1. must to know in clinical chemistry 001


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1. must to know in clinical chemistry 001

  1. 1. 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 accuracy Difference of >2: not in agreement w/ other labQC 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 analyte Altered proteinPrecision 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 V-mask(CUSUM) Earliest indication of systematic errors (trend)Youden/Twin Plot Compare results obtained from diff. labShewhart Levey-Jennings Graphic representation of the acceptable limits of variationChartTrend Gradual loss of reliability Cause: Deterioration of reagents (Systematic error)Shift Values: one side or either side of the mean Cause: Improper calibration (Systematic error)Outliers Values: far from the main set of values Highly deviating values Random or systematic errorsKurtosis Degree of flatness or sharpnessPrecision Random errorAccuracy Systematic errorRandom error Causes:(Imprecision; -MislabelingIndeterminate) -Pipetting error -Improper mixing of sample and reagents -Voltage/Temperature fluctuation -Dirty optics Parameters: SD and CVSystematic error Causes:(Inaccuracy/Determinate) -Improper calibration -Deterioration of reagents -Contaminated solution -Sample instability/unstable reagent blanks lec.mt 04 |Page | 1
  2. 2. -Diminishing lamp power -Incorrect sample and reagent volume Parameter: MeanMultirule Shewhart Control rules + Control chartprocedureTest 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 development Assessment and monitoring Quality improvementQuality Patient Care Test request forms, clear instruction for patient prep., specimen handling…Reference Range/ Interval At least 120 individuals should be tested in each age and sex categoryRange/ Reference Values 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 arc Xenon lamp Hydrogen lampIR Merst glower Globar (Silicone carbide)Stray light Wavelength outside the band Most common cause of loss of linearityDiffraction gratings Most commonly used monochromator Cutting groovesPrisms 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/ Simplest detectorphotocell/ photovoltaic cell No external voltage For filter photometersPhototube Contains anode and cathode Req external voltagePhotomultiplier tube Most common type lec.mt 04 |Page | 2
  3. 3. Most sensitive UV and visible regionGalvanometer/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  sample One beam  reference soln or blank (corrects for variation in light source intensity)Double-beam in space 2 photodetectors (sample beam and reference beam)Double-beam in time 1 photodetector Monochromatic light  sample cuvet and reference 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: Excitation Lt. source and cuvette: Flame For excited ions (Na+, K+)Cesium and Lithium Internal standards (FEP) Correct variations in flameLithium Preferred internal std Potent antidepressantAAS Meas. light absorbed by atoms dissociated by heat Principle: Dissociation (unionized, unexcited, ground state) Lt. source: Hollow-cathode lamp For unexcited trace metals (Ca++ and Mg++) More sensitive than FEPAtomizer (nebulizer) Convert ions  atomsChopper Modulate the light sourceLanthanum/Strontium Complex with phosphatechloride Avoid calcium interferenceVolumetric (Titrimetric) Unknown sample is made to react with a known solution in the presence of an indicatorTurbidimetry Light blocked Meas. abundant large particles (Proteins) Depend on specimen concentration and particle sizeNephelometry Meas. amt of Ag-Ab complexes Scattered light Depends on wavelength and particle sizeElectrophoresis 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 lec.mt 04 |Page | 3
  4. 4. Isoelectric focusing Molecules migrate through a pH gradient pH = pI For isoenzymes: same size, different chargeDensitometry 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 movesGas chromatography Separation of steroids, barbiturates, blood, alcohol, and lipids Volatile compounds Specimens  vaporized Mobile phase: Inert gasesGas 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 proteins Ex: Dextran and agaroseHydrophobic gel Gel permeation Separation of triglyceride and fatty acid Ex: SephadexIon exchange Separation depends on the sign and ionic charge densitychromatographyPartition chromatography Based on relative solubility in an organic solvent (nonpolar) and an aqueous solvent (polar)Affinity chromatography For lipoproteins, CHO and glycated hemoglobinsAdsorption Based on differences between the adsorption and desorption of solutes at thechromatography surfaces of a solid particleFluorometry/Molecular Det. amt. of lt. emitted by a molecule after excitation by electromagneticLuminescence Spectro. radiation Lt. sources: Mercury arc and Xenon lamp (UV) Lt. detector: Photomultiplier tubes 2 monochromators: Primary filter – selects wavelength absorbed by the solution to be measured Secondary filter – prevents incident light from striking the photodetector Sensitivity: 1000x than spectroQuenching Major disadvantage of fluorometry pH and temperature changes, chemical contaminants, UVL changes lec.mt 04 |Page | 4
  5. 5. 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 thermally 6x stronger than borosilicateVycor (Corning) For high thermal, drastic heat and shock Can be heated to 900OCFlint glass Soda-lime glass + Calcium, Silicon, Sodium oxides Easy to melt For making disposable glasswaresTD: 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 ring Pasteur: w/o consideration of a specific volume Automatic macro-/micropipetsGraduated or measuring Serological: w/ graduations to the tip (blowout)pipet Mohr: w/o graduations to the tip (self-draining) Bacteriologic Ball, Kolmer and Kahn Micropipettes: <1 mLMicropipettes TC pipets: Sahli-Hellige pipet Lang-Levy pipet RBC and WBC pipets Kirk and Overflow pipetsAir displacement pipet Piston: suction Disposable tipPositive 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 Cleaning solution for glasswares(H2SO4 + K2Cr2O4)Continuous flow analyzer Common reaction vessel Air bubbles: separates and cleans Glass coil: mix Examples: “STS” Simultaneous Multiple Analyzer (SMA) Technicon Autoanalyzer II SMACCentrifugal analyzer Acceleration and deceleration of the rotor Advantage: Batch analysis Examples: “RICC” Cobas-Bio (Roche) IL Monarch CentrifiChem lec.mt 04 |Page | 5
  6. 6. RotoChemDiscrete Analyzer Most popular Req. vol: 2-6 μL Uses positive-displacement pipets Run multiple-tests-one-sample-at-a-time Random access capability (STAT) Examples: Vitros Dimension Dade Beckman ASTRA System (4 & 8) Hitachi Bayer Advia Roche Cobas Integra 800 Roche Analytics P Module Automated Clinical Analyzer (ACA) Star (Dade) Dupont ACA Abbott ABA-100 Bichromatic Analyzer ABA-200 VP Analyzer American Monitor KDA Olympus DemandThin-Film Analyzers 4 or 5 layers:(Dry slide technology) -Spreading layer -Scavenger layer - Ascorbate Oxidase -Reagent layer -Indicator layer -Support layer Colored reaction  Reflectance spectrophotometry Examples: “KV2(75)” Kodak Ektachem Vitros 750XRC Vitros 550XRCCarry over Transport of quantity of analyte or rgt from one specimen rxn into another, and contaminating a subsequent oneBatch testing All samples loaded at the same time Single test is conducted on each sampleParallel testing One specimen More than one test is analyzedRandom access testing Any sample Any test Any sequence STATSequential 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 GH Urea Urinary protein (Proteinuria) Fatty acid Testosterone lec.mt 04 |Page | 6
  7. 7. CPK (muscle) Creatinine (muscle) Lactate LH LD (muscle) ACP Aldolase (muscle) AST ALT Ammonia Pyruvate Prolactin Decreased: GlucoseFist clenching Increased: “LPP” Lactate Potassium PhosphateFasting 8-16 hours: Glucose Lipids Lipoproteins Increased: Bilirubin (48 hours) Triglyceride (72 hours)Basal state collection Glucose Cholesterol Triglyceride ElectrolytesDiet Increased: “GLUC2H” Glucose Lipids Urea (High protein diet) Caffeine: increases glucose Catecholamines 5-HIAA (From Serotonin)Turbidity/Lactescence Triglyceride >400mg/dLIcterisia Bilirubin: 25.2 mg/dLIcteric samples Interfere with: "TACGu” Total Protein Albumin Cholesterol GlucoseUpright/supine (lying) Preferred positionposition Patient should be seated/supine at least 20 mins before blood collection to prevent hemodilution or hemoconcentrationSupine  Sitting/Standing Vasoconstriction  Reduced plasma volume Increased: “ECA” Enzymes Calcium AlbuminSitting  Supine Hemoconcentration lec.mt 04 |Page | 7
  8. 8. Increased: “P(u)BLIC” Proteins BUN Lipids Iron CalciumStanding  Supine Hemodilution Decreased: “TLC” Triglycerides Lipoproteins CholesterolProlonged standing Increased: K+ (muscles)Prolonged bedrest Decreased: Albumin (Fluid retention)Tourniquet Recommended: 1 minute applicationProlonged tourniquet app. Hemoconcentration Anaerobiosis Increased: “C2LEA2K” Calcium Cholesterol Lactate Enzymes Ammonia Albumin K+Tobacco smoking (Nicotine) Increased: “TUNG2C3” Triglycerides Urea Nonesterified fatty acid Glucose GH Catecholamines Cortisol CholesterolAlcohol ingestion Increased: “THUG” Triglycerides Hypoglycemia (chronic alcoholism) Uric acid/Urates GGTAmmonia Increases by 100-200μg/L/cigarStress (anxiety) Increased: “LAGIC” Lactate Albumin Glucose Insulin CholesterolDrugs Medications affecting plasma volume can affect protein, BUN, iron, calcium Hepatotoxic drugs: increased liver function enzymes Diuretics: decreased sodium and potassiumDiurnal variation "CA3PI2TG” Cortisol ACTH ACP lec.mt 04 |Page | 8
  9. 9. Aldosterone Prolactin Iron Insulin Thyroxine GH 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 site Not exceed 1 minuteNeedle Bevel up 15-30O angle Length: 1 or 1.5 inch (Butterfly needle: ½ to ¾ inch)After blood collection Cotton  site Apply pressure for 3-5 minutesBP cuff as tourniquet Inflate to 60 mmHgBenzalkonium chloride Disinfectant for ethanol testing(Zephiran) Dilution – 1:750IV line on both arms Discontinue IV for 2 minutes Collect sample below the IV site Initial sample (5mL)  discardIV fluid contamination Increased: Glucose (10% contam. w/ 5% dextrose  increased bld glucose by 500 mg/dL) Chloride Potassium Sodium Decreased: Urea CreatinineRenin 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 lec.mt 04 |Page | 9
  10. 10. -Aldolase -ALT -AST -Albumin -Mg2+ -Phosphorus -Iron -Total protein Affects bilirubin levels Inhibits lipaseRefrigeration/Chilling Required for: “ABCGLRP2”(Low temp) Ammonia Blood gases Catecholamines Gastrin Lactic acid Renin PTH Pyruvate Decreased: LD 4 and 5 Increased: ALPPhotosensitive analytes Bilirubin Beta-carotene Folate Porphyrins Vitamins A and B6Oxalate Insoluble salt 1-2 mg/mL bloodCitrate Non-ionized form 3.2-3.8 g/dL (1:9 ratio)EDTA Chelation 1-2 mg/mL blood Versene: disodium salt Sequestrene: Dipotassium saltFluoride Weakly dissociated calcium component 2 mg/mL blood: anti-glycolytic 10 mg/mL blood: anticoagulantHeparin A.k.a. Mucoitin polysulfuric acid Universal anticoagulant Antithrombin 0.2 mg/mL bloodLithium heparin For glucose, BUN, ionized calcium, electrolyte studies (K+: best) and creatinineOrange top tube Additive: ThrombinRoyal blue top tube Additives: None; Na2EDTA Sodium heparinBrown top tube Lead testingTan top tube Lead testingBlack top tube Additive: Buffered sodium citrate lec.mt 04 |Page | 10
  11. 11. 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 M = % w/v  10Molarity GMWNormality N = _Grams of solute_ EW x volume (L)Equivalent weight (EW) EW = __MW___ valenceTo prepare a normal Grams of solute = Normality x EW x Volume (L)solution of solidsTo convert % w/v to N = w/v  10Normality EWNormality 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  μmol/L)0.01 Conversion factor for phospholipid (g/dL to g/L)2.27 Conversion factor for folateAnalytical reagent (AR) For qualitative and quantitative analysesgrade For accuracy Established by American Chemical Society (ACS) Uses: Trace metal analysis and preparation of standard solutionsUltrapure reagents Additional purification steps Ex: Spectrograde, nanograde, HPLC grade Uses: Chromatography, atomic absorption, immunoassaysChemically Pure (CP) or Indicates that the impurity limitations are not stated lec.mt 04 |Page | 11
  12. 12. Pure Grade Purity is delivered by meas. of melting point or boiling pointTechnical/Commercial In manufacturinggrade Never used in clin. lab. testingUnited States For human consumptionPharmacopoeia (USP) and Not applicable for lab. analysisNational Formulery (NF) Purpose: For drug manufacturingPreparation of reagent Filtration (1st)  Distillation, Ion exchange, Reverse Osmosisgrade waterType I Rgt Water Min. interference Max. water purity Used immediately For ultramicrochemical analyses, measurements of nanogram or subnanogram concentrations, tissue or cell methods (microscopy) and preparation of standard solutions Uses: FEP, AAS, blood gases and pH, enzyme studies, electrolyte testing, HPLC, trace metal and iron studiesType II Rgt Water For clinical laboratory use (hematology, microbiology, immunology, chemistry) For prep. of rgts and QC materialsType III For washing glasswares For urinalysis, parasitology and histologyDistilled 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 Req. manuf. to indicate lot no., physical or biological health hazard of the chem..Health Act (OSHA) rgts, and precautions for safe use and storageCollege of American Recommends that a lab. document culture growth, pH and specific waterPathologists (CAP) resistance on reagent grade waterTests for water purity Microbiological content pH Resistivity Chemical oxygen demand Ammonia Ions MetalsDetergent-contaminated Alkaline pHwaterHard 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 Highly purified(IUPAC) Measured directly to produce a substance of exact known concentrationSecondary standard Low purity Concentration 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 lec.mt 04 |Page | 12
  13. 13. Class B fire Flammable liquids: grease, gasoline, paints, oil Extinguisher: Dry chemical (ABC), carbon dioxide (BC), halon foam (BC)Class C fire Electrical equipment and motor switches Extinguisher: Dry chemical (ABC), Carbon dioxide (BC), halon (BC)Class D fire Flammable metals: mercury, magnesium, sodium, lithium Extinguisher: Metal X Fought be fire fighters onlyClass E fire Detonation (Arsenal fire) Allowed to burn out and nearby materials protectedStandard 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 To disinfect and clean bench topsbleach (10%) In contact with the area for at least 20 minutes HBV: 10 minutes HIV: 2 minutesPoisonous vapors Chloroform Methanol Carbon tetrachloride Bromide Ammonia Formaldehyde MercuryFlammable and Acetonecombustible solvents Ethanol Toluene Methanol Xylene Benzene Isopropanol HeptaneFlammable 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” Glucagon ACTH GH lec.mt 04 |Page | 13
  14. 14. Cortisol Human Placental Lactogen Epinephrine ThyroxineHyperglycemia Electrolyte Imbalance:(≥126 mg/dL) Decreased: Sodium, Bicarbonate Increased: PotassiumHypoglycemia 50-55 mg/dL = Symptoms ≤50 mg/dL = DiagnosticWhipple’s triad Low blood glucose concentration(Hypoglycemia) Typical symptoms Symptoms alleviated by glucose administration6:1 Ratio of BHA to AA in severe DM (Normal = 1:1)Type 1 DM IDDM Juvenile Onset Brittle Ketosis-prone 80-90% reduction of beta-cells  Symptomatic Type 1 DM HLA-DR3 and DR4 (+) Glutamic acid decarboxylase (GAD65) (+) Insulin autoantibodies (+) Microalbuminuria: 50-200 mg/24 hours = Diabetic nephropathy (-) C-peptideComplications of Type I DM Microvascular disorders: Nephropathy Neuropathy RetinopathyType 2 NIDDM Adult type/Maturity Onset Stable Ketosis-resistant Receptor-deficient Insulin resistance: relative insulin deficiency Strong genetic predisposition Geneticist’s nightmare If untreated  glucose: >500 mg/dL  nonketotic hyperosmolar comaGestational DM Screening: 1hr GCT (50g) – bet. 24 and 28 weeks of gestation Confirmatory: 3-hr GTT (100g) Infants: at risk for respiratory distress syndrome, hypocalcemia, hyperbilirubinemia After giving birth, evaluate 6-12 weeks postpartum Converts to DM w/in 10 years in 30-40% of casesOGTT (GDM) FBS = ≥95 mg/dL 1-Hr = ≥ 180 mg/dL 2-Hr = ≥ 155 mg/dL 3-Hr = ≥ 140 mg/dL GDM = 2 plasma values of the above glucose levels are exceededImpaired fasting glucose FBS = 100-125 mg/dL(Pre-diabetes)Impaired glucose tolerance FBS = <126 mg/dL 2-Hr OGTT = 140-199 mg/dL lec.mt 04 |Page | 14
  15. 15. FBS WB = 15% lower than in serum or plasma VB = 7 mg/dL lower than capillary and arterial bloodCSF glucose 60-70% of the plasma glucosePeritoneal fluid glucose Same with plasma glucosePlasma glucose increases Fasting: 2 mg/dL/decadew/ age Postprandial: 4 mg/dL/decade Glucose challenge: 8-13 mg/dL/decadew/in 1 hour Separate serum/plasma from the cells(Preferably w/in 30 mins)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 Ferricyanide ---(Glucose)--> Ferrocyanidemethod (Hagedorn-Jensen) (Yellow) (Colorless)Ortho-toluidine Schiff’s base(Dubowski method)Glucose oxidase Measures beta-D-glucose (65%)Mutarotase Converts alpha-D-glucose (35%) to beta-D-glucose (65%)NADH/NADPH Absorbance at 340nmPolarographic glucose Consumption of oxygen on an oxygen-sensing electrodeoxidase O2 consumption α glucose concentrationHexokinase method Most specific method Reference method Uses G-6-PDG-6-PD Most specific enzyme rgt for glucose testingInterfering substances False-decreased(Glucose oxidase) Bilirubin Uric acid AscorbateHemolysis (>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 minsIVGTT For patients with gastrointestinal disorders (malabsorption) Glucose: 0.5 g/kg body weight Given w/in 3 mins 1st blood collection: after 5 mins of IV glucoseRequirements for OGTT Ambulatory Fasting: 8-14 hours Unrestricted diet of 150g CHO/day for 3 days Do not smoke or drink alcoholGlucose load 75 g = adult (WHO std) 100 g = pregnant 1.75 g glucose/kg BW = childrenHbA1c 2-3 months Glucose = beta-chain of HbA1 lec.mt 04 |Page | 15
  16. 16. 1% increase in HbA1c = 35 mg/dL increase in plasma glucose 18-20% = prolonged hyperglycemia 7% = cutoff Specimen: EDTA whole blood Test: Affinity chromatography (preferred)IDA and older RBCs High HbA1cRBC lifespan disorders Low HbA1cFructosamine 2-3 weeks(Glycosylated albumin/ Useful for patients w/ hemolytic anemias and Hgb variantsplasma protein ketoamine) Not used in cases of low albumin Specimen: SerumGalactosemia Congenital deficiency of 1 of 3 enzymes in galactose metabolism Galactose-1-phosphate uridyl transferase (most common) Galactokinase Uridine diphosphate galactose-4-epimeraseEssential fructosuria Autosomal recessive Fructokinase deficiencyHereditary fructose Defective fructose-1,6-biphosphate aldolase B activityintoleranceFructose-1,6-biphosphate Failure of hepatic glucose generation by gluconeogenic precursors such asdeficiency lactate and glycerolGlycogen Storage Disease Autosomal recessive Defective glycogen metabolism Test: IVGTT (Type I GSD)Ia = Von Gierke Glucose-6-Phosphatase deficiency (most common worldwide)II = Pompe Alpha-1,4-glucosidase deficiency (most common in the Philippines)III = Cori Forbes Debrancher enzyme 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 AcetoacetatetestNitroprusside 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 RBS = <140 mg/dL(Carbohydrates) FBS = 70-100 mg/dL HbA1c = 3-6% Fructosamine = 205-285 μmol/L 2-Hr PPBS = <140 mg/dL GTT: 30 mins = 30-60 mg/dL above fasting lec.mt 04 |Page | 16
  17. 17. 1-Hr = 20-50 mg/dL above fasting 2-Hr = 5-15 mg/dL above fasting 3-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 lecithin Not derived from glycerol but from sphingosine (amino alcohol)Forms of phospholipids 70% Lecithin/Phosphatidyl choline 20% Sphingomyelin 10% CephalinTLC + Densitometric Method for L/S ratioquantitationMicroviscosity Measured by fluorescence polarizationCholesterol Not a source of fuel Not affected by fasting 70% Cholesterol ester (plasma/serum) 30% Free cholesterol (plasma/serum and RBC)LCAT Esterification of cholesterolApo A-1 Activator of LCATCholesterol increases after 2 mg/dL/year between 50 and 60 years oldthe age of 50Liebermann Burchardt Cholestadienyl Monosulfonic acid Green end colorSalkowski Cholestadienyl Disulfonic acid Red end colorColor developer mixture Glacial acetic acid(Cholesterol) Acetic anhydride Conc. H2SO4One-step method Colorimetry (Pearson, Stern and Mac Gavack)Two-step method Color. + Extraction (Bloor’s)Three-step method Color. + Extract. + Saponification (Abell-Kendall)Four-step method Color. +Extract. + Sapon. + Precipitation (Schaenheimer Sperry, Parekh and Jung)Abell, Levy and Brodie mtd CDC reference method for cholesterol:(Chemical method) -Hydrolysis/saponification (Alc. KOH) -Hexane extraction -Colorimetry (Liebermann-Burchardt)Triglycerides Most insoluble lipid Main storage lipid in man (adipose tissue) – 95% Fasting: 12 hoursTriglyceride increases after 2 mg/dL/year between 50 and 60 years oldthe age of 50Van Handel & Zilversmith Chromotropic acid(Colorimetric) (+) Blue color compoundHantzsch Condensation Diacetyl acetone(Fluorometric) (+) Diacetyl lutidine compoundModified Van Handel and CDC reference method for triglycerides:Zilversmith -Alkaline hydrolysis(Chemical method) -Chloroform extraction  extract treated w/ silicic acid -Color reaction w/ chromotropic acid – meas. HCHO lec.mt 04 |Page | 17
  18. 18. (+) Pink coloredFatty acids Short chain = 4-6 C atoms Medium chain = 8-12 C atoms Long chain = >12 C atoms Saturated = w/o double bonds Unsaturated = w/ double bonds Substrate for gluconeogenesis Most is bound to albuminPalmitic acid 16:0Stearic acid 18:0Oleic acid 18:1Linoleic acid 18:2Arachidonic acid 20:4Lipoprotein lipase Hydrolyzes TAG in lipoproteins, releasing fatty acid and glycerol(Lipemia clearing factor)Hepatic lipase Hydrolyzes TAG and phospholipids from HDL Hydrolyzes lipids on VLDL and IDLEndothelial 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 intestine SG: <0.95 80-95% TAG (exogenous) Apo B-48 (Major) EP: OriginVLDL Secreted by the liver SG: 0.95-1.006 65% TAG (endogenous) Apo B-100 (Major) EP: pre-betaLDL Synthesized by the liver SG: 1.006-1.063 50% CE Apo B-100 (Major) EP: beta Cholesterol transport: LiverTissues Target of cholesterol lowering therapy Better marker for CHD riskHDL Smallest but dense SG: 1.063-1.21 45-55% protein 26-32% phospholipid Apo A-1 (Major) EP: alpha Produced by the liver and intestine Reverse cholesterol transport: TissueLiverIDL Product of VLDL catabolism Seen in Type 3 hyperlipoproteinemia (Apo E-III def.; beta-VLDL) SG: 1.006-1.019Lp(a) Sinking pre-beta lipoprotein SG: 1.045-1.080 lec.mt 04 |Page | 18
  19. 19. Apo B-100 EP: pre-beta (VLDL) UC: like LDL Independent risk factor for atherosclerosisLpX Found in obstructive jaundice (cholestasis) and LCAT deficiency 90% FC and PL Apo C and albuminBeta-VLDL Floating beta-lipoprotein SG: <1.006 EP: beta (LDL) UC: like VLDL Found in type 3 hyperlipoproteinemia (Apo E-III def; IDL) Rich in cholesterol content than VLDLLipoprotein methodologies Specimen: sample from serum separator tubes (preferred) EDTA plasma: choice for research studies of LPP fractions Fasting state: TAG  VLDL Nonfasting state: TAG  CMUltracentrifugation Reference method for LPP quantitation Reagent: Potassium bromide (SG: 1.063) Ultracentrifugation of plasma for 24 hours Expressed in Svedberg unitsElectrophoresis Electrophoretic pattern: (+) HDL VLDL  LDL  CM (Origin) (-) Agarose gel: sensitive medium VLDL: migrates w/ alpha2-globulin (pre-beta)Chemical precipitation Uses polyanions (heparin and divalent cations) and polyethylene glycol Dextran sulfate-Mg2+ Heparin-Mn2+3-step procedure: CDC Reference method for HDLUltracentrifugationPrecipitationAbell-Kendall assayBeta quantification + Method for LDLUltracentrifugation + Sample: EDTA plasmaChemical precipitationImmunoturbidimetric 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/dLDe 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 lec.mt 04 |Page | 19
  20. 20. Abetalipoproteinemia Autosomal recessive(Basses-Kornzweig syn.) Defective apo B synthesis Deficient fat soluble vitaminsNiemann-Pick disease Sphingomyelinase deficiencyTangier’s disease Deficiency of HDL (1-2 mg/dL) Defects in the gene for the ABCA1 transporterLPL deficiency TAG = 10,000 mg/dL(Chylomicronemia) Do not develop premature coronary disease (CM are not atherogenic) Abdominal pain and pancreatitisLCAT deficiency Fish-eye disease Low HDLTay-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 xanthomasType 4 Hypertriglyceridemia Increased: VLDL (TAG)Type 5 Increased: VLDL (Endo.TAG), CM (Exo.TAG)Normal Values Cholesterol:(Lipids) Desirable = <200 mg/dL Borderline high = 200-239 mg/dL High = >240 mg/dL Triglycerides: Desirable = <150 mg/dL Borderline high = 150-199 mg/dL High = 200-499 Very high = >500 mg/dL HDL: Low = <40 mg/dL (Cutoff) High = >60 mg/dL LDL: Optimal = <100 mg/dL Near/above optimal = 100-129 mg/dL Borderline high = 130-159 mg/dL High = 160-189 mg/dL Very high = >190 mg/dL ProteinsProteis First rank of importanceProteins Amphoteric: positive and negative charges Effective blood buffers Synthesized by the liver except immunoglobulins (plasma cells) Provide 12-20% of total daily body energy requirement Composed of 50-70% of the cell’s dry weight lec.mt 04 |Page | 20
  21. 21. Primary structure Amino acid sequence Det. the identity of protein, molecular structure, function binding capacity, recognition abilitySecondary structure Winding of polypeptide chain Specific 3-D conformations: alpha-helix, beta-pleated sheath, bend formTertiary structure Actual 3-D configuration Folding pattern Physical and chemical properties of proteinsQuarternary 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-CoA Leucine LysineSimple proteins Hydrolysis  Amino acids Fibrous: fibrinogen, troponins, collagen Globular: hemoglobin, plasma proteins, enzymes, peptide hormonesConjugated proteins Protein (apoprotein) + nonprotein moiety (prosthetic group) Metalloproteins: ferritin, ceruloplasmin, hemoglobin, flavoproteins Lipoproteins: VLDL, HDL, LDL, CM Glycoproteins: haptoglobin, alpha1-antitrypsin (10-40% CHO) Mucoproteins or proteoglycans: Mucin (CHO > CHON) Nucleoproteins: Chromatin (combined w/ nucleic acids)Nitrogen balance Balance bet. anabolism and 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 activity 90% of alpha1-globulin bandAlpha1-fetoprotein Gestational marker Tumor marker: hepatic and gonodal cancers Screening test for fetal conditions (Spx: maternal serum) Amniotic fluid: confirmatory test Increased: Hepatoma, spina bifida, neural tube defects Decreased: Down Syndrome (Trisomy 21)Alpha1-acid glycoprotein/ Low pI (2.7)orosomucoid Negatively charged even in acid solutionAlpha1-antichymotrypsin Acute phase reactant Binds and inactivates PSA Increased: Alzheimer’s disease, AMI, infection, malignancy, burnsHaptoglobin (alpha2) Acute phase reactant Binds free hemoglobin (alpha chain)Ceruloplasmin (alpha2) Copper binding (6-8 atoms of copper are attached to it) Has enzymatic activities lec.mt 04 |Page | 21
  22. 22. Decreased: Wilson’s disease (copper  skin, liver, brain, cornea [Kayser- Fleisher rings])Alpha2-macroglobulin Larges major nonimmunoglobulin protein Increased: Nephrotic syndrome (10x) Forms a complex w/ PSAGroup-specific component Affinity w/ vitamin D and actin(Gc)-globulin (bet. alpha1and alpha2)Hemopexin (beta) Binds free hemeBeta2-microglobulin HLA Filtered by glomeruli but reabsorbedTransferrin/Siderophilin Negative acute phase reactant(beta) Major component of beta2-globulin fraction Pseudoparaproteinemia in severe IDA Increased: Hemochromatosis (bronze-skin), IDAComplement (beta) C3: majorFibrinogen (bet. beta and Acute phase reactantgamma) Between beta and gamma globulinsCRP (gamma) General scavenger molecule Undetectable in healthy individuals hsCRP: warning test to persons at risk of CADImmunoglobulins (gamma) Synthesized by the plasma cells IgG>IgA>IgM>IgD>IgEMyoglobin Marker: Ischemic muscle cells, chest pain (angina), AMITroponins Most important marker for AMITnT (Tropomyosin-binding Specific for heart musclesubunit) Det. unstable angina (angina at rest)TnI (Inhibitory subunit or Only found in the myocardiumActin-binding unit) Greater cardiac specificity than TnT Highly specific for AMI 13x more abundant in the myocardium than CK-MB Very sensitive indicator of even minor amount of cardiac necrosisTnC 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 Myoglobinuria Bence-Jones proteinuriaPostrenal proteinuria Urinary tract infection, bleeding, malignancyMicroalbuminuria Type 1 DM Albumin excretion ≥30 mg/g creatinine (cutoff: DM) but ≤300 mg/g creatinine Microalbuminuria: 2 out of 3 specimens submitted are w/ abnormal findings (w/in 6 months)CSF Oligoclonal banding 2 or more IgG bands in the gamma region: Multiple sclerosis Encephalitis Neurosyphilis Guillain-Barre syndrome Neoplastic disorders lec.mt 04 |Page | 22
  23. 23. Serum Oligoclonal banding Leukemia Lymphoma Viral infectionsAlkaptonuria Ochronosis (tissue pigmentation)Homocystinuria Impaired activity of cystathione beta-synthetase Elevated homocysteine and methionine in blood and urine Screen: Modified Guthrie test (Antagonist: L-methionine sulfoximine)MSUD Markedly reduced or absence of alpha-ketoacid decarboxylase 4 mg/dL of leucine is indicative of MSUD Screen: Modified Guthrie test (Antagonist: 4-azaleucine) Diagnostic: Amino acid analysis (HPLC)PKU Deficiency of tetrahydrobiopterin (BH4)  elevated blood phenylalanineNormal Values Total protein = 6.5-8.3 g/dL(Proteins) Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL Kidney Function TestsTests for GFR Clearance: -Inulin clearance -Creatinine clearance -Urea clearance Phenolsulfonphthalein dye test Cystatin CTests for Renal Blood Flow BUN Creatinine Uric acidTests Measuring Tubular Excretion:Function -Para-amino hippurate test (Diodrast test) -Phenolsulfonphthalein dye test Concentration: -Specific gravity -OsmolalityGFR Decreases by 1.0 mL/min/year after age 20-30 years 150 L of glomerular filtrate is produced dailyInulin clearance Reference methodCreatinine clearance Best alternative method Measure of the completeness of a 24-hour urine collection Excretion: 1.2-1.5 g creatinine/dayUrea clearance Demonstrate progression of renal disease or response to therapyCystatin C Low MW protease inhibitor FilteredNot secretedCompletely reabsorbed (PCT) Indirect estimates of GFR Its presence in urine denotes damage to PCTBUN Synthesized from Ornithine or Kreb’s Henseleit cycle First metabolite to elevate in kidney diseases Better indicator of nitrogen intake and state of hydration2.14 BUN  Urea (mg/dL)Fluoride or citrate Inhibit ureaseThiosemicarbazide Enhance color development (BUN mtd)Ferric ionsDiacetyl monoxime method Yellow diazine derivativeUrease method Routinely used Urease: prepared from jack beans lec.mt 04 |Page | 23
  24. 24. Urea ---(Urease)--> NH4 + Berthelot reagent (Measure ammonia)Coupled urease Glutamate dehydrogenase method UV enzymatic methodIsotope dilution mass Reference methodspectrometry For research purposesNPN 45% Urea 20% Amino acid 20% Uric acid 5% Creatinine 1-2% Creatine 0.2% AmmoniaCreatinine Derived from alpha-methyl guanidoacetic acid (creatine) Produced by 3 amino acids (methionine, arginine, lysine) Most commonly used to monitor renal functionEnzymatic methods Creatinine Aminohydrolase – CK method(Creatinine) Creatinase-Hydrogen Peroxide method – benzoquinonemine dye (red) Creatininase (a.k.a. creatinine aminohydrolase)Direct Jaffe method Formation of red tautomer of creatinine picrateInterferences (Direct Jaffe) Falsely increased: Ascorbate Glucose Uric acid Alpha-keto acidsFolin Wu Method (+) Red orange tautomerLloyd’s or Fuller’s Earth True measure of creatininemethod Sensitive and specific Uses 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, CHF Increased: BUN Normal: CreatinineRenal azotemia True renal disease Decreased GFR Striking BUN level but slowly rising creatinine value BUN = >100 mg/dL Creatinine = >20 mg/dL Uric acid = >12 mg/dLPost renal azotemia Urinary tract obstruction Decreased GFR Nephrolithiasis, cancer or tumors of GUT Creatinine = normal or slightly increasedUremia Marked elevation of urea, accompanied by acidemia and electrolyte imbalance (K+ elevation) of renal failure Normocytic, normochromic anemia Uremic frost (dirty skin) Edema lec.mt 04 |Page | 24
  25. 25. Foul breath Urine-like sweatUric acid From purine (adenine and guanine) catabolism Excretion: 1g/dayHyperuricemia -Gout -Increased nuclear metabolism (leukemia, lymphoma, MM, polycythemia, hemolytic and megaloblastic anemia) – Tx: Allopurinol -Chronic renal disease -Lesch-Nyhan syndrome (HGPRT deficiency)Hypouricemia Fanconi’s syndrome Wilson’s disease Hodgkin’s diseaseMethods (Uric acid) Stable for 3 days Potassium oxalate cannot be used Major interferences: Ascorbate and bilirubinPhosphotungstic acid mtd Uric acid + Phosphotungstic acid ---(NaCN/NaCO3)--> Tungsten blue + AllantoinNaCN Folin Newton Brown BenedictNaCO3 Archibald Henry CarawayLagphase Incubation period after the addition of an alkali to inactivate non-uric acid reactantsUricase method Simplest and most specific method Candidate reference method Uric acid (Absorbance at 293nm) ---[Uricase]--> Allantoin (No absorbance) Decrease in absorbance α uric acid concentrationPara-amino hippurate test Measures renal plasma flow Reference method for tubular functionPhenolsulfonphthalein dye Measures excretion of dye proportional to renal tubular masstest 6 mg of PSP is administered IVConcentration tests Collecting tubules and loops of Henle Specimen: 1st morning urineSpecific gravity Affected by solute number and mass SG >1.050: X-ray dye and mannitol 1.010 = SG of ultrafiltrate in Bowman’s spaceOsmolality Total number solute particles present/kg of solvent (moles/kg solvent) Affectted only by number of solutes present Urine osmolality = due to urea Serum osmolality = due to sodium and chloride Det. by Colligative properties: Freezing point (incr. osm. = decr. FP) Vapor pressure (incr. osm. = decr. VP) Osmotic pressure (incr. osm. = incr. OP) Boiling point (incr. osm. = incr. BP)Direct methods Freezing point osmometry = popular method(Osmolality) Vapor pressure osmometry (Seebeck effect)Incr. plasma osmolality Incr. vasopressin (H2O reabsorption)  decr. plasma osmolalityTubular failure Increased: BUN, creatinine, calcium Decreased: Phosphate lec.mt 04 |Page | 25
  26. 26. Osmolal gap Difference between measured and calculated osmolality Sensitive indicator of alcohol or drug overdoseOsmolal gap: >12 mOsm/kg DKA Drug overdose Renal failureNormal Values Creatinine Clearance:(Kidney Function Tests) Male = 85-125 mL/min Female = 75-112 mL/min BUN = 8-23 mg/dL Creatinine = 0.5-1.5 mg/dL Uric acid: Male = 3.5-7.2 mg/dL Female = 2.6-6.0 mg/dL Renal plasma flow (PAH) = 600-700 mL/min Renal blood flow (PSP) = 1200 mL/min SG = 1.005-1.030 Osmolality: Serum = 275-295 mOsm/kg Urine (24-hr) = 300-900 mOsm/kg [<290 mOsm/kg = kidney damage] Urine osmolality: Serum osmolality = 1:1 to 3:1 [>1:1 = Glomerular disease] [1.2:1 = loss of renal concentrating ability] [<1:1 = Diabetes Insipidus] Liver Function 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 Drugsmetabolism Ammonia  Urea  ExcretedExcretory and Secretory Bile acids: cholic acid and chenodeoxycholic acidfunctions Bile salts: bile acids + amino acids (glycine and taurine)Storage function Vitamins GlycogenTest measuring the Hepatic Total Protein Determination:Synthetic Ability -Kjeldahl method -Biuret method -Folin-Ciocalteu (Lowry) method -UV absorption method -Electrophoresis -Refractometry -Turbidimetric and Nephelometric methods -Salt fractionation Prothrombin Time (Vitamin K Response Test)Test measuring Bilirubin Assay:Conjugation/Excretion -Evelyn and Malloy methodFunction -Jendrassik and Grof Bromsulfonphthalein (BSP) Dye Excretion test lec.mt 04 |Page | 26
  27. 27. Test for Detoxification Enzyme tests: ALP, AST, ALT, 5’NT, GGT, OCT, LAP, LDHFunction Ammonia: -Kjeldahl (Digestion) method -Nesslerization reaction -Berthelot reactionPlasma protein 0.2-0.4 g/dL higher than serum due to fibrinogenKjeldahl (Digestion) mtd Standard reference method Measurement of nitrogen content Serum + Tungstic acid  PFF 1g N2 = 6.54g protein 15.1-16.8% = N2 content of proteins Rgt: H2SO4 End product: NH3Biuret method Most widely used method (IFCC recommended) Req. at least 2 peptide bonds and an alkaline medium Rgts: Alkaline CuSO4 Rochelle salt (NaK Tartrate) NaOH KI End product: Violet color (545nm)Folin-Ciocalteu (Lowry) Highest analytical sensitivitymethod Oxidation of phenolic compounds (tyrosine, tryptophan, histidine) Rgts: Phenol (or phosphotungstic-molybdic acid) Biuret (color enhancer) End product: Blue colorElectrophoresis 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- Inflammationglobulin band spikesPolyclonal 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 SSAnephelometric methods TCASalt fractionation Salt: Sodium sulfateAlbumin Soluble: Water Moderately concentrated salt solution Concentrated salt solution Insoluble: Hydrocarbon solvents Highly concentrated salt solution Saturated salt solution lec.mt 04 |Page | 27
  28. 28. Globulin Soluble: Hydrocarbon solvents Weak salt solution Insoluble: Water Saturated salt solution Concentrated salt solutionProthrombin 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 inflammationBilirubin Derived from hemoglobin myoglobin, catalase and cytochrome oxidaseHeme oxygenase Protoporphyrin  BiliverdinBiliverdin reductase Biliverdin B1Urobilinogen Deconjugated bilirubinBilirubin 1 Non-polar bilirubin Free/Slow bilirubinBilirubin 2 Polar bilirubin One-minute/prompt bilirubin Regurgitative bilirubinDelta bilirubin Bilirubin tightly bound to albumin Delta bilirubin = TB-DB+IBJaundice Bilirubin >2 or 3 mg/dLPre-hepatic jaundice Hemolytic B1 = increased B2 = normal UG = increased UB = negativeHepatic jaundice Hepatocellular B1 = increased B2 = increased UG = increased UB = positive ALT = increased AST = increasedPost-hepatic jaundice Obstructive B1 = normal B2 = increased UG = decreased/negative UB = positive lec.mt 04 |Page | 28
  29. 29. ALP = increased GGT = increased Cholesterol = increasedGilbert’s syndrome Bilirubin transport deficit (uptake) B1 = increased B2 = decreasedCrigler-Najjar syndrome Conjugation deficit Type I = total UDPGT deficiency Type II = partial UDPGT deficiency B1 = increased B2 = decreased Danger: Kernicterus Bile is colorlessDubin-Johnson syndrome & Bilirubin excretion deficitRotor syndrome Blockade of excretion into the canaliculi TB = increased B2 = increasedLucey-Driscoll syndrome Circulating inhibitor of bilirubin conjugation B1 = increasedMethods (Bilirubin) Free from hemolysis and lipemia Store in the dark Measured ASAP or w/in 2-3 hoursVan 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 azobilirubin Affected by hemolysisJendrassik and Grof Candidate reference method Accelerator: Caffeine sodium benzoate Buffer: Sodium acetate Ascorbic acid: terminates the initial reaction and destroys the excess diazo rgt Not falsely elevated by hemolysis Total bilirubin is measured 15 minutes after adding methanol or caffeine solnBilirubin 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 method Collection: -After 45 mins (+/- 5% dye retention)Ammonia From deamination of amino acids Elevated levels are neurotoxic and often associated w/ encephalopathy and acetaminophen poisoning Diagnosis of hepatic failure and Reye’s syndrome In severe liver disorder: NH3  circulation  brain (conv. to glutamine)  increases pH  compromise the Kreb’s cycle  Coma due to lack of ATP for the brain lec.mt 04 |Page | 29
  30. 30. Methods (Ammonia) Specimen: Heparin or EDTA plasma Fasting is required Avoid smoking Prolonged standing of specimen: increased NH3 due to deamination Place on iced water immediately Avoid hemolysisKjeldahl (Digestion) Specimen  PFFmethod N2 ----------(hot conc. H2SO4 + CuSO4 + Hg + Selenium)----------> NH3Nesslerization 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 Total protein = 6.5-8.3 g/dL(Liver Function Tests) Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL α1-globulin = 0.1-0.3 g/dL α2-globulin = 0.6-1.0 g/dL β-globulin = 0.7-1.1 g/dL γ-globulin = 0.8-1.6 g/dL Total bilirubin = 0.2-1.0 mg/dL Indirect bilirubin = 0.2-0.8 mg/dL Direct bilirubin = 0-0.2 mg/dL Urobilinogen: Urine = 0.1-1.0 Ehrlich units/2hrs (or 0.54 Ehrlich units/day) Stool = 75-275 Ehrlich units/100g feces (or 75-400 Ehrlich units/24hrs) Ammonia = 19-60 μg/dL 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 =  VelocityActivators Inorganic ions Alters spatial configuration of the enzyme for proper substrate binding Ex. Ca2+ (#1 activator), Zn2+ (LDH), Cl- (AMS), Mg2+ (CK, ALP)Metalloenzymes Inorganic ion attached to a molecule Ex. Catalase, cytochrome oxidaseInhibitors 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 lec.mt 04 |Page | 30
  31. 31. Temperature 37’C = optimum temperature for enzyme activity Temperature = Reaction rate (movement of molecules)40-50’C Denaturation of enzymes60-65’C Inactivation of enzymesTemperature coefficient For every 10OC increase in temperature, there will be a two-fold increase in(Q10) enzyme activitypH Most physiologic reactions occur in the pH range of 7-8Storage Enzymes: -20’C = for longer period of time Substrate and Coenzymes: 2-8’C LDH (LD4 & 5): Room temperatureHemolysis Mostly increases enzyme concentrationLactescence or milky Decreases enzyme concentrationspecimenEnzyme nomenclature 1st digit: classification 2nd and 3rd digits: subclass 4th digit(s): serial numberEnzyme classification “OTHLIL” Oxidoreductases Transferases Hydrolases Lyases Isomerases LigasesOxidoreductases Redox reaction Dehydrogenases: -Cytochrome oxidase -LDH -MDH -Isocitrate dehydrogenase -G-6-PDTransferases Transfer of a chemical group other than hydrogen from 1 substrate to another Kinases, Transaminases, Aminotransferases: -CK -GGT -AST -ALT -OCTHydrolases Hydrolysis/splitting by addition of water Esterases: -ACP -ALP -CHS -LPS Peptidases: -Trypsin -Pepsin -LAP Glycosidases: -AMS -GalactosidasesLyases Removal of groups w/o hydrolysis (product contains double bonds) Aldolase lec.mt 04 |Page | 31
  32. 32. Decarboxylases: -Glutamate decarboxylase -Pyruvate decarboxylase -Tryptophan decarboxylaseIsomerases Intramolecular arrangements Glucose phosphate isomerase Ribose phosphate isomeraseLigases Joining of 2 substrate molecules SynthasesActive site Water-free cavity Where the substrate interactsAllosteric site Cavity other than the active site May bind regulatory moleculesProsthetic group Coenzyme that is bound tightly to the enzymeHoloenzyme Apoenzyme + Prosthetic groupZymogen/proenzyme Inactive form of enzymeEmil Fisher’s/Lock and Key Shape of the key (substrate) must fit into the lock (enzyme)theoryKochland’s/Induced fit Based on the substrate binding to the active site of the enzymetheory Acceptable theoryEnzyme kinetics Enzymes catalyze reactions by lowering the activation energy level that the substrate must reach for the reaction to occurAbsolute 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 Change in substrate concentrationactivity Change in product concentration Change in coenzyme concentrationInternational 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 405nm Electrophoresis: (+) Liver  Bone (Regan)  Placenta  Intestine (-) Heat fractionation: (Δ Stable) Regan  Placenta  Intestine  Liver  Bone (Δ Labile)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 recommended 2. Bessy, Lowry and Brock (PNPP) 3. Bodansky, Shinowara, Jones, Reinhart = BGP (beta glycerophosphate) 4. King and Armstrong = PP (phenylphosphate) 5. Klein, Babson & Read = Buffered PPP (phenolphthalein phosphate) 6. Huggins and Talalay = PPDP (phenolphthalein diphosphate) lec.mt 04 |Page | 32