTotal CO2:WB arterial = 19-24 mmol/LWB venous = 22-26 mmol/LHCO3- = 21-28 mEq/LpO2 = 81-100 mmHg[Hypoxemia:] -Mild (61-80 mmHg)-Moderate (41-60 mmHg)-Severe (40 mmHg or less)O2 saturation = 94-100%

EndocrinologyEndocrine Hormone blood circulation specific receptorParacrine Hormone interstitial space adjacent cellAutocrine Hormone self-regulationJuxtacrine Hormone direct cell-to-cell contactExocrine Hormone gutNeurocrine Hormone neurons extracellular spaceNeuroendocrine Hormone neurons nerve endingsGlycoproteins FSH, hCG, TSH, LHPolypeptides ACTH, ADH, GH, angiotensin, calcitonin, CCK, gastrin, glucagons, insulin, MSH,

oxytocin, PTH, PRL, somatostatinSteroids Precursor: cholesterol

Aldosterone, cortisol, estrogen, progesterone, testosterone, vitamin DAmines Derived from amino acids

Catecholamines, T3, T4Hypothalamus Connected to the posterior pituitary by the infundibulum stalk

Hypophyseal hormones: TRH, GnRH, GH-IH, GH-RH, PIFPineal gland Melatonin: decreases pigmentation of the skinPituitary gland Master Gland

Located in the sella turcica or Turkish saddle


Anterior Pituitary(Adenohypophysis)

True endocrine glandHormones: PRL, GH, FSH, LH, TSH, MSH, ACTH

GH (Somatotropin) Most abundant of all pituitary hormonesStructurally similar to PRL and HPLMarkedly elevated during deep sleep

Dwarfism Decreased GHAcromegaly Increased GHGH deficiency tests 1. Insulin tolerance test = Gold standard (Confirmatory test)

2. Arginine stimulation test = 2nd confirmatory testTests for Acromegaly 1. Somatomedin C or insulin-like growth factor I (Screening)

-Increased: Acromegaly-Decreased: GH deficiency2. OGTT (Confirmatory)-75g glucose

FSH SpermatogenesisLH Helps Leydig cells to produce testosterone (male)

Ovulation (female)Synthesis of androgens, estrogens, and progesterone

TSH (Thyrotropin) Stimulates thyroid gland to produce T3 and T4Increased: 1’ hypothyroidism, 2’ hyperthyroidismDecreased: 1’ hyperthyroidism, 2’ hypothyroidism, 3’ hypothyroidism

ACTH (Corticotropin) Highest: 6-8 AMLowest: 6-11 PMNot allowed to have contact with glass because it adheres to glass surfaceCollect blood in plastic tubes

Prolactin Initiation and maintenance of lactationInhibited by DopamineHighest: 4AM and 8AM, and 8PM and 10PMIncreased: Menstrual irregularity, infertility, amenorrhea, galactorrhea

Panhypopituitarism From pituitary tumor (adenoma) or IschemiaPituitary ischemia(Shechan’s)

Hemorrhage or shock in a pregnant female at the time of deliver

Posterior pituitary(Neurohypophysis)

Release but not produce oxytocin and vasopressin

Oxytocin Uterine contraction and milk ejectionADH/AVP (Arginine vasopressin)

H2O reabsorption (DCT and CD)Stimulus: Increased plasma osmolality (>295 mOsm/kg), decreased blood vol.Promotes factor VII and vWF release

Overnight water deprivation test (Conc. test)

Diagnostic test for ADH

Neurogenic DI True Diabetes InsipidusFailure of the pituitary gland to secrete ADH

Nephrogenic DI Failure of the kidneys to respond to normal or elevated ADHSIADH Syndrome of inappropriate ADH

Sustained production of ADHDecreased urine volumeLow plasma osmolalityLow serum electrolytes

Thyroid Gland Butterfly-shaped2 lobes = connected by the isthmus

Follicle Fundamental structural unit of the thyroid glandFollicular cells Secrete T3 and T4


Parafollicular or C cells Secrete calcitoninThyroglobulin Preformed matrix containing tyrosyl groups

Stored in the follicular colloid of the thyroid glandThyroid hormone Biosynthesis

1. Trapping of Iodine2. Iodination: I2 Tyrosine ring MIT and DIT3. Condensation: MIT+DIT=T3 / DIT+DIT=T44. Release: T3/T4 Blood circulation5. Transport of T3/T4 by proteins

Protein-bound hormones Metabolically inactiveBiologically inertDo not enter cellsStorage sites

Free hormones (FT3/FT4) Physiologically activeReadily enters cells

Reverse T3 (rT3) From removal of one iodine from T4 (product of T4 metabolism)Metabolically inactive

I2 intake <50 g/dayμ Deficiency of hormone secretionT3 3,5,3’-Triiodothyronine

Most active thyroid hormonal activity75-80% is produced from the tissue deiodination of T4Diagnosis of T3 thyrotoxicosis

T4 3,5,3’5’-TetraiodothyroninePrincipal secretory productAll originated in the thyroid gland

TBG Transports 70-75 of TT4, and majority of T3TBPA (Transthyretin) Transports 15-20% of TT4

No affinity for T3TBA Transports T3 and 10% of T4Thyroid autoantigens TPO

TgTSHR

Thyroid disorders Screening is recommended when a person reaches 35 yrs old and every 5 yrs thereafter

Primary hyperthyroidism T3 and T4 TSH

Secondary hyperthyroidism T3 and T4 TSH

T3 Thyrotoxicosis (Plummer’s disease)

T3N-T4 TSH

Graves’ disease(Diffuse toxic goiter)

1’ HyperthyroidismMost common cause of thyrotoxicosis (autoimmune)Women > MenAnti-TSH receptor

Riedel’s thyroiditis Thyroid woody or stony-hard massSubclinical hyperthyroidism

No symptomsN-T3 and T4 TSH

Subacute granulomatous/ Subacute nonsuppurative/ De Quervain’s thyroditis

HyperthyroidismPainful thyroiditisNeck pain, low-grade fever(-) anti-TPO, ESR and Tg


Hypothyroidism Treatment: LevothyroxinePrimary hypothyroidism T3 and T4

TSHHashimoto’s disease (Chronic autoimmune thyroiditis)

Most common cause of 1’ hypothyroidismThyroid is replaced by a nest of lymphoid tissue (T cells)Goiter(+) anti-TPO TSH

Myxedema coma Severe form of 1’ hypothyroidismPeculiar nonpitting swelling of the skinSkin is infiltrated by mucopolysaccharides“Puffy” face, thin eyebrows

Secondary hypothyroidism T3 and T4 TSH

Tertiary hypothyroidism T3 and T4 TSH TRH

Congenital hypothyroidism(Cretinism)

Mental retardation (child)Screening: T4Confirmatory: TSH

Subclinical hypothyroidism N-T3 and T4 TSH

TRH stimulation test Most specific and sensitive test for diagnosing thyroid diseaseConfirm borderline cases and euthyroid Graves’ disease: 1’ hypothyroidism: Hyperthyroidism

Radioactive Iodine Uptake (RAIU)

Measure the ability of the thyroid gland to trap iodine

Thyroglobulin (Tg) assay Postoperative marker of thyroid cancer: Untreated and metastatic differentiated thyroid cancer, hyperthyroidism: Hypothyroidism, thyrotoxicosis factitia

rT3 Assess borderline or conflicting laboratory resultsFree Thyroxine Index (FT4I)

Indirectly assesses the level of FT4 in bloodEquilibrium relationship of bound T4 and FT4Reference method: Equilibrium dialysisFT4I = TT4 x T3U(%) or TT4 x THBR 100

TT3, FT3, FT4 FT4 test: differentiates drug induced TSH elevation and hypothyroidismTT3 or FT3: confirm hyperthyroidismReference method (FT4): Equilibrium dialysis

T3 Uptake test Measures the number of available binding sites of the thyroxine binding proteins (TBG) TBG = T3U TBG = T3U

TBG test Confirm results of FT3 or FT4 or abnormalities in the relationship of TT4 and THBR testEstrogen: TBGAndrogen: TBG

Fine-needle aspiration Most accurate tool in the evaluation of thyroid nodulesRecombinant Human TSH Test patients w/ thyroid cancers for the presence of residual or recurrent dis.Tanned Erythrocyte Hemagglutination method

Test for anti-Tg disorders


Serum calcitonin test Marker for familial medullary thyroid carcinomaFT4 and TSH Best indicators of thyroid statusFT3 and FT4 More specific indicators of thyroid function than meas. of total hormone

Not affected by TBGEuthyroid sick syndrome Acutely ill but without thyroid disease

T3 and T4N/ TSH rT3

Parathyroid gland 4 parathyroid glandsSmalles endocrine gland

PTH Hypercalcemic hormone Ca2+ (bone resorption and renal reabsorption) and Mg2+

iPO4

1’ hyperparathyroidism Defective: Parathyroid glandMost common cause of hypercalcemiaParathyroid adenoma PTH and iCa2+

HypercalciuriaPhosphaturia HypophosphatemiaIf goes undetected severe demineralization (osteitis fibrosa cystica)

2’ hyperparathyroidism In response to Ca2+

Hyperplasia of all 4 glandsCauses: Vit. D deficiency and chronic renal failure PTH Ca2+

3’ hyperparathyroidism Occurs w/ 2’ hyperparathyroidism ( Ca2+)Autonomous function of hyperplastic PT glands or PT adenoma PO4

Calcium phosphates precipitate in soft tissuesHypoparathyroidism Accidental injury of the PT glands (neck) during surgery

Autoimmune parathyroid destruction PTH = Ca2+

Hyperparathyroidism AcidosisHypoparathyroidism AlkalosisAdrenal glands Pyramid-shaped

Above the kidneysAdrenal cortex = outer (yellow)Adrenal medulla = inner (dark mahogany)Has prime effects on blood pressure

Adrenal cortex Major site of steroid hormone productionG cells: convert cholesterol pregnenolone

CPPP ring 17-carbon skeleton derived from cholesterol3 layers (Adrenal cortex) 1. Zona Glomerulosa = Mineralocorticoids (Aldosterone)

2. Zona Fasciculata = Glucocorticoids (Cortisol)3. Zona Reticularis = Weak androgens (androstenedione, DHEA)

Cortisol Gluconeogenesis hyperglycemiaThe only adrenal hormone that inhibit the secretion of ACTHAnti-inflammatory and immunosuppressiveDiurnal: 6-8AM / 10PM-12AMUrinary metabolites: 17-OHCS and 17-KGS


Porter-Silber method Meas. 17-OHCSRgt: DNPH in H2SO4 + Alcohol(+) Yellow

Zimmerman reaction Meas. 17-KGSRgt: m-dinitrobenzene(+) Reddish purpleOxidation procedure: Norymberski (Na+ bismuthate)

Pisano method For quantitating metanephrines and normetanephrinesKober reaction For estrogen

Rgt: H2SO4 + hydroquinone(+) Reddish brown color

Cushing’s syndrome(Hypercortisolism)

Excessive production of cortisol and ACTHOveruse of corticosteroidsBuffalo humpHyperglycemiaHypertensionHypercholesterolemia Lymphocytes

Screening tests (Cushing’s) 1. 24-hour urine free cortisol test2. Overnight dexamethasone suppression tests = Most widely used (1mg)3. Salivary cortisol test

Confirmatory tests (Cushing’s)

1. Low-dose dexamethasone suppression test (0.5mg)2. Midnight plasma cortisol3. CRH stimulation test

Addison’s disease(1’ Hypocorticolism)

Primary adrenal insufficiency Cortisol and aldosterone ACTH(+) HyperpigmentationScreen: ACTH Stimulation Test

2’ Hypocorticolism Secondary adrenal insufficiencyHypothalamic-pituitary insufficiency ACTHTest: ACTH Stimulation test

ACTH Stimulation test(Corsyntropin stimulation test)

Corsyntropin: synthetic coritsol and aldosterone stimulatorDifferentiates:2’ adrenal insufficiency (ACTH) from3’ adrenal insufficiency ( ACTH)

Metyrapone test Metyrapone: inhibitor of 11 -hydroxylaseβMeasures the ability of the pituitary gland to respond to declining levels of circulating cortisol, thereby secrete ACTHAlternative diagnostic or confirmatory test for 2’ or 3’ adrenal insufficiency(+): ACTH

24-hour urine free cortisol Most sensitive and specific screening test for excess cortisol production because plasma cortisol is affected by diurnal variationMethods: HPLC or GC-MS

HPLC-MS Reference method for measuring urinary free cortisolITT (Insulin tolerance test) Gold standard for 2’ and 3’ hypocorticolism

Confirms borderline response to ACTH stimulation testSerum ACTH Differentiates:

Cushing’s disease (ACTH)Cushing’s syndrome (0-ACTH)

ACTH 17-OHCS and 17-KS


Congenital Adrenal Hyperplasia

Enzyme deficiencies:1.) 21-hydroxylase = most common2.) 11 -hydroxylase = 2β nd most common3.) 3 -hydroxysteroid dehydrogenase-isomeraseβ4.) C-17,20-lyase/17α-hydroxylase Cortisol ACTH Androgens (hirsutism, virilization, amenorrhea, pseudohermaphroditism)

Aldosterone (Aldo) Electro-regulating hormone Na+ and Cl-

K+ and H+

at night18-hydroxysteroid dehydrogenase: enzyme needed for aldosterone synthesis

Conn’s disease(1’ hyperaldosteronism)

Aldosterone-secreting adrenal adenomaScreen: Plasma Aldo conc./Plasma renin activity ratio (PAC/PRA ratio)-(+): >50 ratioConfirm: Saline suppression test-(+): >5 ng/dL aldosterone

2’ Hyperaldosteronism Excessive production of reninLiddle’s syndrome Pseudohyperaldosteronism

Resembles 1’ aldosteronism clinically Aldosterone(-) Hypertension

Bartter’s syndrome Bumetanide-sensitive chloride channel mutation Aldosterone and Renin

Gitelman’s syndrome Thiazide-sensitive transporter mutation Aldosterone

Hypoaldosteronism Destruction of the adrenal glandsGlucocorticoid deficiency21-hydroxylase deficiency

Postural stimulation test Test for aldosteroneFlorinef Synthetic mineralocorticoidWeak androgens Precursors for the production of more potent androgens and estrogens

Precursors: Pregnenolone and 17-OH pregnenoloneExamples: DHEA and androstenedioneBound to steroid hormone binding globulin (SHBG): Virilization (pseudohermaphroditism)

DHEA (Dehydroepiandrosterone)

Principal adrenal androgenConverted to estrone

Adrenal medulla Chromaffin cells: secrete catecholaminesPrecursor: L-tyrosineNorepinephrine/Epinephrine ---(Monoamine oxidase and Catechol-0-methyl-transferase)---> Metanephrines and VMA

9:1 Norepinephrine: Epinephrine ratioNorepinephrine Primary amine

in CNSMetabolites:-3-methoxy-4-hydroxyphenylglycol (MHPG) = Major metabolite-VMA

Epinephrine Secondary amineMost abundant medullary hormone“Flight or fight hormone”


Metabolites:-Vanillylmandelic acid (VMA) = Major metabolite-Metanephrines-Normetanephrines-HVA

Dopamine Primary amineFrom the decarboxylation of 3,4-Dihydroxyphenylalanine (DOPA)Major metabolite: Homovanillic acid (HVA)

Pheochromocytoma Tumors of the adrenal medullaCatecholaminesClassic “Spells”: tachycardia, headache, chest tightness, sweating, hypertension

Clonidine test Differentiates:Pheochromocytoma (Catecholamines not suppressed) fromNeurogenic hypertension (50% decreased in catecholamines)

Neuroblastoma Norepinephrine (Children) urinary HVA, VMA or both and dopamine

Methods (Catecholamines) Specimen: 24-hr urine and plasma1. Chromatography: HPLC or GC-MS2. RIA: sensitive screening test->2000pg/mL = diagnostic for pheochromocytoma

Estrogens Estrone = Postmenopausal womenEstradiol = Premenopausal women (most potent, secreted by the ovary)Estriol = Pregnancy (placenta)

Markers for Down Syndrome

AFPUnconjugated EstriolhCG Inhibin A

Karyotyping or FISH typing Test for Down syndrome (amniotic fluid)Progesterone Produced mainly by the corpus luteum

Det. whether ovulation has occurredLuteal phase

Tests for menstrual cycle dysfunction and anovulation

EstrogenProgesteroneFSHLH

Tests for female infertility hCGPRLFT4TSHFSHLHEstradiolProgesterone

Pancreas (Exocrine) Digestive enzymes (AMS, LPS)Acinus: functional secretory unit

Pancreas (Endocrine) Hormones:Alpha cells (20-30%) = glucagonBeta cells (60-70%) = insulinDelta cells (2-8%) = somatostatin

hCG Produced by the syncytiotrophoblasts (placenta)Maintain progesterone production by the corpus luteum

Human placental lactogen Stimulates development of mammary gland


(HPL) Increases maternal plasma glucose levelsDiagnosis of intrauterine growth retardation

Gastrin Secreted by G cells (stomach)Stimulates parietal cells to secrete HClStimulus: Amino acidZollinger-Ellison syndromePernicious anemia

Serotonin(5-hydroxytryptamine)

Synthesized by argentaffin cells (GIT)Metabolite: 5-HIAA

5-HIAA Diagnostic marker for carcinoid syndromeTest: Ehrlich’s aldehyde test = (+) purple color

Somatostatin A.k.a. GH-IHInhibitor of GH, glucagon and insulin

1’ amenorrhea Menstruation having never occurred2’ amenorrhea Absence of menses for 6 monthsCushing’s disease Abnormal increased secretion of ACTHCushing’s syndrome Chronic excessive production of cortisol by the adrenal cortex

-Large doses of glucocorticoids-Pituitary tumor (ACTH) = most common cause

Gynecomastia Development of breast tissue in malesHirsutism Excessive hair growth w/ a male distribution pattern in a female

Most common endocrine disorder in womenMullerian agenesis Congenital malformation or absence of the fallopian tubes, uterus or vagina

N-FSH, LH and testosteroneNonthyroidal illness Illness that do not directly involve the thyroid glandSipples syndrome (MEN II) Medullary carcinoma of the thyroid

PheochromocytomaParathyroid adenoma

Stein-Leventhal syndrome Mild hirsutism w/ normal menses to excessive hirsutism w/ amenorrheaThyroid stones A.k.a. thyroid crisis

Life-threateningUncontrolled thyrotoxicosis

Normal Values(Endocrinology)

T3:Adult = 80-200 ng/dLChildren 1-14 y.o. = 105-215 ng/dLT4:Adult = 5.5-12.5 g/dLμNeonate = 11.8-22.6 g/dLμT3U = 25-35%

Therapeutic Drug MonitoringMixed function oxidase (MFO) system

Biochemical pathway responsible for the greatest portion of drug metabolism

Intravenous route 100% bioavailabilityLiberation Drug ReleaseAbsorption Drug Blood (most: by passive diffusion)Distribution Drug TissuesMetabolism Drug Chemical modificationExcretion Drug metabolites excretedBioavailable fraction (f) Fraction of the dose that reaches the bloodVd of a drug Dilution of the drug after it has been distributed in the body


First-pass hepatic metabolism

Drugs Liver Decreased bioavailability

First order elimination Linear relationship bet. the amt. of drug eliminated per hour and the blood level of drug

Pharmacodynamics Relationship bet. drug concentration at the target site and response of the tissues

Pharmacokinetics Relationship bet. drug dose and drug blood levelPharmacogenomics Study of genes that affect the performance of a drug in an individualTherapeutic index Ratio bet. the minimum toxic and maximum therapeutic serum conc.Trough concentration Lowest concentration of a drug obtained in the dosing interval

Drawn immediately (or 30 mins) before the next dosePeak concentration Highest concentration of a drug obtained in the dosing interval

Drawn one hour after an orally administered dose (except digoxin)Cardioactive DrugsClass I Rapid Na+ channel blockers (Procainamide, Lidocaine, Quinidine)Class II Beta receptor blockers (Propanolol)Class III K+ channel blockers (Amiodarone)Class IV Ca2+ channel blockers (Verapamil)Digoxin Tx: CHFLidocaine (Xylocaine) Local anesthetic

1’ product of hepatic metabolism: MEGX (monoethylglycinexylidide)Quinidine Common formulations: Quinidine sulfate and Quinidine gluconateProcainamide (Pronestyl) Hepatic metabolite: NAPA (N-acetylprocainamide)

Toxic effect: reversible lupus-like syndromeDisopyramide Substitute for quinidine

Anticholinergic effectsPropanolol Tx: angina pectorisAmiodarone (Cordarone) Iodine-containing drugVerapamil Tx: angina, hypertension, supraventricular arrhythmiasAntibioticsAminoglycosides Tx: Gram (-) bacterial infections

Nephrotoxic and ototoxicVancomycin Tx: Gram (+) cocci and bacilli

Toxic effects:“Red man syndrome”Nephrotoxic and ototoxic

Antiepileptic DrugsPhenobarbital Long acting barbiturate

Enhances bilirubin metabolismInactive proform: Primidone

Phenytoin (Dilantin) Injectable proform: fosphenytoinValproic acid (Depakene) Tx: petit mal and grand malCarbamazepine (Tegretol) Tx: grand malEthosuximide (Zarontin) Drug of choice for controlling petit mal seizureGabapentin (Neurontin) Similar to neurotransmitter GABAOthers (Antiepileptic) Topiramate

Lamotrigine (Lamictal)Felbamate

Psychoactive DrugsLithium Tx: Bipolar disorders (Manic depression)Tricyclic antidepressantas (TCA)

ImipramineAmitriptyline


DoxepinNortriptylineTradazoneMajor metabolite: Desipramine

Fluoxetine (Prozac) Blocks reuptake of serotoninTx: Obsessive-compulsive disorders

BronchodilatorTheophylline Tx: Asthma and other COPDAnti-inflammatory and Analgesic DrugsSalicylates/Aspirin (Acetylsalicylic acid)

Antiplatelet (inhibits cyclooxygenase)Method: Trinder assay

Acetaminophen (Tylenol) HepatotoxicIbuprofen Lower risk of toxicity than salicylates and acetaminophenNeuroleptics (Antipsychotic major tranquilizers)Neuroleptics Block the action of dopamine and serotonin

Tx: Schizophrenia2 classes:-Phenothiazines (chlorpromazine)-Butyrophenones (haloperidol)Examples:-Risperdal-Olonzapine (Zyprexa)-Quetiapine (Seroquel)-Aripiprazole (Abilify)

Immunosuppressants CyclosporineTacrolimus (FK-506)Rapamycin (Sirolimus)Mycophenolate mofetilLefluamide

Chemotherapeutic agents BusulfanMethotrexate

ToxicologyToxic AgentsAlcohols (%w/v) Common CNS depressants0.01-0.05 No obvious impairment, some changes observable on performance testing0.03-0.12 Mild euphoria, decr. inhibitions, some impairment of motor skills0.09-0.25 Decr. inhibitions, loss of critical judgment, memory impairment, decr. rxn time0.18-0.30 Mental confusion, dizziness, strongly impaired motor skills (slurred speech)0.27-0.40 Unable to stand/walk, vomiting, impaired consciousness0.35-0.50 Coma and possible death≥0.10 Presumptive evidence of driving under influence of alcoholEthanol (Grain alcohol) Most common abused drug

Ethanol Acetic acidMajor metabolic pathway:Ethanol ------(Alcohol Dehydrogenase)------> AcetaldehydeTesting: Use benzalkonium chloride as antiseptic

Methanol (Wood alcohol) Cause blindnessMethanol Formaldehyde Formic acid (liver)

Isopropanol(Rubbing alcohol)

Liver metabolism:Isopropanol Acetone

Ethylene glycol(1,2-ethanediol)

Antifreezing agentEthylene glycol Oxalic acid and glycolic acid


(+) Monohydrate calcium oxalate crystalsCarbon Monoxide Colorless, odorless, tasteless gas

Has 210x greater affinity than O2 for Hgb“Cherry-red” color of the face and bloodSpecimen: EDTA whole bloodMethod: Co-oximetry (HbCO measurement)

Cyanide Binds to iron (ferric and ferrous) containing substances like hemoglobin and cytochrome oxidase“Odor of bitter almonds”Antidote: Sodium thiosulfate, amyl and sodium nitrite

Arsenic “Odor of garlic”“Metallic taste”Hair and nails: “Mees lines”Method: Reinsch test (Flat black)

Cadmium Significant environmental pollutant(+) GGT in urine sample

Lead Blocks D-ALA synthase and Ferrocheletase“Wrist drop or Foot drop” manifestationTx: EDTA and dimercaptosuccinic acid (DMA) – remove leadFree erythrocyte protoporphyrin(+) Basophilic stippling (course)

Mercury Amalgamate: mix or merge w/ other substancesSpecimen:-Whole blood (organic mercury)-Urine (inorganic mercury)Method: Reinsch test (Silvery gray)

Drugs of AbuseOpiates Morphine

CodeineHeroinMethadone

Tranquilizers Diazepam (Valium)Oxazepam

Barbiturates:Sedative Hypnotics

PhenobarbitalPentobarbitalAmobarbital

Dopaminergic pathway stimulants

CocaineBenzoylecgonineAmphetamine

Hallucinogens PhencyclidineLysergic acid diethylamideTetrahydrocannabinolMethaqualone

Amphetamines Increase mental alertness (“Uppers”)MDMA (methylenedioxymethamphetamine) = ecstasyMethamphetamine HCl = shabu

Annabolic steroids Improves athletic performance by increasing muscle massCannabinoids Marijuana and hashishTetrahydrocannabinol (THC)

Psycoactive substance of marijuanaUrinary metabolite: 11-nor-deltatetrahydrocannabinol (THC-COOH)

Cocaine (Crack) Alkaloid saltAdmin: Insufflation of IV or by inhalation/snorting


Derived from coca plant (erythroxylon)Cardiac toxicityProzac: inhibit the action of cocaineUrine metabolite: benzoylecgonine

Opiates From opium poppyHeroinMorphineCodeineMethadoneMajor metabolites: N-acetylmorphine (heroin) and morphineAntagonist: Nalaxone (Narcan)

Phencyclidine(Angel dust or angel hair)

HallucinogenAdmin: Ingestion or inhalationMajor metabolite: Phencyclidine HCl

Sedative hypnotics Barbiturates (Secobarbital, pentobarbital, Phenobarbital)Benzodiazopines: Diazepam (Valium), Lorazepam (Ativan), Chlordiazepoxide (Librium)Major metabolite (barbiturates): Secobarbial

Lysergic acid diethylamide (LSD, Lysergide)

“Undulating vision”“Bad trip” – panic reactions

Methaqualone (Quaalude) Pyramidal signs (Hypertonicity, hyperreflexia, myoclonus)Vitamins

Vitamins Water soluble: B1, B2, B3, B5, B6, B9, B12, Biotin, C, CarnitineFat soluble: A, D, E, K

Vitamin A CN: RetinolDef: Night blindness

Vitamin E CN: TocopherolDef: Mild hemolytic anemia, RBC fragility

Vitamin D2

Vitamin D3

CN: Ergocalciferol, Cholecalciferol (D2), 1,25-dihydroxycholecalciferol (D3)Def: Rickets (young), Osteomalacia (adult)

Vitamin K CN: Phylloquinones, MenaquinonesDef: Hemorrhage

Vitamin B1 CN: ThiamineDef: Beriberi, Wernicke-Korsakoff syndrome

Vitamin B2 CN: RiboflavinDef: Angular stomatitis, dermatitis, photophobia

Vitamin B3 CN: Niacin/Niacinamide/Nicotinic acid/NicotinamideDef: Pellagra (dermatitis, disorientation, weight loss)

Vitamin B5 CN: Panthotenic acidDef: Depressed immune system, muscle weakness

Vitamin B6 CN: Pyridoxine, PyridoxalDef: Facial seborrhea

Vitamin B9 CN: Folic acid, Pteroylglutamic acidDef: Megaloblastic anemia

Vitamin B12 CN: CyanocobalaminDef: Megaloblastic anemia, neurologic abnormalities

Vitamin C CN: Ascorbic acidDef: Scurvy

Biotin Def: DermatitisCarnitine Def: Muscle weakness, fatigue


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1. must to know in clinical chemistry 001