Must to Know in Clinical Chemistry

MUST TO KNOW IN CLINICAL CHEMISTRY(From CC by Rodriguez) Quality Control Practicability Method is easily repeated Reliability Maintain accuracy and precision Intralab/Interlab QC Daily monitoring of accuracy and precision Interlab/External QC Proficiency testing (Reference lab) Long-term accuracy Difference of >2: not in agreement w/ other lab QC materials Available for a min. of 1 yr Bovine control materials Preferred (Human: biohazard) Not for immunochem, dye-binding and bilirubin Matrix effect Improper product manufacturing Unpurified analyte Altered protein Precision study First step in method evaluation Nonlab. Personnel 29% of errors (lab results) SD Dispersion of values from the mean CV Index of precision Relative magnitude of variability (%) Variance SD2 Measure of variability Inferential statistics Compare means or SD of 2 groups of data T-test Means of 2 groups of data F-test SD of 2 groups of data Cumulative Sum Graph (CUSUM) V-mask Earliest indication of systematic errors (trend) Youden/Twin Plot Compare results obtained from diff. lab Shewhart Levey-Jennings Chart Graphic representation of the acceptable limits of variation Trend Gradual loss of reliability Cause: Deterioration of reagents (Systematic error) Shift Values: one side or either side of the mean Cause: Improper calibration (Systematic error) Outliers Values: far from the main set of values Highly deviating values Random or systematic errors Kurtosis Degree of flatness or sharpness Precision Random error Accuracy Systematic error Random error Causes: (Imprecision; Indeterminate) -Mislabeling -Pipetting error -Improper mixing of sample and reagents -Voltage/Temperature fluctuation -Dirty optics Parameters: SD and CV Systematic error Causes: (Inaccuracy/Determinate) -Improper calibration -Deterioration of reagents -Contaminated solution -Sample instability/unstable reagent blanks -Diminishing lamp power lec.mt 04 |Page | 1 -Incorrect sample and reagent volume Parameter: Mean Multirule Shewhart procedure Control rules + Control chart Test method Westgard: at least 40 samples Reference method Westgard: preferably 100 samples Analytical Run Control and patient specimens assayed, evaluated, and report together Physiologic Limit Referred to as absurd value POCT Performed by nonlab personnel Quality Assurance Tripod: Program development Assessment and monitoring Quality improvement Quality Patient Care Test request forms, clear instruction for patient prep., specimen handling… Reference Range/ Interval At least 120 individuals should be tested in each age and sex category Range/ Reference Values Analytical Methods Wavelength Distance bet 2 successive peaks (nm) Lower frequency = Longer wavelength (Ex. Red) Higher frequency = Shorter wavelength (Ex. Violet) Spectrophotometric meas. Meas. light intensity in a narrower wavelength Photometric measurement Meas. light intensity w/o consideration of wavelength Multiple wavelength (uses filter only) LASER Light Amplification by Stimulated Emission of Radiation Light source for spectrophotometry Visible region Tungsten light bulb Mercury arc UV Deuterium lamp Mercury arc Xenon lamp Hydrogen lamp IR Merst glower Globar (Silicone carbide) Stray light Wavelength outside the band Most common cause of loss of linearity Diffraction gratings Most commonly used monochromator Cutting grooves Prisms Rotatable Nickel sulfate Prevents stray light Cutoff filter Anti-stray light Bandpass ½ peak transmittance Alumina silica glass cuvet Most commonly used cuvet Quartz/plastic cuvet UV Borosilicate glass cuvet Strong bases Photodetector Converts transmitted light into photoelectric energy Barrier layer cell/ photocell/ Simplest detector photovoltaic cell No external voltage For filter photometers Phototube Contains anode and cathode Req external voltage Photomultiplier tube Most common type Most sensitive UV and visible region lec.mt 04 |Page | 2 Galvanometer/Ammeter Meter or read-out device Absorbance A = abc (a = absorptivity; b = length of light (1cm); c = concentration) A = 2 – log%T Double beam spectro. Splits monochromatic light into two components: One beam  sample One beam  reference soln or blank (corrects for variation in light source intensity) Double-beam in space 2 photodetectors (sample beam and reference beam) Double-beam in time 1 photodetector Monochromatic light  sample cuvet and reference cuvet Dydimium filter 600 nm Holmium oxide filter 360 nm Reagent blank Color of reagents Sample blank Optical interference (Hgb) FEP Meas. light emitted by a single atom burned in a flame Principle: Excitation Lt. source and cuvette: Flame For excited ions (Na+, K+) Cesium and Lithium Internal standards (FEP) Correct variations in flame Lithium Preferred internal std Potent antidepressant AAS Meas. light absorbed by atoms dissociated by heat Principle: Dissociation (unionized, unexcited, ground state) Lt. source: Hollow-cathode lamp For unexcited trace metals (Ca++ and Mg++) More sensitive than FEP Atomizer (nebulizer) Convert ions  atoms Chopper Modulate the light source Lanthanum/Strontium chloride Complex with phosphate Avoid calcium interference Volumetric (Titrimetric) Unknown sample is made to react with a known solution in the presence of an indicator Turbidimetry Light blocked Meas. abundant large particles (Proteins) Depend on specimen concentration and particle size Nephelometry Meas. amt of Ag-Ab complexes Scattered light Depends on wavelength and particle size Electrophoresis Migration of charged particles in an electric field Iontophoresis Migration of small charged ions Zone electrophoresis Migration of charged macromolecules Endosmosis Movement of buffer ions and solvent relative to the fixed support Ex: gamma globulins Cellulose acetate Molecular size Agarose gel Electrical charge Polyacrylamide gel Charge and molecular size 20 fractions (ex. isoenzymes) Electrophoretic mobility Directly proportional to net charge Inversely proportional to molecular size & viscosity of the supporting medium Isoelectric focusing Molecules migrate through a pH gradient lec.mt 04 |Page | 3 pH = pI For isoenzymes: same size, different charge Densitometry Scan & quantitate electrophoretic pattern Capillary electrophoresis Electro-osmotic flow Southern blot DNA Northern blot RNA Western blot Proteins Chromatography Separation by specific differences in physical-chemical characteristics of the different constituents Paper chromatography Fractionation of sugar and amino acid Sorbent: Whatman paper TLC Screening: Drugs Retention factor (Rf) value Relative distance of migration from the point of application Rf = Distance leading edge of component moves Total distance solvent front moves Gas chromatography Separation of steroids, barbiturates, blood, alcohol, and lipids Volatile compounds Specimens  vaporized Mobile phase: Inert gases Gas Solid chromatography Differences in absorption at the solid phase surfaces Gas Liquid chromatography Differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase Mass Spectrometry Fragmentation and ionization GC-MS Gold standard for drug testing MS/MS Detect 20 inborn errors of metabolism from a single blood spot HPLC Most widely used liquid chromatography Fractionation of drugs, hormones, lipids, carbohydrates and proteins Hydrophilic gel Gel filtration Separation of enzymes, antibodies and proteins Ex: Dextran and agarose Hydrophobic gel Gel permeation Separation of triglyceride and fatty acid Ex: Sephadex Ion exchange 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 hemoglobins Adsorption chromatography Based on differences between the adsorption and desorption of solutes at the surfaces of a solid particle Fluorometry/Molecular Det. amt. of lt. emitted by a molecule after excitation by electromagnetic Luminescence Spectro. radiation Lt. sources: Mercury arc and Xenon lamp (UV) Lt. detector: Photomultiplier tubes 2 monochromators: Primary filter – selects wavelength absorbed by the solution to be measured Secondary filter – prevents incident light from striking the photodetector Sensitivity: 1000x than spectro Quenching Major disadvantage of fluorometry pH and temperature changes, chemical contaminants, UVL changes Instrumentation lec.mt 04 |Page | 4 Borosilicate glasswares For heating and sterilization Ex: Pyrex and Kimax Boron-free/Soft glasswares High resistance to alkali Corex (Corning) Special alumina-silicate glass Strengthened chemically than thermally 6x stronger than borosilicate Vycor (Corning) For high thermal, drastic heat and shock Can be heated to 900OC Flint glass Soda-lime glass + Calcium, Silicon, Sodium oxides Easy to melt For making disposable glasswares TD: To deliver Exact amount TC: To contain Does not disperse the exact volume Blowout w/ etched rings on top of pipet Self-draining w/ o etched rings Drain by gravity Transfer pipet Volumetric: for non-viscous fluid; self-draining Ostwald folin: for viscous fluid; w/ etched ring Pasteur: w/o consideration of a specific volume Automatic macro-/micropipets Graduated or measuring pipet Serological: w/ graduations to the tip (blowout) Mohr: w/o graduations to the tip (self-draining) Bacteriologic Ball, Kolmer and Kahn Micropipettes: <1 mL Micropipettes TC pipets: Sahli-Hellige pipet Lang-Levy pipet RBC and WBC pipets Kirk and Overflow pipets Air displacement pipet Piston: suction Disposable tip Positive displacement pipet Piston  barrel (like a hypodermic syringe) Dispenser/Dilutor pipet Liquid: common reservoir  dispense repeatedly Distilled H2O Calibrating medium for TD pipettes Mercury Calibrating medium for TC pipettes Acid dichromate Cleaning solution for glasswares (H2SO4 + K2Cr2O4) Continuous flow analyzer Common reaction vessel Air bubbles: separates and cleans Glass coil: mix Examples: “STS” Simultaneous Multiple Analyzer (SMA) Technicon Autoanalyzer II SMAC Centrifugal analyzer Acceleration and deceleration of the rotor Advantage: Batch analysis Examples: “RICC” Cobas-Bio (Roche) IL Monarch CentrifiChem RotoChem lec.mt 04 |Page | 5 Discrete Analyzer Most popular Req. vol: 2-6 μL Uses positive-displacement pipets Run multiple-tests-one-sample-at-a-time Random access capability (STAT) Examples: Vitros Dimension Dade Beckman ASTRA System (4 & 8) Hitachi Bayer Advia Roche Cobas Integra 800 Roche Analytics P Module Automated Clinical Analyzer (ACA) Star (Dade) Dupont ACA Abbott ABA-100 Bichromatic Analyzer ABA-200 VP Analyzer American Monitor KDA Olympus Demand Thin-Film Analyzers 4 or 5 layers: (Dry slide technology) -Spreading layer -Scavenger layer - Ascorbate Oxidase -Reagent layer -Indicator layer -Support layer Colored reaction  Reflectance spectrophotometry Examples: “KV2(75)” Kodak Ektachem Vitros 750XRC Vitros 550XRC Carry over Transport of quantity of analyte or rgt from one specimen rxn into another, and contaminating a subsequent one Batch testing All samples loaded at the same time Single test is conducted on each sample Parallel testing One specimen More than one test is analyzed Random access testing Any sample Any test Any sequence STAT Sequential testing Multiple tests analyzed one after another on a given specimen Open reagent system System other than manufacturer’s reagents can be utilized for measurement Closed reagent system The operator can only use the manufacturer’s reagents Patient Preparation Exercise Increased: GU2FT C2L3A5P2 GH Urea Urinary protein (Proteinuria) Fatty acid Testosterone lec.mt 04 |Page | 6 CPK (muscle) Creatinine (muscle) Lactate LH LD (muscle) ACP Aldolase (muscle) AST ALT Ammonia Pyruvate Prolactin Decreased: Glucose Fist clenching Increased: “LPP” Lactate Potassium Phosphate Fasting 8-16 hours: Glucose Lipids Lipoproteins Increased: Bilirubin (48 hours) Triglyceride (72 hours) Basal state collection Glucose Cholesterol Triglyceride Electrolytes Diet Increased: “GLUC2H” Glucose Lipids Urea (High protein diet) Caffeine: increases glucose Catecholamines 5-HIAA (From Serotonin) Turbidity/Lactescence Triglyceride >400mg/dL Icterisia Bilirubin: 25.2 mg/dL Icteric samples Interfere with: "TACGu” Total Protein Albumin Cholesterol Glucose Upright/supine (lying) position Preferred position Patient should be seated/supine at least 20 mins before blood collection to prevent hemodilution or hemoconcentration Supine  Sitting/Standing Vasoconstriction  Reduced plasma volume Increased: “ECA” Enzymes Calcium Albumin Sitting  Supine Hemoconcentration lec.mt 04 |Page | 7 Increased: “P(u)BLIC” Proteins BUN Lipids Iron Calcium Standing  Supine Hemodilution Decreased: “TLC” Triglycerides Lipoproteins Cholesterol Prolonged standing Increased: K+ (muscles) Prolonged bedrest Decreased: Albumin (Fluid retention) Tourniquet Recommended: 1 minute application Prolonged tourniquet app. Hemoconcentration Anaerobiosis Increased: “C2LEA2K” Calcium Cholesterol Lactate Enzymes Ammonia Albumin K+ Tobacco smoking (Nicotine) Increased: “TUNG2C3” Triglycerides Urea Nonesterified fatty acid Glucose GH Catecholamines Cortisol Cholesterol Alcohol ingestion Increased: “THUG” Triglycerides Hypoglycemia (chronic alcoholism) Uric acid/Urates GGT Ammonia Increases by 100-200μg/L/cigar Stress (anxiety) Increased: “LAGIC” Lactate Albumin Glucose Insulin Cholesterol Drugs Medications affecting plasma volume can affect protein, BUN, iron, calcium Hepatotoxic drugs: increased liver function enzymes Diuretics: decreased sodium and potassium Diurnal variation "CA3PI2TG” Cortisol ACTH ACP lec.mt 04 |Page | 8 Aldosterone Prolactin Iron Insulin Thyroxine GH Specimen Collection and Handling Sleeping patients Must be awakened before blood collection Unconscious patients Ask nurse or relative Identification bracelet Venipuncture Median Cubital (1st)  Cephalic (2nd)  Basilic (3rd) Tourniquet Velcro or Seraket type 3-4 inches above the site Not exceed 1 minute Needle Bevel up 15-30O angle Length: 1 or 1.5 inch (Butterfly needle: ½ to ¾ inch) After blood collection Cotton  site Apply pressure for 3-5 minutes BP cuff as tourniquet Inflate to 60 mmHg Benzalkonium chloride Disinfectant for ethanol testing (Zephiran) Dilution – 1:750 IV line on both arms Discontinue IV for 2 minutes Collect sample below the IV site Initial sample (5mL)  discard IV fluid contamination Increased: Glucose (10% contam. w/ 5% dextrose  increased bld glucose by 500 mg/dL) Chloride Potassium Sodium Decreased: Urea Creatinine Renin blood level Collected after a 3-day diet, from a peripheral vein Basal state collection Early morning blood collection 12 hours after the last ingestion of food Lancet 1.75mm: preferred length to avoid penetrating the bone Incision (Skin puncture) <2.0mm (infants and children) 2-3mm (adults) 1.5-2.4mm Distance from the skin surface to bone or cartilage (middle finger) Arterialized capillary blood Earlobe: Preferred site Lateral plantar heel surface: most commonly used site Flea Minute metal filling which may be inserted into the capillary tube before collecting blood to help mix the specimen while the blood is entering the tube Indwelling umbilical artery Best site for blood gas analysis (newborns) 1000-3000 RCF for 10 mins Centrifugation requirement Hemolysis Increased: “KLA6MP ITC2” -K+ -LDH (150x) lec.mt 04 |Page | 9 -ACP -ALP -Aldolase -ALT -AST -Albumin -Mg2+ -Phosphorus -Iron -Total protein Affects bilirubin levels Inhibits lipase Refrigeration/Chilling Required for: “ABCGLRP2” (Low temp) Ammonia Blood gases Catecholamines Gastrin Lactic acid Renin PTH Pyruvate Decreased: LD 4 and 5 Increased: ALP Photosensitive analytes Bilirubin Beta-carotene Folate Porphyrins Vitamins A and B6 Oxalate Insoluble salt 1-2 mg/mL blood Citrate Non-ionized form 3.2-3.8 g/dL (1:9 ratio) EDTA Chelation 1-2 mg/mL blood Versene: disodium salt Sequestrene: Dipotassium salt Fluoride Weakly dissociated calcium component 2 mg/mL blood: anti-glycolytic 10 mg/mL blood: anticoagulant Heparin A.k.a. Mucoitin polysulfuric acid Universal anticoagulant Antithrombin 0.2 mg/mL blood Lithium heparin For glucose, BUN, ionized calcium, electrolyte studies (K+: best) and creatinine Orange top tube Additive: Thrombin Royal blue top tube Additives: None; Na2EDTA Sodium heparin lec.mt 04 |Page | 10 Brown top tube Lead testing Tan top tube Lead testing Black top tube Additive: Buffered sodium citrate For ESR Respinning gel tubes Increases potassium Thixotropic gel Gel separator (SG: 1.04) Serum: (SG: 1.03) RBC: (SG: 1.05) Laboratory Mathematics % w/v Grams of solute = % solution desired x total volume desired 100 % v/v mL of solute = % solution desired x total volume desired 100 % w/w Grams of solute = % solution desired x grams of the total solution 100 Molarity M= _grams of solute_______ GMW x volume of solution Moles Mol = weight (grams) GMW To prepare a molar solution Grams of solute = Molarity x GMW of the solute x Volume (L) desired To convert % w/v to 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 Grams of solute = Normality x EW x Volume (L) solids To convert % w/v to Normality N = w/v  10 EW Normality N = Molarity x Valence Molarity M = Normality valence Molality m = Grams of solute__ MW x kg of solvent Milliequivalents mEq/L = mg/dL  10  valence MW Millimoles mmol/L = mg/dL  10 MW Ratio Ratio = _Volume of solute_ Volume of solvent Dilution Dilution = __Volume of solute__ Volume of solution 0.179 Conversion factor for iron (mg/dL  μmol/L) 0.01 Conversion factor for phospholipid (g/dL to g/L) 2.27 Conversion factor for folate Analytical reagent (AR) grade For qualitative and quantitative analyses For accuracy Established by American Chemical Society (ACS) Uses: Trace metal analysis and preparation of standard solutions Ultrapure reagents Additional purification steps lec.mt 04 |Page | 11 Ex: Spectrograde, nanograde, HPLC grade Uses: Chromatography, atomic absorption, immunoassays Chemically Pure (CP) or Pure Indicates that the impurity limitations are not stated Grade Purity is delivered by meas. of melting point or boiling point Technical/Commercial grade In manufacturing Never used in clin. lab. testing United States Pharmacopoeia For human consumption (USP) and National Formulery Not applicable for lab. analysis (NF) Purpose: For drug manufacturing Preparation of reagent grade Filtration (1st)  Distillation, Ion exchange, Reverse Osmosis water Type I Rgt Water Min. interference Max. water purity Used immediately For ultramicrochemical analyses, measurements of nanogram or subnanogram concentrations, tissue or cell methods (microscopy) and preparation of standard solutions Uses: FEP, AAS, blood gases and pH, enzyme studies, electrolyte testing, HPLC, trace metal and iron studies Type II Rgt Water For clinical laboratory use (hematology, microbiology, immunology, chemistry) For prep. of rgts and QC materials Type III For washing glasswares For urinalysis, parasitology and histology Distilled water Purified to remove almost all organic materials Deionized water Free from mineral salts; removed by ion exchange processes Organic material may still be present Occupational Safety and Health Req. manuf. to indicate lot no., physical or biological health hazard of the Act (OSHA) chem.. rgts, and precautions for safe use and storage College of American Pathologists Recommends that a lab. document culture growth, pH and specific water (CAP) resistance on reagent grade water Tests for water purity Microbiological content pH Resistivity Chemical oxygen demand Ammonia Ions Metals Detergent-contaminated water Alkaline pH Hard water Contains calcium, iron and other dissolved elements NCCLS Now: Clinical and Laboratory Standards Institute (CLSI) Dilute solution Relatively little solute Concentrated solution Large quantity of solute in solution Saturated solution Excess of undissolved solute particles Super saturated solution Greater concentration of undissolved solute particles than does a saturated solution of the same substance Primary standard Highly purified (IUPAC) Measured directly to produce a substance of exact known concentration Secondary standard Low purity Concentration is determined by comparison w/ a primary standard Laboratory Safety lec.mt 04 |Page | 12 National Fire Protection Association (NFPA) Classification of Fires Class A fire Ordinary combustibles: paper, cloth, rubbish, plastics, wood Extinguisher: Water (A), Dry chemical (ABC), loaded steam Class B fire Flammable liquids: grease, gasoline, paints, oil Extinguisher: Dry chemical (ABC), carbon dioxide (BC), halon foam (BC) Class C fire Electrical equipment and motor switches Extinguisher: Dry chemical (ABC), Carbon dioxide (BC), halon (BC) Class D fire Flammable metals: mercury, magnesium, sodium, lithium Extinguisher: Metal X Fought be fire fighters only Class E fire Detonation (Arsenal fire) Allowed to burn out and nearby materials protected Standard Hazards Identification System (Diamond-shaped color coded symbol) Blue quadrant Health hazard Red quadrant Flammable hazard Yellow quadrant Reactivity/Stability hazard White quadrant Other special information Chemical spills 1st step: assist/evacuate personnel 1:10 dilution of chlorine bleach To disinfect and clean bench tops (10%) In contact with the area for at least 20 minutes HBV: 10 minutes HIV: 2 minutes Poisonous vapors Chloroform Methanol Carbon tetrachloride Bromide Ammonia Formaldehyde Mercury Flammable and combustible Acetone solvents Ethanol Toluene Methanol Xylene Benzene Isopropanol Heptane Flammable liquids Flash point below 37.8OC Combustible liquids Flash point at or above 37.8OC Strong acids or bases Neutralized before disposal Water should NEVER be added to concentrated acid Ether Deteriorate over time  hazardous Forms explosive peroxides Benzidine Known carcinogen Fumehoods Ventilation: velocity of 100-120 ft/min Safety showers Deliver 30-50 gal/min of H2O at 20-50 psi Carbohydrates Glycol aldehyde The simplest carbohydrate Sucrose Most common nunreducing sugar Pancreas Exocrine: Enzymes (AMS, LPS) Endocrine: Hormones (Insulin, glucagon, somatostatin) Hyperglycemic Hormones “GAG CHET” lec.mt 04 |Page | 13 Glucagon ACTH GH Cortisol Human Placental Lactogen Epinephrine Thyroxine Hyperglycemia Electrolyte Imbalance: (≥126 mg/dL) Decreased: Sodium, Bicarbonate Increased: Potassium Hypoglycemia 50-55 mg/dL = Symptoms ≤50 mg/dL = Diagnostic Whipple’s triad Low blood glucose concentration (Hypoglycemia) Typical symptoms Symptoms alleviated by glucose administration 6:1 Ratio of BHA to AA in severe DM (Normal = 1:1) Type 1 DM IDDM Juvenile Onset Brittle Ketosis-prone 80-90% reduction of beta-cells  Symptomatic Type 1 DM HLA-DR3 and DR4 (+) Glutamic acid decarboxylase (GAD65) (+) Insulin autoantibodies (+) Microalbuminuria: 50-200 mg/24 hours = Diabetic nephropathy (-) C-peptide Complications of Type I DM Microvascular disorders: Nephropathy Neuropathy Retinopathy Type 2 NIDDM Adult type/Maturity Onset Stable Ketosis-resistant Receptor-deficient Insulin resistance: relative insulin deficiency Strong genetic predisposition Geneticist’s nightmare If untreated  glucose: >500 mg/dL  nonketotic hyperosmolar coma Gestational DM Screening: 1hr GCT (50g) – bet. 24 and 28 weeks of gestation Confirmatory: 3-hr GTT (100g) Infants: at risk for respiratory distress syndrome, hypocalcemia, hyperbilirubinemia After giving birth, evaluate 6-12 weeks postpartum Converts to DM w/in 10 years in 30-40% of cases OGTT (GDM) FBS = ≥95 mg/dL 1-Hr = ≥ 180 mg/dL 2-Hr = ≥ 155 mg/dL 3-Hr = ≥ 140 mg/dL GDM = 2 plasma values of the above glucose levels are exceeded Impaired fasting glucose (Pre- FBS = 100-125 mg/dL lec.mt 04 |Page | 14 diabetes) Impaired glucose tolerance FBS = <126 mg/dL 2-Hr OGTT = 140-199 mg/dL FBS WB = 15% lower than in serum or plasma VB = 7 mg/dL lower than capillary and arterial blood CSF glucose 60-70% of the plasma glucose Peritoneal fluid glucose Same with plasma glucose Plasma glucose increases w/ age Fasting: 2 mg/dL/decade Postprandial: 4 mg/dL/decade Glucose challenge: 8-13 mg/dL/decade w/in 1 hour Separate serum/plasma from the cells (Preferably w/in 30 mins) 5-7%/hr Glycolysis at room temperature 1-2 mg%/hr Glycolysis at refrigerated temperature Copper reduction methods Cupric  Cuprous  Cuprous oxide Folin Wu Cuprous ions + phosphomolybdate  phosphomolybdenum blue Nelson-Somogyi Cuprous ions + arsenomolybdate  arsenomolybdenum blue Neocuproine method Cuprous ions + neocuproine  Cuprous-neocuproine complex (yellow) Benedict’s method Reducing substances in blood and urine Alkaline Ferric Reduction Ferricyanide ---(Glucose)--> Ferrocyanide method (Hagedorn-Jensen) (Yellow) (Colorless) Ortho-toluidine Schiff’s base (Dubowski method) Glucose oxidase Measures beta-D-glucose (65%) Mutarotase Converts alpha-D-glucose (35%) to beta-D-glucose (65%) NADH/NADPH Absorbance at 340nm Polarographic glucose oxidase Consumption of oxygen on an oxygen-sensing electrode O2 consumption α glucose concentration Hexokinase method Most specific method Reference method Uses G-6-PD G-6-PD Most specific enzyme rgt for glucose testing Interfering substances False-decreased (Glucose oxidase) Bilirubin Uric acid Ascorbate Hemolysis (>0.5 g/dL Hgb) Major interfering substance in hexokinase method (false-decreased) Dextrostics Cellular strip Strip w/ glucose oxidase, peroxidase and chromogen OGTT Janney-Isaacson method (Single dose) = most common Exton Rose (Double dose) Drink the glucose load within 5 mins IVGTT For patients with gastrointestinal disorders (malabsorption) Glucose: 0.5 g/kg body weight Given w/in 3 mins 1st blood collection: after 5 mins of IV glucose Requirements for OGTT Ambulatory Fasting: 8-14 hours Unrestricted diet of 150g CHO/day for 3 days Do not smoke or drink alcohol Glucose load 75 g = adult (WHO std) 100 g = pregnant lec.mt 04 |Page | 15 1.75 g glucose/kg BW = children HbA1c 2-3 months Glucose = beta-chain of HbA1 1% increase in HbA1c = 35 mg/dL increase in plasma glucose 18-20% = prolonged hyperglycemia 7% = cutoff Specimen: EDTA whole blood Test: Affinity chromatography (preferred) IDA and older RBCs High HbA1c RBC lifespan disorders Low HbA1c Fructosamine 2-3 weeks (Glycosylated albumin/ plasma Useful for patients w/ hemolytic anemias and Hgb variants protein ketoamine) Not used in cases of low albumin Specimen: Serum Galactosemia Congenital deficiency of 1 of 3 enzymes in galactose metabolism Galactose-1-phosphate uridyl transferase (most common) Galactokinase Uridine diphosphate galactose-4-epimerase Essential fructosuria Autosomal recessive Fructokinase deficiency Hereditary fructose intolerance Defective fructose-1,6-biphosphate aldolase B activity Fructose-1,6-biphosphate Failure of hepatic glucose generation by gluconeogenic precursors such as deficiency lactate and glycerol Glycogen Storage Disease Autosomal recessive Defective glycogen metabolism Test: IVGTT (Type I GSD) Ia = Von Gierke Glucose-6-Phosphatase deficiency (most common worldwide) II = Pompe Alpha-1,4-glucosidase deficiency (most common in the Philippines) III = Cori Forbes Debrancher enzyme deficiency IV = Andersen Brancher enzyme deficiency V = McArdle Muscle phosphorylase deficiency VI = Hers Liver phosphorylase deficiency VII = Tarui Phosphofructokinase deficiency XII = Fanconi-Bickel Glucose transporter 2 deficiency CSF glucose Collect blood glucose at least 60 mins (to 2 hrs) before the lumbar puncture (Because of the lag in CSF glucose equilibrium time) < 0.5 Normal CSF : serum glucose ratio C-peptide Formed during conversion of pro-insulin to insulin 5:1 to 15:1 Normal C-peptide : insulin ratio D-xylose absorption test Differentiate pancreatic insufficiency from malabsorption (low blood or urine xylose) Gerhardt’s ferric chloride test Acetoacetate Nitroprusside test 10x more sensitive to acetoacetate than to acetone Acetest tablets Acetoacetate and acetone Ketostix Detects acetoacetate better than acetone KetoSite assay Detects beta-hydroxybutyrate but not widely used Normal Values RBS = <140 mg/dL (Carbohydrates) FBS = 70-100 mg/dL HbA1c = 3-6% Fructosamine = 205-285 μmol/L 2-Hr PPBS = <140 mg/dL lec.mt 04 |Page | 16 GTT: 30 mins = 30-60 mg/dL above fasting 1-Hr = 20-50 mg/dL above fasting 2-Hr = 5-15 mg/dL above fasting 3-Hr = fasting level or below Lipids Phospholipids Most abundant lipid Amphipathic: polar (hydrophilic head) and nonpolar (hydrophobic side chain) Sphingomyelin Reference material during 3rd trimester of pregnancy Concentration is constant as opposed to lecithin Not derived from glycerol but from sphingosine (amino alcohol) Forms of phospholipids 70% Lecithin/Phosphatidyl choline 20% Sphingomyelin 10% Cephalin TLC + Densitometric Method for L/S ratio quantitation Microviscosity Measured by fluorescence polarization Cholesterol Not a source of fuel Not affected by fasting 70% Cholesterol ester (plasma/serum) 30% Free cholesterol (plasma/serum and RBC) LCAT Esterification of cholesterol Apo A-1 Activator of LCAT Cholesterol increases after the 2 mg/dL/year between 50 and 60 years old age of 50 Liebermann Burchardt Cholestadienyl Monosulfonic acid Green end color Salkowski Cholestadienyl Disulfonic acid Red end color Color developer mixture Glacial acetic acid (Cholesterol) Acetic anhydride Conc. H2SO4 One-step method Colorimetry (Pearson, Stern and Mac Gavack) Two-step method Color. + Extraction (Bloor’s) Three-step method Color. + Extract. + Saponification (Abell-Kendall) Four-step method Color. +Extract. + Sapon. + Precipitation (Schaenheimer Sperry, Parekh and Jung) Abell, Levy and Brodie mtd CDC reference method for cholesterol: (Chemical method) -Hydrolysis/saponification (Alc. KOH) -Hexane extraction -Colorimetry (Liebermann-Burchardt) Triglycerides Most insoluble lipid Main storage lipid in man (adipose tissue) – 95% Fasting: 12 hours Triglyceride increases after the 2 mg/dL/year between 50 and 60 years old age of 50 Van Handel & Zilversmith Chromotropic acid (Colorimetric) (+) Blue color compound Hantzsch Condensation Diacetyl acetone (Fluorometric) (+) Diacetyl lutidine compound Modified Van Handel and CDC reference method for triglycerides: lec.mt 04 |Page | 17 Zilversmith -Alkaline hydrolysis (Chemical method) -Chloroform extraction  extract treated w/ silicic acid -Color reaction w/ chromotropic acid – meas. HCHO (+) Pink colored Fatty acids Short chain = 4-6 C atoms Medium chain = 8-12 C atoms Long chain = >12 C atoms Saturated = w/o double bonds Unsaturated = w/ double bonds Substrate for gluconeogenesis Most is bound to albumin Palmitic acid 16:0 Stearic acid 18:0 Oleic acid 18:1 Linoleic acid 18:2 Arachidonic acid 20:4 Lipoprotein lipase Hydrolyzes TAG in lipoproteins, releasing fatty acid and glycerol (Lipemia clearing factor) Hepatic lipase Hydrolyzes TAG and phospholipids from HDL Hydrolyzes lipids on VLDL and IDL Endothelial lipase Hydrolyzes phospholipids and TAG in HDL Apolipoprotein Protein component of lipoprotein Amphipathic helix – ability of proteins to bind to lipids Chylomicrons Largest and least dense Produced by the intestine SG: <0.95 80-95% TAG (exogenous) Apo B-48 (Major) EP: Origin VLDL Secreted by the liver SG: 0.95-1.006 65% TAG (endogenous) Apo B-100 (Major) EP: pre-beta LDL Synthesized by the liver SG: 1.006-1.063 50% CE Apo B-100 (Major) EP: beta Cholesterol transport: LiverTissues Target of cholesterol lowering therapy Better marker for CHD risk HDL Smallest but dense SG: 1.063-1.21 45-55% protein 26-32% phospholipid Apo A-1 (Major) EP: alpha Produced by the liver and intestine Reverse cholesterol transport: TissueLiver IDL Product of VLDL catabolism Seen in Type 3 hyperlipoproteinemia (Apo E-III def.; beta-VLDL) lec.mt 04 |Page | 18 SG: 1.006-1.019 Lp(a) Sinking pre-beta lipoprotein SG: 1.045-1.080 Apo B-100 EP: pre-beta (VLDL) UC: like LDL Independent risk factor for atherosclerosis LpX Found in obstructive jaundice (cholestasis) and LCAT deficiency 90% FC and PL Apo C and albumin Beta-VLDL Floating beta-lipoprotein SG: <1.006 EP: beta (LDL) UC: like VLDL Found in type 3 hyperlipoproteinemia (Apo E-III def; IDL) Rich in cholesterol content than VLDL Lipoprotein methodologies Specimen: sample from serum separator tubes (preferred) EDTA plasma: choice for research studies of LPP fractions Fasting state: TAG  VLDL Nonfasting state: TAG  CM Ultracentrifugation Reference method for LPP quantitation Reagent: Potassium bromide (SG: 1.063) Ultracentrifugation of plasma for 24 hours Expressed in Svedberg units Electrophoresis Electrophoretic pattern: (+) HDL VLDL  LDL  CM (Origin) (-) Agarose gel: sensitive medium VLDL: migrates w/ alpha2-globulin (pre-beta) Chemical precipitation Uses polyanions (heparin and divalent cations) and polyethylene glycol Dextran sulfate-Mg2+ Heparin-Mn2+ 3-step procedure: CDC Reference method for HDL Ultracentrifugation Precipitation Abell-Kendall assay Beta quantification + Method for LDL Ultracentrifugation + Chemical Sample: EDTA plasma precipitation Immunoturbidimetric assay Measures Lipoprotein (a) LDL Cholesterol Total Cholesterol – HDL – VLDL Friedewald method Most commonly used VLDL = TAG/2.175 (mmol/L) VLDL = TAG/5 (mg/dL) Not applicable if TAG is >400 mg/dL De Long method VLDL = TAG/2.825 (mmol/L) VDL = TAG/6.5 (mg/dL) Apo A-1 Activates LCAT Apo B-100 LDL  LDL receptor Apo B-48 CM (major) Not recognized by LDL receptor Apo C-II Activates LPL Apo D Activates LCAT lec.mt 04 |Page | 19 Apo E Apo E-4: associated w/ high LDL, higher risk of CHD and Alzheimer’s disease Apo(a) Lp(a) Homologous to plasminogen Abetalipoproteinemia Autosomal recessive (Basses-Kornzweig syn.) Defective apo B synthesis Deficient fat soluble vitamins Niemann-Pick disease Sphingomyelinase deficiency Tangier’s disease Deficiency of HDL (1-2 mg/dL) Defects in the gene for the ABCA1 transporter LPL deficiency TAG = 10,000 mg/dL (Chylomicronemia) Do not develop premature coronary disease (CM are not atherogenic) Abdominal pain and pancreatitis LCAT deficiency Fish-eye disease Low HDL Tay-Sachs disease Hexosaminidase A deficiency Fredrickson Classification Type 1 LPL deficiency (Chylomicronemia) Increased: CM (TAG) Type 2a Familial hypercholesterolemia Increased: LDL (cholesterol) Type 2b Combined hyperlipidemia (most common primary hyperlipidemia) Increased: LDL (cholesterol), VLDL (TAG) Type 3 Dysbetalipoproteinemia Increased: IDL, (+) beta-VLDL (+) Apo E-II (+) Eruptive and palmar xanthomas Type 4 Hypertriglyceridemia Increased: VLDL (TAG) Type 5 Increased: VLDL (Endo.TAG), CM (Exo.TAG) Normal Values Cholesterol: (Lipids) Desirable = <200 mg/dL Borderline high = 200-239 mg/dL High = >240 mg/dL Triglycerides: Desirable = <150 mg/dL Borderline high = 150-199 mg/dL High = 200-499 Very high = >500 mg/dL HDL: Low = <40 mg/dL (Cutoff) High = >60 mg/dL LDL: Optimal = <100 mg/dL Near/above optimal = 100-129 mg/dL Borderline high = 130-159 mg/dL High = 160-189 mg/dL Very high = >190 mg/dL Proteins Proteis First rank of importance Proteins Amphoteric: positive and negative charges Effective blood buffers lec.mt 04 |Page | 20 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 Primary structure Amino acid sequence Det. the identity of protein, molecular structure, function binding capacity, recognition ability Secondary structure Winding of polypeptide chain Specific 3-D conformations: alpha-helix, beta-pleated sheath, bend form Tertiary structure Actual 3-D configuration Folding pattern Physical and chemical properties of proteins Quarternary structure Association of 2 or more polypeptide chains  protein Albumin No quarternary structure Glucogenic amino acids Alanine (pyruvate) Arginine (alpha-ketoglutarate) Aspartate (oxaloacetate) Ketogenic amino acids Degraded to acetyl-CoA Leucine Lysine Simple proteins Hydrolysis  Amino acids Fibrous: fibrinogen, troponins, collagen Globular: hemoglobin, plasma proteins, enzymes, peptide hormones Conjugated proteins Protein (apoprotein) + nonprotein moiety (prosthetic group) Metalloproteins: ferritin, ceruloplasmin, hemoglobin, flavoproteins Lipoproteins: VLDL, HDL, LDL, CM Glycoproteins: haptoglobin, alpha1-antitrypsin (10-40% CHO) Mucoproteins or proteoglycans: Mucin (CHO > CHON) Nucleoproteins: Chromatin (combined w/ nucleic acids) Nitrogen balance Balance bet. anabolism and catabolism Negative nitrogen balance Catabolism > anabolism Excessive tissue destruction Positive nitrogen balance Anabolism > catabolism Growth and repair processes Prealbumin (Transthyretin) Transports thyroxine and retinol (Vit. A) Landmark to confirm that the specimen is really CSF Albumin Maintains osmotic pressure Negative acute phase reactant Alpha1-antitrypsin Acute phase reactant Major inhibitor of protease activity 90% of alpha1-globulin band Alpha1-fetoprotein Gestational marker Tumor marker: hepatic and gonodal cancers Screening test for fetal conditions (Spx: maternal serum) Amniotic fluid: confirmatory test Increased: Hepatoma, spina bifida, neural tube defects Decreased: Down Syndrome (Trisomy 21) Alpha1-acid glycoprotein/ Low pI (2.7) orosomucoid Negatively charged even in acid solution Alpha1-antichymotrypsin Acute phase reactant Binds and inactivates PSA Increased: Alzheimer’s disease, AMI, infection, malignancy, burns lec.mt 04 |Page | 21 Haptoglobin (alpha2) Acute phase reactant Binds free hemoglobin (alpha chain) Ceruloplasmin (alpha2) Copper binding (6-8 atoms of copper are attached to it) Has enzymatic activities Decreased: Wilson’s disease (copper  skin, liver, brain, cornea [Kayser- Fleisher rings]) Alpha2-macroglobulin Larges major nonimmunoglobulin protein Increased: Nephrotic syndrome (10x) Forms a complex w/ PSA Group-specific component (Gc)- Affinity w/ vitamin D and actin globulin (bet. alpha1 and alpha2) Hemopexin (beta) Binds free heme Beta2-microglobulin HLA Filtered by glomeruli but reabsorbed Transferrin/Siderophilin (beta) Negative acute phase reactant Major component of beta2-globulin fraction Pseudoparaproteinemia in severe IDA Increased: Hemochromatosis (bronze-skin), IDA Complement (beta) C3: major Fibrinogen (bet. beta and Acute phase reactant gamma) Between beta and gamma globulins CRP (gamma) General scavenger molecule Undetectable in healthy individuals hsCRP: warning test to persons at risk of CAD Immunoglobulins (gamma) Synthesized by the plasma cells IgG>IgA>IgM>IgD>IgE Myoglobin Marker: Ischemic muscle cells, chest pain (angina), AMI Troponins Most important marker for AMI TnT (Tropomyosin-binding Specific for heart muscle subunit) Det. unstable angina (angina at rest) TnI (Inhibitory subunit or Actin- Only found in the myocardium binding unit) Greater cardiac specificity than TnT Highly specific for AMI 13x more abundant in the myocardium than CK-MB Very sensitive indicator of even minor amount of cardiac necrosis TnC Binds calcium ions and regulate muscle contractions Glomerular proteinuria Most common and serious type Often called albuminuria Tubular proteinuria Defective reabsorption Slightly increased albumin excretion Overload proteinuria Hemoglobinuria Myoglobinuria Bence-Jones proteinuria Postrenal proteinuria Urinary tract infection, bleeding, malignancy Microalbuminuria Type 1 DM Albumin excretion ≥30 mg/g creatinine (cutoff: DM) but ≤300 mg/g creatinine Microalbuminuria: 2 out of 3 specimens submitted are w/ abnormal findings (w/in 6 months) CSF Oligoclonal banding 2 or more IgG bands in the gamma region: Multiple sclerosis Encephalitis lec.mt 04 |Page | 22 Neurosyphilis Guillain-Barre syndrome Neoplastic disorders Serum Oligoclonal banding Leukemia Lymphoma Viral infections Alkaptonuria Ochronosis (tissue pigmentation) Homocystinuria Impaired activity of cystathione beta-synthetase Elevated homocysteine and methionine in blood and urine Screen: Modified Guthrie test (Antagonist: L-methionine sulfoximine) MSUD Markedly reduced or absence of alpha-ketoacid decarboxylase 4 mg/dL of leucine is indicative of MSUD Screen: Modified Guthrie test (Antagonist: 4-azaleucine) Diagnostic: Amino acid analysis (HPLC) PKU Deficiency of tetrahydrobiopterin (BH4)  elevated blood phenylalanine Normal Values Total protein = 6.5-8.3 g/dL (Proteins) Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL Kidney Function Tests Tests for GFR Clearance: -Inulin clearance -Creatinine clearance -Urea clearance Phenolsulfonphthalein dye test Cystatin C Tests for Renal Blood Flow BUN Creatinine Uric acid Tests Measuring Tubular Excretion: Function -Para-amino hippurate test (Diodrast test) -Phenolsulfonphthalein dye test Concentration: -Specific gravity -Osmolality GFR Decreases by 1.0 mL/min/year after age 20-30 years 150 L of glomerular filtrate is produced daily Inulin clearance Reference method Creatinine clearance Best alternative method Measure of the completeness of a 24-hour urine collection Excretion: 1.2-1.5 g creatinine/day Urea clearance Demonstrate progression of renal disease or response to therapy Cystatin C Low MW protease inhibitor FilteredNot secretedCompletely reabsorbed (PCT) Indirect estimates of GFR Its presence in urine denotes damage to PCT BUN Synthesized from Ornithine or Kreb’s Henseleit cycle First metabolite to elevate in kidney diseases Better indicator of nitrogen intake and state of hydration 2.14 BUN  Urea (mg/dL) Fluoride or citrate Inhibit urease Thiosemicarbazide Enhance color development (BUN mtd) lec.mt 04 |Page | 23 Ferric ions Diacetyl monoxime method Yellow diazine derivative Urease method Routinely used Urease: prepared from jack beans Urea ---(Urease)--> NH4 + Berthelot reagent (Measure ammonia) Coupled urease Glutamate dehydrogenase method UV enzymatic method Isotope dilution mass Reference method spectrometry For research purposes NPN 45% Urea 20% Amino acid 20% Uric acid 5% Creatinine 1-2% Creatine 0.2% Ammonia Creatinine Derived from alpha-methyl guanidoacetic acid (creatine) Produced by 3 amino acids (methionine, arginine, lysine) Most commonly used to monitor renal function Enzymatic methods Creatinine Aminohydrolase – CK method (Creatinine) Creatinase-Hydrogen Peroxide method – benzoquinonemine dye (red) Creatininase (a.k.a. creatinine aminohydrolase) Direct Jaffe method Formation of red tautomer of creatinine picrate Interferences (Direct Jaffe) Falsely increased: Ascorbate Glucose Uric acid Alpha-keto acids Folin Wu Method (+) Red orange tautomer Lloyd’s or Fuller’s Earth method True measure of creatinine Sensitive and specific Uses adsorbent to remove interferences (UA, Hgb, Bili) Lloyd’s reagent Sodium aluminum silicate Fuller’s earth reagent Aluminum magnesium silicate Jaffe reagent (Alk. picrate) Satd. picric acid + 10% NaOH Kinetic Jaffe method Popular, inexpensive, rapid and easy to perform Requires automated equipment Azotemia Elevated urea and creatinine in blood Pre-renal azotemia Decreased GFR but normal renal function Dehydration, shock, CHF Increased: BUN Normal: Creatinine Renal azotemia True renal disease Decreased GFR Striking BUN level but slowly rising creatinine value BUN = >100 mg/dL Creatinine = >20 mg/dL Uric acid = >12 mg/dL Post renal azotemia Urinary tract obstruction Decreased GFR Nephrolithiasis, cancer or tumors of GUT Creatinine = normal or slightly increased Uremia Marked elevation of urea, accompanied by acidemia and electrolyte lec.mt 04 |Page | 24 imbalance (K+ elevation) of renal failure Normocytic, normochromic anemia Uremic frost (dirty skin) Edema Foul breath Urine-like sweat Uric acid From purine (adenine and guanine) catabolism Excretion: 1g/day Hyperuricemia -Gout -Increased nuclear metabolism (leukemia, lymphoma, MM, polycythemia, hemolytic and megaloblastic anemia) – Tx: Allopurinol -Chronic renal disease -Lesch-Nyhan syndrome (HGPRT deficiency) Hypouricemia Fanconi’s syndrome Wilson’s disease Hodgkin’s disease Methods (Uric acid) Stable for 3 days Potassium oxalate cannot be used Major interferences: Ascorbate and bilirubin Phosphotungstic acid mtd Uric acid + Phosphotungstic acid ---(NaCN/NaCO3)--> Tungsten blue + Allantoin NaCN Folin Newton Brown Benedict NaCO3 Archibald Henry Caraway Lagphase Incubation period after the addition of an alkali to inactivate non-uric acid reactants Uricase method Simplest and most specific method Candidate reference method Uric acid (Absorbance at 293nm) ---[Uricase]--> Allantoin (No absorbance) Decrease in absorbance α uric acid concentration Para-amino hippurate test Measures renal plasma flow Reference method for tubular function Phenolsulfonphthalein dye test Measures excretion of dye proportional to renal tubular mass 6 mg of PSP is administered IV Concentration tests Collecting tubules and loops of Henle Specimen: 1st morning urine Specific gravity Affected by solute number and mass SG >1.050: X-ray dye and mannitol 1.010 = SG of ultrafiltrate in Bowman’s space Osmolality Total number solute particles present/kg of solvent (moles/kg solvent) Affectted only by number of solutes present Urine osmolality = due to urea Serum osmolality = due to sodium and chloride Det. by Colligative properties: Freezing point (incr. osm. = decr. FP) Vapor pressure (incr. osm. = decr. VP) Osmotic pressure (incr. osm. = incr. OP) lec.mt 04 |Page | 25 Boiling point (incr. osm. = incr. BP) Direct methods (Osmolality) Freezing point osmometry = popular method Vapor pressure osmometry (Seebeck effect) Incr. plasma osmolality Incr. vasopressin (H2O reabsorption)  decr. plasma osmolality Tubular failure Increased: BUN, creatinine, calcium Decreased: Phosphate Osmolal gap Difference between measured and calculated osmolality Sensitive indicator of alcohol or drug overdose Osmolal gap: >12 mOsm/kg DKA Drug overdose Renal failure Normal Values Creatinine Clearance: (Kidney Function Tests) Male = 85-125 mL/min Female = 75-112 mL/min BUN = 8-23 mg/dL Creatinine = 0.5-1.5 mg/dL Uric acid: Male = 3.5-7.2 mg/dL Female = 2.6-6.0 mg/dL Renal plasma flow (PAH) = 600-700 mL/min Renal blood flow (PSP) = 1200 mL/min SG = 1.005-1.030 Osmolality: Serum = 275-295 mOsm/kg Urine (24-hr) = 300-900 mOsm/kg [<290 mOsm/kg = kidney damage] Urine osmolality: Serum osmolality = 1:1 to 3:1 [>1:1 = Glomerular disease] [1.2:1 = loss of renal concentrating ability] [<1:1 = Diabetes Insipidus] Liver Function Tests Liver Receives 15 mL of blood per minute Lobule: anatomic unit Synthetic function Proteins, CHO, lipids, LPP, clotting factors, ketone bodies, enzymes Albumin: 12g/day Conjugation function Bilirubin metabolism Bilirubin: 200mg/day Detoxification and Drug Drugs metabolism Ammonia  Urea  Excreted Excretory and Secretory Bile acids: cholic acid and chenodeoxycholic acid functions Bile salts: bile acids + amino acids (glycine and taurine) Storage function Vitamins Glycogen Test measuring the Hepatic Total Protein Determination: Synthetic Ability -Kjeldahl method -Biuret method -Folin-Ciocalteu (Lowry) method -UV absorption method -Electrophoresis -Refractometry -Turbidimetric and Nephelometric methods -Salt fractionation lec.mt 04 |Page | 26 Prothrombin Time (Vitamin K Response Test) Test measuring Bilirubin Assay: Conjugation/Excretion Function -Evelyn and Malloy method -Jendrassik and Grof Bromsulfonphthalein (BSP) Dye Excretion test Test for Detoxification Function Enzyme tests: ALP, AST, ALT, 5’NT, GGT, OCT, LAP, LDH Ammonia: -Kjeldahl (Digestion) method -Nesslerization reaction -Berthelot reaction Plasma protein 0.2-0.4 g/dL higher than serum due to fibrinogen Kjeldahl (Digestion) mtd Standard reference method Measurement of nitrogen content Serum + Tungstic acid  PFF 1g N2 = 6.54g protein 15.1-16.8% = N2 content of proteins Rgt: H2SO4 End product: NH3 Biuret method Most widely used method (IFCC recommended) Req. at least 2 peptide bonds and an alkaline medium Rgts: Alkaline CuSO4 Rochelle salt (NaK Tartrate) NaOH KI End product: Violet color (545nm) Folin-Ciocalteu (Lowry) method Highest analytical sensitivity Oxidation of phenolic compounds (tyrosine, tryptophan, histidine) Rgts: Phenol (or phosphotungstic-molybdic acid) Biuret (color enhancer) End product: Blue color Electrophoresis MI: elevated APRs (AAT, HPG, a1-x) Gamma-spike Monoclonal gammopathy (multiple myeloma) Beta-gamma bridging In serum: Hepatic cirrhosis (IgA) In plasma: normal (fibrinogen) Alpha2-globulin band spike Nephrotic syndrome Alpha1-globulin flat curve Juvenile cirrhosis (AAT deficiency) Alpha1, alpha2, beta-globulin Inflammation band spikes Polyclonal gammopathy Chronic inflammation (RA, malignancy) Small spikes in beta region IDA (transferrin) Free hemoglobin “Blip” in the late alpha2 or early beta region Refractometry Refractive index Turbidimetric and SSA nephelometric methods TCA Salt fractionation Salt: Sodium sulfate Albumin Soluble: Water Moderately concentrated salt solution Concentrated salt solution lec.mt 04 |Page | 27 Insoluble: Hydrocarbon solvents Highly concentrated salt solution Saturated salt solution Globulin Soluble: Hydrocarbon solvents Weak salt solution Insoluble: Water Saturated salt solution Concentrated salt solution Prothrombin time Differentiates intrahepatic disorder (prolonged PT) from extrahepatic obstructive liver disease (normal PT) Albumin Inversely proportional to the severity of the liver disease Hepatic cirrhosis Low total protein + low albumin Bromcresol green Most commonly used dye for albumin Bromcresol purple Most specific dye for albumin Other dyes for albumin Hydroxyazobenzene benzoic acid (HABA) Methyl orange (MO) Nephrotic syndrome Albumin excretion: 20-30 g/day Analbuminemia (-) albumin Bisalbuminemia EP: 2 albumin bands Therapeutic drugs in serum Inverted A/G ratio Hepatic cirrhosis (IgA) Multiple Myeloma (IgG) Waldenström’s macroglobulinemia (IgM) Chronic inflammation Bilirubin Derived from hemoglobin myoglobin, catalase and cytochrome oxidase Heme oxygenase Protoporphyrin  Biliverdin Biliverdin reductase Biliverdin B1 Urobilinogen Deconjugated bilirubin Bilirubin 1 Non-polar bilirubin Free/Slow bilirubin Bilirubin 2 Polar bilirubin One-minute/prompt bilirubin Regurgitative bilirubin Delta bilirubin Bilirubin tightly bound to albumin Delta bilirubin = TB-DB+IB Jaundice Bilirubin >2 or 3 mg/dL Pre-hepatic jaundice Hemolytic B1 = increased B2 = normal UG = increased UB = negative Hepatic jaundice Hepatocellular B1 = increased B2 = increased UG = increased UB = positive ALT = increased lec.mt 04 |Page | 28 AST = increased Post-hepatic jaundice Obstructive B1 = normal B2 = increased UG = decreased/negative UB = positive ALP = increased GGT = increased Cholesterol = increased Gilbert’s syndrome Bilirubin transport deficit (uptake) B1 = increased B2 = decreased Crigler-Najjar syndrome Conjugation deficit Type I = total UDPGT deficiency Type II = partial UDPGT deficiency B1 = increased B2 = decreased Danger: Kernicterus Bile is colorless Dubin-Johnson syndrome & Bilirubin excretion deficit Rotor syndrome Blockade of excretion into the canaliculi TB = increased B2 = increased Lucey-Driscoll syndrome Circulating inhibitor of bilirubin conjugation B1 = increased Methods (Bilirubin) Free from hemolysis and lipemia Store in the dark Measured ASAP or w/in 2-3 hours Van den Berg reaction Diazotization of bilirubin Evelyn and Malloy method Accelerator: Methanol Diazo rgts: Diazo A (0.1% Sulfanilic acid + HCl) Diazo B (0.5% Sodium nitrite) Diazo blank (1.5% HCl) (+) pink to purple azobilirubin Affected by hemolysis Jendrassik and Grof Candidate reference method Accelerator: Caffeine sodium benzoate Buffer: Sodium acetate Ascorbic acid: terminates the initial reaction and destroys the excess diazo rgt Not falsely elevated by hemolysis Total bilirubin is measured 15 minutes after adding methanol or caffeine soln Bilirubin Absorbs light maximally at 450nm Rosenthal White method Double collection method Collection: -After 5 mins (50% dye retention) -After 30 mins (0% dye retention) Mac Donald method Single collection method Collection: -After 45 mins (+/- 5% dye retention) lec.mt 04 |Page | 29 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 Methods (Ammonia) Specimen: Heparin or EDTA plasma Fasting is required Avoid smoking Prolonged standing of specimen: increased NH3 due to deamination Place on iced water immediately Avoid hemolysis Kjeldahl (Digestion) method Specimen  PFF N2 ----------(hot conc. H2SO4 + CuSO4 + Hg + Selenium)----------> NH3 Nesslerization of ammonia NH3 + K2Hg2I2 ----------(Gum Ghatti)----------> NH2Hg2I2 End color: Yellow (low to moderate N2) Orange brown (high N2) Berthelot reaction NH3 + Phenol + Hypochlorite -----(Na Nitroprusside)-----> Indophenol blue Normal Values Total protein = 6.5-8.3 g/dL (Liver Function Tests) Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL α1-globulin = 0.1-0.3 g/dL α2-globulin = 0.6-1.0 g/dL β-globulin = 0.7-1.1 g/dL γ-globulin = 0.8-1.6 g/dL Total bilirubin = 0.2-1.0 mg/dL Indirect bilirubin = 0.2-0.8 mg/dL Direct bilirubin = 0-0.2 mg/dL Urobilinogen: Urine = 0.1-1.0 Ehrlich units/2hrs (or 0.54 Ehrlich units/day) Stool = 75-275 Ehrlich units/100g feces (or 75-400 Ehrlich units/24hrs) Ammonia = 19-60 μg/dL Enzymes Enzyme concentration Serum  Enzyme concentration =  reaction rate Substrate concentration Reagent If enzyme > substrate,  substrate =  reaction rate Saturation kinetics When substrate concentration reaches a maximal value, higher concentration of substrate no longer results in increased rate of reaction Cofactors Nonprotein entities Coenzymes Organic compound Ex. NADP  Coenzyme =  Velocity Activators Inorganic ions Alters spatial configuration of the enzyme for proper substrate binding Ex. Ca2+ (#1 activator), Zn2+ (LDH), Cl- (AMS), Mg2+ (CK, ALP) Metalloenzymes Inorganic ion attached to a molecule Ex. Catalase, cytochrome oxidase Inhibitors Interferes with the enzymatic reactions lec.mt 04 |Page | 30 Competitive inhibitor Binds to the active site of an enzyme Reversible (Substrate > Inhibitor) Noncompetitive inhibitor Binds to the allosteric site (cofactor site) Irreversible Uncompetitive inhibitor Binds to the enzyme-substrate complex  Substrate = ES = Inhibition Isoenzymes Same catalytic reactions but slightly different molecular structures Fractionation of isoenzymes Temperature 37’C = optimum temperature for enzyme activity Temperature = Reaction rate (movement of molecules) 40-50’C Denaturation of enzymes 60-65’C Inactivation of enzymes Temperature coefficient (Q10) For every 10OC increase in temperature, there will be a two-fold increase in enzyme activity pH Most physiologic reactions occur in the pH range of 7-8 Storage Enzymes: -20’C = for longer period of time Substrate and Coenzymes: 2-8’C LDH (LD4 & 5): Room temperature Hemolysis Mostly increases enzyme concentration Lactescence or milky specimen Decreases enzyme concentration Enzyme nomenclature 1st digit: classification 2nd and 3rd digits: subclass 4th digit(s): serial number Enzyme classification “OTHLIL” Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases Oxidoreductases Redox reaction Dehydrogenases: -Cytochrome oxidase -LDH -MDH -Isocitrate dehydrogenase -G-6-PD Transferases Transfer of a chemical group other than hydrogen from 1 substrate to another Kinases, Transaminases, Aminotransferases: -CK -GGT -AST -ALT -OCT Hydrolases Hydrolysis/splitting by addition of water Esterases: -ACP -ALP -CHS -LPS Peptidases: lec.mt 04 |Page | 31 -Trypsin -Pepsin -LAP Glycosidases: -AMS -Galactosidases Lyases Removal of groups w/o hydrolysis (product contains double bonds) Aldolase Decarboxylases: -Glutamate decarboxylase -Pyruvate decarboxylase -Tryptophan decarboxylase Isomerases Intramolecular arrangements Glucose phosphate isomerase Ribose phosphate isomerase Ligases Joining of 2 substrate molecules Synthases Active site Water-free cavity Where the substrate interacts Allosteric site Cavity other than the active site May bind regulatory molecules Prosthetic group Coenzyme that is bound tightly to the enzyme Holoenzyme Apoenzyme + Prosthetic group Zymogen/proenzyme Inactive form of enzyme Emil Fisher’s/Lock and Key Shape of the key (substrate) must fit into the lock (enzyme) theory Kochland’s/Induced fit theory Based on the substrate binding to the active site of the enzyme Acceptable theory Enzyme kinetics Enzymes catalyze reactions by lowering the activation energy level that the substrate must reach for the reaction to occur Absolute specificity Enzyme combines w/ only 1 substrate and catalyzes only 1 reaction Group specificity Enzymes combine w/ all the substrates in a chemical group Bond specificity Enzymes reacting w/ specific chemical bonds Zero-order reaction Reaction rate depends only on enzyme concentration Independent on substrate concentration First-order reaction Reaction rate is directly proportional to substrate concentration Independent on enzyme concentration Measurement of enzyme activity Change in substrate concentration Change in product concentration Change in coenzyme concentration International Unit 1 micromole of substrate/minute Katal Unit 1 mole of substrate/second Nonkinetic assay Absorbance is made at 10-second intervals for 100 seconds Alkaline Phosphatase pH = 10.5 405nm Electrophoresis: (+) Liver  Bone (Regan)  Placenta  Intestine (-) Heat fractionation: (Δ Stable) Regan  Placenta  Intestine  Liver  Bone (Δ Labile) Phenylalanine Inhibits Regan, placental and intestinal ALP L-leucine Inhibits Nagao ALP Levamisole Inhibits liver and bone ALP lec.mt 04 |Page | 32 3M urea Inhibits bone ALP Methods (ALP) Low temperature = Increased ALP 1. Bowers and McComb (PNPP) – IFCC recommended 2. Bessy, Lowry and Brock (PNPP) 3. Bodansky, Shinowara, Jones, Reinhart = BGP (beta glycerophosphate) 4. King and Armstrong = PP (phenylphosphate) 5. Klein, Babson & Read = Buffered PPP (phenolphthalein phosphate) 6. Huggins and Talalay = PPDP (phenolphthalein diphosphate) 7. Moss = ANP (alpha naphthol phosphate) Increased ALP Sprue Hyperparathyroidism Rickets (children) and osteomalacia (adults) Acid Phosphatase pH = 5.5 405nm Sources: Prostate (major), RBC, platelets, bone Prostatic ACP Inhibited by L-tartrate ions RBC ACP Inhibited by cupric and formaldehyde ions Methods (ACP) Room temperature (1-2 hrs) = decreased ACP Thymolphthalein monophosphate = specific substrate, substrate of choice (endpoint) Alpha-naphthyl phosphate = preferred for continuous monitoring methods 1. Gutman and Gutman = PP 2. Shinowara = PNPP 3. Babsonm Read and Phillips = ANP (continuous monitoring) 4. Roy and Hillman = Thymolphthalein monophosphate (endpoint) Aspartate Aminotransferase pH 7.5 (AST/SGOT) 340nm Sources: Cardiac tissue > Liver > Skeletal muscle > Kidney, pancreas, RBCs Alanine Aminotransferase pH 7.5 (ALT/SGPT) 340nm Major Source: Liver Methods (AST and ALT) 1. Karmen method = Kinetic 2. Reitman and Frankel = Endpoint -Color developer: DNPH -Color intensifier: 0.4N NaOH Increased Transaminases DeRitis ratio (ALT:AST) >1.0 = Acute hepatitis (Highest) 20x = viral or toxic hepatitis Moderate elevation = chronic hepatitis, hepatic cancer, IM Slight elevation = Hepatic cirrhosis, alcoholic hepatitis, obstructive jaundice Amylase Smallest enzyme (appears in urine) Earliest pancreatic marker P3: most predominant pancreatic AMS isoenzyme in AP Isoenzymes: S-type (ptyalin): anodal P-type (amylopsin): cathodal Methods (AMS) Samples w/ high activity of AMS should be diluted w/ NaCl to prev. inactivation Salivary AMS = inhibited by wheat germ lectin Substrate: Starch Saccharogenic Reducing sugars produced lec.mt 04 |Page | 33 Classic reference method (SU) Amyloclastic Degradation of starch Chromogenic Increase in color intensity Coupled-enzyme Continuous-monitoring technique Lipase Late marker (AP) Most specific pancreatic marker Methods (LPS) Substrate: Olive oil/Triolein 1. Cherry Crandal (Reference method) 2. Tietz and Fiereck 3. Peroxidase coupling (most commonly used method) Lactate dehydrogenase Lacks specificity RBC: 150x LDH than in serum Sources: LD1 (α-HBD) and LD2 = Heart, RBC, Kidneys LD3 = pancreas, lungs, spleen LD4 an LD5 = liver and muscle LD6 = alcohol dehydrogenase Methods (LDH) 1. Wacker method (forward/direct) = pH 8.8, 340 nm, most commonly used 2. Wrobleuski LaDue (reverse/indirect) = pH 7.2, 2x faster 3. Wrobleuski Cabaud 4. Berger Broida 10-fold increase (LDH) Hepatic carcinoma and toxic hepatitis 2-3x URL Viral hepatitis and cirrhosis Creatine Kinase Isoenzymes: CK-BB = most anodal, brain CK-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, 340nm 2. Oliver-Rosalki/ Rosalki & Hess (reverse/indirect) = most commonly used method, faster reaction; pH 6.8, 340nm Adenylate kinase Inside RBCs Interferes w/ CK assay Inhibited by adenosine monophosphate N-acetylcysteine Activate CK Liver cells and RBC Do not contain CK Cleland’s reagent and Partially restore lost activity of CK glutathione Electrophoresis Reference method for CK CK relative index (CKI) CKI (%) = CK-MB/Total CK x 100 Aldolase Isoenzymes: Aldolase A = Skeletal muscles Aldolase B = WBC, liver, kidney Aldolase C = brain tissue 5’ Nucleotidase Marker for hepatobiliary diseases and infiltrative lesions of the liver Methods: 1. Dixon and Purdon 2. Campbell, Belfield and Goldberg GGT Located in the canaliculi of the hepatic cells lec.mt 04 |Page | 34 Differentates the source of an elevated ALP level Sensitive indicator of occult alcoholism Increased: Obstructive jaundice Alcoholic hepatitis (most sensitive) Methods (GGT) Substrate: gamma-glutamyl-p-nitroanilide 1. Szass 2. Rosalki and Tarrow 3. Orlowski Cholinesterase/ Monitor effects of relaxants (succinylcholine) after surgery Pseudocholinesterase Marker for organophosphate poisoning (Low CHS) Methods: 1. Ellman technic 2. Potentiometric Angiotensin-Converting Enzyme A.k.a. peptidyldipeptidase A or Kininase II Converts angiotensin I  angiotensin II (lungs) Indicator of neuronal dysfunction (Alzheimer’s disease – CSF) Ceruloplasmin Ferrooxidase enzyme Ornithine 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/L Prostatic ACP = 0-3.5 ng/mL AST = 5-37 U/L ALT = 6-37 U/L AMS = 60-180 SU/dL (95-290 U/L) LPS = 0-1.0 U/mL LDH: Forward = 100-225 U/L Reverse = 80-280 U/L Acute Myocardial Infarction Markers M T y o g l o b i n Rise 1 - 3 h Peak 5 - 1 2 lec.mt 04 |Page | 35 h Normalize 1 7d 8 - 3 0 h Acute Pancreatitis Markers A m y l a s e Rise 2 - 1 2 h Peak 2 4 h Normalize 3 - 5 d Electrolytes Electroneutrality Equal no. of cations and anions Balance of charges 40-75% Average water content of the human body ECF 1/3 of total body water ICF 2/3 of total body water Normal plasma 93% water (Plasma: 13% > Whole blood) 7% solutes: (Increased in dehydration) -Proteins -Glucose -NPN -Lipids -Ions Vasopressin deficiency Excretion of 10-20L H2O everyday Volume and Osmotic regulation Sodium Potassium Chloride Electrolytes EC = Na+ > Cl- > HCO3- > Ca2+(5th) > iPO4 IC = K+ > Mg2+(4th) Myocardial rhythm and Potassium contractility Calcium lec.mt 04 |Page | 36 Neuromuscular excitability Magnesium Cofactors (enzyme) Calcium Magnesium (CK) Zinc Chloride (AMS) Potassium ATPase ion pump Magnesium Production and use of ATP from Magnesium glucose Phosphate Acid-base balance Bicarbonate Replication of DNA and Magnesium translation of mRNA Sodium Major contributor of osmolality (92%, together w/ Chloride and Bicarbonate) 100 mg/dL glucose = 1.6 mmol/L sodium Aldosterone Sodium Potassium =  Magnesium Atrial natriuretic factor  Sodium Hypernatremia Excess water loss Decreased water intake Hyperaldosteronism (Conn’s disease) Hypothalamic disease (Chronic hypernatremia) Hyponatremia Renal failure SIADH (increased water retention) Marked hemolysis (dilutional effect) <125 mmol/L = severe neuropsychiatric symptoms Thirst Major defense against hyperosmolality and hypernatremia 1-2% water deficit = severe thirst 150-160 mEq/L Na+ = Moderate deficit of water >165 mEq/L Na+ = Severe water deficit Pseudohyponatremia Hyperlipidemia (turbidity) (artifactual) Hyperproteinemia Methods (Na+) 1. FEP 2. AAS 3. ISE = Glass aluminum silicate 4. Colorimetry = Albanese Lein Potassium Concentration in RBC is 105 mmol/L Reciprocal relationship with H+ Specimen Considerations (K+) 0.5% hemolysis =  0.5 mmol/L Gross hemolysis =  30% Serum K+ > Plasma K+ by 0.1-0.7 mmol/L because of platelets (clot) 10-20% in muscle activity 0.3-1.2 mmol/L = mild to moderate exercise 2-3 mmol/L = vigorous exercise; fist clenching Hyperkalemia Decreased resting membrane potential  incr. contractility  lack of muscle excitability Decreased renal excretion (Dehydration, renal failure, Addison’s disease) Acidosis (DM) Muscle injury Spironolactone Hypokalemia Increased resting membrane potential  arrhythmia lec.mt 04 |Page | 37 Leads to hypomagnesemia Vomiting Diuretics Cushing’s syndrome Alkalosis Insulin overdose pH and K+  pH by 0.1 =  K+ by 0.2-1.7 mmol/L Methods (K+) Lithium heparin plasma = preferred 1. FEP 2. AAS 3. ISE = Valinomycin gel 4. Colorimetry = Lockhead and Purcell Chloride Chief counter ion of sodium in ECF Specimen Considerations (Cl-) Chloride methods measure bromide and iodide Cl- = HCO3- Methods (Cl-) 1. Schales and Schales: -Mercurimetric titration -Diphenylcarbazone -Excess Hg++ -(+) Blue violet 2. Whiterhorn Titration method -Mercuric thiocyanate -Reddish complex 3. Ferric perchlorate 4. Cotlove chloridometer -Coulometric amperometric titration -Excess Ag++ 5. ISE -Ion exchange membrane -Tri-n-octylpropylammonium chloride decanol Hyperchloremia Renal tubular acidosis Metabolic acidosis Diabetes insipidus (Dehydration) Prolonged diarrhea Hypochloremia Prolonged vomiting (HCl) Aldosterone deficiency (Na+ = Cl- = K+) Metabolic alkalosis (HCO3- = Cl-) Marked hemolysis (dilutional effect) Calcium 99%  Bones 1%  ECF Absorbed in the duodenum Absorption is favored at an acidic pH 3 Forms of Calcium 50% = Free/Ionized/Unbound/Active Calcium 40% = 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) lec.mt 04 |Page | 38 Cancer Hyperthyroidism Milk-alkali syndrome Hypocalcemia Tetany Alkalosis (Ca2+: from Blood  Bones) Acute pancreatitis (Ca2+: binds to damage pancreatic tissues) Primary hypocalcemia Low PTH Parathyroid gland disease Secondary hypocalcemia High PTH Renal failure ( excretion) Methods (Ca2+) 1. Clark Collip precipitation method -(+) Oxalic acid -Renal calculi 2. Ferro Ham Chloranilic acid precipitation method -(+)Chloranilic acid 3. Colorimetric = Ortho-Cresolphthalein complexone dyes -Dye: Arzeno III -8-hydroxyquinoline = chelates (inhibits) Mg2+ 4. EDTA titration method (Bachra, Dawer and Sobel) 5. AAS = Reference method 6. ISE = Liquid membrane 7. FEP Inorganic Phosphorus 85%  Bones 15%  ECF (iPO4) Maximally absorbed in the jejunum (Ca2+: duodenum) Trancellular shift: Once absorbed inside cells, it no longer comes out  used for energy production Dirunal variation:  late morning,  evening Organic phosphate = principal anion within cells Inorganic phosphate = part of the blood buffer (Measured in the clin.lab.) 3 Forms of Inorganic Phosphorus 55% = Free 35% = Complexed with ions 10% = Protein-bound PTH  PO4 =  Ca2+ Calcitonin  PO4 =  Ca2+ Growth hormone  PO4 (renal reabsorption) Practical considerations Fasting is required (Nonfasting:  PO4) Hyperphosphatemia Hypoparathyroidism Renal failure Hypervitaminosis D Hypophosphatemia Alcohol abuse = most common cause Primary hyperparathyroidism Avitaminosis 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%  Bones 46%  Muscles and soft tissues 1%  Serum and RBC Vasodilator 3 Forms of Magnesium 55% = Free/Ionized/Physiologically active lec.mt 04 |Page | 39 30% = Protein-bound 10% = Complexed with ions PTH Mg2+ =  Ca2+ =  PO4 Aldosterone (& Thyroxine) Mg2+ =  K+ =  Na+ Hypermagnesemia Addison’s disease Chronic renal failure Hypomagnesemia Acute renal failure Chronic alcoholism Methods (Mg2+) 1. Calmagite -(+) Reddish-violet complex 2. Formazen dye method -(+) Colored complex 3. Magnesium Thymol blue method -(+) Colored complex 4. AAS = reference method 5. 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 cell Anion Gap Difference between unmeasured anions and unmeasured cations QC for ISE Increased AG Uremia/renal failure Ketoacidosis Lactic acidosis Methanol poisoning Ethanol poisoning Ethylene glycol poisoning Salicylate poisoning Decreased AG Hypoalbuminemia Hypercalcemia Hyperlipidemia Multiple myeloma Cystic Fibrosis (Mucoviscidosis) Defective gene: Cystic fibrosis transmembranous conductance regulator (Chromosome 7) Miconeum ileus (Infants) Foul-smelling stool URT infection  Na+ and Cl- Pilocarpine Sweat inducer Gibson & Cooke pilocarpine Reference method (Sweat sodium and chloride) iontophoresis Iron Prooxidant 3-5g = Total body iron Ferrous = Hgb Ferric = Transferrin and Ferritin Methods (Iron) 1. Colorimetric = HCl and Ferrozine -(+) Blue color 2. Anodic stripping voltammetry Increased iron Hemochromatosis lec.mt 04 |Page | 40 Viral hepatitis Non-IDA Decreased iron IDA Malnutrition Chronic infection TIBC UIBC + Serum Iron Increased: IDA, hepatitis, iron-supplemented pregnancy Decreased: Non-IDA, nephrosis UIBC TIBC – Serum iron Measure of reserve iron binding capacity of transferrin % Transferrin Saturation Index of iron storage Increased: Iron overdose, hemochromatosis, sideroblastic anemia Decreased: IDA (lowest), malignancy, chronic infection Transferrin TIBC (μg/dL) x 0.70 = mg/dL Note Sodium 1/α Potassium Potassium 1/α Hydrogen ion Potassium α Magnesium Magnesium α Calcium Calcium 1/α Inorganic phosphate Chloride 1/α Bicarbonate Normal Values (Electrolytes) Sodium: Serum = 135-145 mmol/L [Critical: 160 mmol/L and 120 mmol/L] CSF = 136-150 mmol/L Potassium: Serum = 3.5-5.2 mmol/L [Critical: 6.5 mmol/L and 2.5 mmol/L] Chloride: Serum = 98-107 mmol/L Sweat = 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/dL Child = 4.5-5.5 mg/dL Magnesium: Serum = 1.2-2.1 mEq/L Anion Gap: w/ K+ = 10-20 mmol/L w/o K+ = 7-16 mmol/L Iron: Male = 50-160 μg/dL Female = 45-150 μg/dL TIBC: Adult = 245-425 μg/dL >40 y.o. = 10-250 μg/dL NB and Child = 100-200 μg/dL % Transferrin Saturation = 20-50% Blood Gases and pH lec.mt 04 |Page | 41 Regulation of Acid-Base balance Lungs and Kidneys CO2 + H2O <--(Carbonic anhydrase)--> H2CO3 H2CO3 <-------(Carbonic anhydrase)--> H+ + HCO3- 20:1 HCO3-: H2CO3 ratio 4:1 HPO4: H2PO4 ratio Expanded Henderson- pH = 6.1 + log [Total CO2 – (pCO2 x 0.03)] Hasselbalch equation pCO2 x 0.03 Chloride-isohydric shift Buffering effect of hemoglobin pCO2 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  acidosis Metabolic Acidosis Causes: -Bicarbonate deficiency -DKA (normochloremic acidosis) -Renal failure -Diarrhea (HCO3-) Compensation: Hyperventilation Compensated:  HCO3- + pCO2 + pH <7.4 Metabolic Alkalosis Causes: -Bicarbonate excess -Vomiting (Cl-) -Hypochloremia -Hypokalemia Compensation: Hypoventilation Compensated:  HCO3- + pCO2 + pH >7.4 Respiratory Acidosis Causes: -CO2 excess (Hypoventilation) -COPD -Drug overdose (morphine, barbiturates, opiates) Compensation: Bicarbonate retention Compensated:  HCO3- +  pCO2 + pH <7.4 Respiratory Alkalosis Causes -CO2 loss (Hyperventilation) Compensation: Bicarbonate excretion Compensated:  HCO3- +  pCO2 + pH >7.4 Full compensation pH  normal range Partial compensation pH  near normal Buffer base All forms of base that will titrate hydrogen ions Methods for Blood Gases and pH Specimen: Arterial blood Blood gas analyzers: meas. pH, pCO2, pO2 Factors affecting Blood gases & For every 1OC above 37OC: pH measurements  pH by 0.015  pO2 by 7%  pCO2 by 3% Bacterial contamination: consume O2 (pO2) Excess heparin (acid MPS) = pH Air exposure (bubbles): pO2 = 4 mmHg/2mins pCO2 = 4 mmHg/2mins Methods 1. Gasometer lec.mt 04 |Page | 42 (Blood gases & pH) a. Van Slyke b. Natelson -Mercury: produce vacuum -Caprylic alcohol: anti-foam reagent -Lactic acid -NaOH -NaHSO3 2. Electrodes a. 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 skin Blood gas QC Min. requirement: -1 sample every 8 hours -3 levels of control (acidosis, normal, alkalosis) every 24 hours Normal Values pH = 7.35-7.45 (Blood gases and pH) pCO2 = 35-45 mmHg Total CO2: WB arterial = 19-24 mmol/L WB venous = 22-26 mmol/L HCO3- = 21-28 mEq/L pO2 = 81-100 mmHg [Hypoxemia:] -Mild (61-80 mmHg) -Moderate (41-60 mmHg) -Severe (40 mmHg or less) O2 saturation = 94-100% Endocrinology Endocrine Hormone  blood circulation  specific receptor Paracrine Hormone  interstitial space  adjacent cell Autocrine Hormone  self-regulation Juxtacrine Hormone  direct cell-to-cell contact Exocrine Hormone  gut Neurocrine Hormone  neurons  extracellular space Neuroendocrine Hormone  neurons  nerve endings Glycoproteins FSH, hCG, TSH, LH Polypeptides ACTH, ADH, GH, angiotensin, calcitonin, CCK, gastrin, glucagons, insulin, MSH, oxytocin, PTH, PRL, somatostatin Steroids Precursor: cholesterol Aldosterone, cortisol, estrogen, progesterone, testosterone, vitamin D Amines Derived from amino acids Catecholamines, T3, T4 Hypothalamus Connected to the posterior pituitary by the infundibulum stalk Hypophyseal hormones: TRH, GnRH, GH-IH, GH-RH, PIF Pineal gland Melatonin: decreases pigmentation of the skin Pituitary gland Master Gland Located in the sella turcica or Turkish saddle Anterior Pituitary True endocrine gland lec.mt 04 |Page | 43 (Adenohypophysis) Hormones: PRL, GH, FSH, LH, TSH, MSH, ACTH GH (Somatotropin) Most abundant of all pituitary hormones Structurally similar to PRL and HPL Markedly elevated during deep sleep Dwarfism Decreased GH Acromegaly Increased GH GH deficiency tests 1. Insulin tolerance test = Gold standard (Confirmatory test) 2. Arginine stimulation test = 2nd confirmatory test Tests for Acromegaly 1. Somatomedin C or insulin-like growth factor I (Screening) -Increased: Acromegaly -Decreased: GH deficiency 2. OGTT (Confirmatory) -75g glucose FSH Spermatogenesis LH Helps Leydig cells to produce testosterone (male) Ovulation (female) Synthesis of androgens, estrogens, and progesterone TSH (Thyrotropin) Stimulates thyroid gland to produce T3 and T4 Increased: 1’ hypothyroidism, 2’ hyperthyroidism Decreased: 1’ hyperthyroidism, 2’ hypothyroidism, 3’ hypothyroidism ACTH (Corticotropin) Highest: 6-8 AM Lowest: 6-11 PM Not allowed to have contact with glass because it adheres to glass surface Collect blood in plastic tubes Prolactin Initiation and maintenance of lactation Inhibited by Dopamine Highest: 4AM and 8AM, and 8PM and 10PM Increased: Menstrual irregularity, infertility, amenorrhea, galactorrhea Panhypopituitarism From pituitary tumor (adenoma) or Ischemia Pituitary ischemia Hemorrhage or shock in a pregnant female at the time of deliver (Shechan’s) Posterior pituitary Release but not produce oxytocin and vasopressin (Neurohypophysis) Oxytocin Uterine contraction and milk ejection ADH/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 Diagnostic test for ADH (Conc. test) Neurogenic DI True Diabetes Insipidus Failure of the pituitary gland to secrete ADH Nephrogenic DI Failure of the kidneys to respond to normal or elevated ADH SIADH Syndrome of inappropriate ADH Sustained production of ADH Decreased urine volume Low plasma osmolality Low serum electrolytes Thyroid Gland Butterfly-shaped 2 lobes = connected by the isthmus Follicle Fundamental structural unit of the thyroid gland Follicular cells Secrete T3 and T4 lec.mt 04 |Page | 44 Parafollicular or C cells Secrete calcitonin Thyroglobulin Preformed matrix containing tyrosyl groups Stored in the follicular colloid of the thyroid gland Thyroid hormone Biosynthesis 1. Trapping of Iodine 2. Iodination: I2  Tyrosine ring  MIT and DIT 3. Condensation: MIT+DIT=T3 / DIT+DIT=T4 4. Release: T3/T4  Blood circulation 5. Transport of T3/T4 by proteins Protein-bound hormones Metabolically inactive Biologically inert Do not enter cells Storage sites Free hormones (FT3/FT4) Physiologically active Readily enters cells Reverse T3 (rT3) From removal of one iodine from T4 (product of T4 metabolism) Metabolically inactive I2 intake <50 μg/day Deficiency of hormone secretion T3 3,5,3’-Triiodothyronine Most active thyroid hormonal activity 75-80% is produced from the tissue deiodination of T4 Diagnosis of T3 thyrotoxicosis T4 3,5,3’5’-Tetraiodothyronine Principal secretory product All originated in the thyroid gland TBG Transports 70-75 of TT4, and majority of T3 TBPA (Transthyretin) Transports 15-20% of TT4 No affinity for T3 TBA Transports T3 and 10% of T4 Thyroid autoantigens TPO Tg TSHR 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  T3 disease) N-T4  TSH Graves’ disease 1’ Hyperthyroidism (Diffuse toxic goiter) Most common cause of thyrotoxicosis (autoimmune) Women > Men Anti-TSH receptor Riedel’s thyroiditis Thyroid  woody or stony-hard mass Subclinical hyperthyroidism No symptoms N-T3 and T4  TSH Subacute granulomatous/ Hyperthyroidism Subacute nonsuppurative/ De Painful thyroiditis Quervain’s thyroditis Neck pain, low-grade fever (-) anti-TPO,  ESR and Tg lec.mt 04 |Page | 45 Hypothyroidism Treatment: Levothyroxine Primary hypothyroidism  T3 and T4  TSH Hashimoto’s disease (Chronic Most common cause of 1’ hypothyroidism autoimmune thyroiditis) Thyroid is replaced by a nest of lymphoid tissue (T cells) Goiter (+) anti-TPO  TSH Myxedema coma Severe form of 1’ hypothyroidism Peculiar nonpitting swelling of the skin Skin is infiltrated by mucopolysaccharides “Puffy” face, thin eyebrows Secondary hypothyroidism  T3 and T4  TSH Tertiary hypothyroidism  T3 and T4  TSH  TRH Congenital hypothyroidism Mental retardation (child) (Cretinism) Screening: T4 Confirmatory: TSH Subclinical hypothyroidism N-T3 and T4  TSH TRH stimulation test Most specific and sensitive test for diagnosing thyroid disease Confirm borderline cases and euthyroid Graves’ disease : 1’ hypothyroidism : Hyperthyroidism Radioactive Iodine Uptake Measure the ability of the thyroid gland to trap iodine (RAIU) Thyroglobulin (Tg) assay Postoperative marker of thyroid cancer : Untreated and metastatic differentiated thyroid cancer, hyperthyroidism : Hypothyroidism, thyrotoxicosis factitia rT3 Assess borderline or conflicting laboratory results Free Thyroxine Index (FT4I) Indirectly assesses the level of FT4 in blood Equilibrium relationship of bound T4 and FT4 Reference method: Equilibrium dialysis FT4I = TT4 x T3U(%) or TT4 x THBR 100 TT3, FT3, FT4 FT4 test: differentiates drug induced TSH elevation and hypothyroidism TT3 or FT3: confirm hyperthyroidism Reference 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 test Estrogen: TBG Androgen: TBG Fine-needle aspiration Most accurate tool in the evaluation of thyroid nodules Recombinant Human TSH Test patients w/ thyroid cancers for the presence of residual or recurrent dis. lec.mt 04 |Page | 46 Tanned Erythrocyte Test for anti-Tg disorders Hemagglutination method Serum calcitonin test Marker for familial medullary thyroid carcinoma FT4 and TSH Best indicators of thyroid status FT3 and FT4 More specific indicators of thyroid function than meas. of total hormone Not affected by TBG Euthyroid sick syndrome Acutely ill but without thyroid disease  T3 and T4 N/ TSH  rT3 Parathyroid gland 4 parathyroid glands Smalles endocrine gland PTH Hypercalcemic hormone  Ca2+ (bone resorption and renal reabsorption) and Mg2+  iPO4 1’ hyperparathyroidism Defective: Parathyroid gland Most common cause of hypercalcemia Parathyroid adenoma  PTH and iCa2+ Hypercalciuria Phosphaturia  Hypophosphatemia If goes undetected  severe demineralization (osteitis fibrosa cystica) 2’ hyperparathyroidism In response to Ca2+ Hyperplasia of all 4 glands Causes: 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 tissues Hypoparathyroidism Accidental injury of the PT glands (neck) during surgery Autoimmune parathyroid destruction  PTH =  Ca2+ Hyperparathyroidism Acidosis Hypoparathyroidism Alkalosis Adrenal glands Pyramid-shaped Above the kidneys Adrenal cortex = outer (yellow) Adrenal medulla = inner (dark mahogany) Has prime effects on blood pressure Adrenal cortex Major site of steroid hormone production G cells: convert cholesterol  pregnenolone CPPP ring 17-carbon skeleton derived from cholesterol 3 layers (Adrenal cortex) 1. Zona Glomerulosa = Mineralocorticoids (Aldosterone) 2. Zona Fasciculata = Glucocorticoids (Cortisol) 3. Zona Reticularis = Weak androgens (androstenedione, DHEA) Cortisol Gluconeogenesis  hyperglycemia The only adrenal hormone that inhibit the secretion of ACTH Anti-inflammatory and immunosuppressive Diurnal:  6-8AM /  10PM-12AM Urinary metabolites: 17-OHCS and 17-KGS lec.mt 04 |Page | 47 Porter-Silber method Meas. 17-OHCS Rgt: DNPH in H2SO4 + Alcohol (+) Yellow Zimmerman reaction Meas. 17-KGS Rgt: m-dinitrobenzene (+) Reddish purple Oxidation procedure: Norymberski (Na+ bismuthate) Pisano method For quantitating metanephrines and normetanephrines Kober reaction For estrogen Rgt: H2SO4 + hydroquinone (+) Reddish brown color Cushing’s syndrome Excessive production of cortisol and ACTH (Hypercortisolism) Overuse of corticosteroids Buffalo hump Hyperglycemia Hypertension Hypercholesterolemia  Lymphocytes Screening tests (Cushing’s) 1. 24-hour urine free cortisol test 2. 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 cortisol 3. CRH stimulation test Addison’s disease Primary adrenal insufficiency (1’ Hypocorticolism)  Cortisol and aldosterone  ACTH (+) Hyperpigmentation Screen: ACTH Stimulation Test 2’ Hypocorticolism Secondary adrenal insufficiency Hypothalamic-pituitary insufficiency  ACTH Test: ACTH Stimulation test ACTH Stimulation test Corsyntropin: synthetic coritsol and aldosterone stimulator (Corsyntropin stimulation test) Differentiates: 2’ adrenal insufficiency (ACTH) from 3’ adrenal insufficiency ( ACTH) Metyrapone test Metyrapone: inhibitor of 11 β-hydroxylase Measures the ability of the pituitary gland to respond to declining levels of circulating cortisol, thereby secrete ACTH Alternative 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 variation Methods: HPLC or GC-MS HPLC-MS Reference method for measuring urinary free cortisol ITT (Insulin tolerance test) Gold standard for 2’ and 3’ hypocorticolism Confirms borderline response to ACTH stimulation test Serum ACTH Differentiates: lec.mt 04 |Page | 48 Cushing’s disease (ACTH) Cushing’s syndrome (0-ACTH) ACTH 17-OHCS and 17-KS Congenital Adrenal Hyperplasia Enzyme deficiencies: 1.) 21-hydroxylase = most common 2.) 11 β-hydroxylase = 2nd most common 3.) 3β-hydroxysteroid dehydrogenase-isomerase 4.) 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 night 18-hydroxysteroid dehydrogenase: enzyme needed for aldosterone synthesis Conn’s disease Aldosterone-secreting adrenal adenoma (1’ hyperaldosteronism) Screen: Plasma Aldo conc./Plasma renin activity ratio (PAC/PRA ratio) -(+): >50 ratio Confirm: Saline suppression test -(+): >5 ng/dL aldosterone 2’ Hyperaldosteronism Excessive production of renin Liddle’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 glands Glucocorticoid deficiency 21-hydroxylase deficiency Postural stimulation test Test for aldosterone Florinef Synthetic mineralocorticoid Weak androgens Precursors for the production of more potent androgens and estrogens Precursors: Pregnenolone and 17-OH pregnenolone Examples: DHEA and androstenedione Bound to steroid hormone binding globulin (SHBG) : Virilization (pseudohermaphroditism) DHEA Principal adrenal androgen (Dehydroepiandrosterone) Converted to estrone Adrenal medulla Chromaffin cells: secrete catecholamines Precursor: L-tyrosine Norepinephrine/Epinephrine ---(Monoamine oxidase and Catechol-0- methyl-transferase)---> Metanephrines and VMA 9:1 Norepinephrine: Epinephrine ratio Norepinephrine Primary amine  in CNS Metabolites: lec.mt 04 |Page | 49 -3-methoxy-4-hydroxyphenylglycol (MHPG) = Major metabolite -VMA Epinephrine Secondary amine Most abundant medullary hormone “Flight or fight hormone” Metabolites: -Vanillylmandelic acid (VMA) = Major metabolite -Metanephrines -Normetanephrines -HVA Dopamine Primary amine From the decarboxylation of 3,4-Dihydroxyphenylalanine (DOPA) Major metabolite: Homovanillic acid (HVA) Pheochromocytoma Tumors of the adrenal medulla Catecholamines Classic “Spells”: tachycardia, headache, chest tightness, sweating, hypertension Clonidine test Differentiates: Pheochromocytoma (Catecholamines not suppressed) from Neurogenic hypertension (50% decreased in catecholamines) Neuroblastoma Norepinephrine (Children)  urinary HVA, VMA or both and dopamine Methods (Catecholamines) Specimen: 24-hr urine and plasma 1. Chromatography: HPLC or GC-MS 2. RIA: sensitive screening test ->2000pg/mL = diagnostic for pheochromocytoma Estrogens Estrone = Postmenopausal women Estradiol = Premenopausal women (most potent, secreted by the ovary) Estriol = Pregnancy (placenta) Markers for Down Syndrome AFP Unconjugated Estriol hCG Inhibin A Karyotyping or FISH typing Test for Down syndrome (amniotic fluid) Progesterone Produced mainly by the corpus luteum Det. whether ovulation has occurred Luteal phase Tests for menstrual cycle Estrogen dysfunction and anovulation Progesterone FSH LH Tests for female infertility hCG PRL FT4 TSH FSH LH Estradiol Progesterone Pancreas (Exocrine) Digestive enzymes (AMS, LPS) Acinus: functional secretory unit Pancreas (Endocrine) Hormones: lec.mt 04 |Page | 50 Alpha cells (20-30%) = glucagon Beta cells (60-70%) = insulin Delta cells (2-8%) = somatostatin hCG Produced by the syncytiotrophoblasts (placenta) Maintain progesterone production by the corpus luteum Human placental lactogen (HPL) Stimulates development of mammary gland Increases maternal plasma glucose levels Diagnosis of intrauterine growth retardation Gastrin Secreted by G cells (stomach) Stimulates parietal cells to secrete HCl Stimulus: Amino acid Zollinger-Ellison syndrome Pernicious anemia Serotonin Synthesized by argentaffin cells (GIT) (5-hydroxytryptamine) Metabolite: 5-HIAA 5-HIAA Diagnostic marker for carcinoid syndrome Test: Ehrlich’s aldehyde test = (+) purple color Somatostatin A.k.a. GH-IH Inhibitor of GH, glucagon and insulin 1’ amenorrhea Menstruation having never occurred 2’ amenorrhea Absence of menses for 6 months Cushing’s disease Abnormal increased secretion of ACTH Cushing’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 males Hirsutism Excessive hair growth w/ a male distribution pattern in a female Most common endocrine disorder in women Mullerian agenesis Congenital malformation or absence of the fallopian tubes, uterus or vagina N-FSH, LH and testosterone Nonthyroidal illness Illness that do not directly involve the thyroid gland Sipples syndrome (MEN II) Medullary carcinoma of the thyroid Pheochromocytoma Parathyroid adenoma Stein-Leventhal syndrome Mild hirsutism w/ normal menses to excessive hirsutism w/ amenorrhea Thyroid stones A.k.a. thyroid crisis Life-threatening Uncontrolled thyrotoxicosis Normal Values T3: (Endocrinology) Adult = 80-200 ng/dL Children 1-14 y.o. = 105-215 ng/dL T4: Adult = 5.5-12.5 μg/dL Neonate = 11.8-22.6 μg/dL T3U = 25-35% Therapeutic Drug Monitoring Mixed function oxidase (MFO) Biochemical pathway responsible for the greatest portion of drug system metabolism Intravenous route 100% bioavailability lec.mt 04 |Page | 51 Liberation Drug  Release Absorption Drug  Blood (most: by passive diffusion) Distribution Drug  Tissues Metabolism Drug  Chemical modification Excretion Drug  metabolites  excreted Bioavailable fraction (f) Fraction of the dose that reaches the blood Vd 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 level Pharmacogenomics Study of genes that affect the performance of a drug in an individual Therapeutic 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 dose Peak concentration Highest concentration of a drug obtained in the dosing interval Drawn one hour after an orally administered dose (except digoxin) Cardioactive Drugs Class 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: CHF Lidocaine (Xylocaine) Local anesthetic 1’ product of hepatic metabolism: MEGX (monoethylglycinexylidide) Quinidine Common formulations: Quinidine sulfate and Quinidine gluconate Procainamide (Pronestyl) Hepatic metabolite: NAPA (N-acetylprocainamide) Toxic effect: reversible lupus-like syndrome Disopyramide Substitute for quinidine Anticholinergic effects Propanolol Tx: angina pectoris Amiodarone (Cordarone) Iodine-containing drug Verapamil Tx: angina, hypertension, supraventricular arrhythmias Antibiotics Aminoglycosides Tx: Gram (-) bacterial infections Nephrotoxic and ototoxic Vancomycin Tx: Gram (+) cocci and bacilli Toxic effects: “Red man syndrome” Nephrotoxic and ototoxic Antiepileptic Drugs Phenobarbital Long acting barbiturate Enhances bilirubin metabolism Inactive proform: Primidone Phenytoin (Dilantin) Injectable proform: fosphenytoin Valproic acid (Depakene) Tx: petit mal and grand mal Carbamazepine (Tegretol) Tx: grand mal Ethosuximide (Zarontin) Drug of choice for controlling petit mal seizure Gabapentin (Neurontin) Similar to neurotransmitter GABA lec.mt 04 |Page | 52 Others (Antiepileptic) Topiramate Lamotrigine (Lamictal) Felbamate Psychoactive Drugs Lithium Tx: Bipolar disorders (Manic depression) Tricyclic antidepressantas (TCA) Imipramine Amitriptyline Doxepin Nortriptyline Tradazone Major metabolite: Desipramine Fluoxetine (Prozac) Blocks reuptake of serotonin Tx: Obsessive-compulsive disorders Bronchodilator Theophylline Tx: Asthma and other COPD Anti-inflammatory and Analgesic Drugs Salicylates/Aspirin Antiplatelet (inhibits cyclooxygenase) (Acetylsalicylic acid) Method: Trinder assay Acetaminophen (Tylenol) Hepatotoxic Ibuprofen Lower risk of toxicity than salicylates and acetaminophen Neuroleptics (Antipsychotic major tranquilizers) Neuroleptics Block the action of dopamine and serotonin Tx: Schizophrenia 2 classes: -Phenothiazines (chlorpromazine) -Butyrophenones (haloperidol) Examples: -Risperdal -Olonzapine (Zyprexa) -Quetiapine (Seroquel) -Aripiprazole (Abilify) Immunosuppressants Cyclosporine Tacrolimus (FK-506) Rapamycin (Sirolimus) Mycophenolate mofetil Lefluamide Chemotherapeutic agents Busulfan Methotrexate Toxicology Toxic Agents Alcohols (%w/v) Common CNS depressants 0.01-0.05 No obvious impairment, some changes observable on performance testing 0.03-0.12 Mild euphoria, decr. inhibitions, some impairment of motor skills 0.09-0.25 Decr. inhibitions, loss of critical judgment, memory impairment, decr. rxn time 0.18-0.30 Mental confusion, dizziness, strongly impaired motor skills (slurred speech) 0.27-0.40 Unable to stand/walk, vomiting, impaired consciousness 0.35-0.50 Coma and possible death ≥0.10 Presumptive evidence of driving under influence of alcohol Ethanol (Grain alcohol) Most common abused drug Ethanol  Acetic acid lec.mt 04 |Page | 53 Major metabolic pathway: Ethanol ------(Alcohol Dehydrogenase)------> Acetaldehyde Testing: Use benzalkonium chloride as antiseptic Methanol (Wood alcohol) Cause blindness Methanol  Formaldehyde  Formic acid (liver) Isopropanol Liver metabolism: (Rubbing alcohol) Isopropanol  Acetone Ethylene glycol Antifreezing agent (1,2-ethanediol) Ethylene glycol  Oxalic acid and glycolic acid (+) Monohydrate calcium oxalate crystals Carbon Monoxide Colorless, odorless, tasteless gas Has 210x greater affinity than O2 for Hgb “Cherry-red” color of the face and blood Specimen: EDTA whole blood Method: 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” manifestation Tx: EDTA and dimercaptosuccinic acid (DMA) – remove lead Free erythrocyte protoporphyrin (+) Basophilic stippling (course) Mercury Amalgamate: mix or merge w/ other substances Specimen: -Whole blood (organic mercury) -Urine (inorganic mercury) Method: Reinsch test (Silvery gray) Drugs of Abuse Opiates Morphine Codeine Heroin Methadone Tranquilizers Diazepam (Valium) Oxazepam Barbiturates: Phenobarbital Sedative Hypnotics Pentobarbital Amobarbital Dopaminergic pathway Cocaine stimulants Benzoylecgonine Amphetamine Hallucinogens Phencyclidine Lysergic acid diethylamide Tetrahydrocannabinol Methaqualone lec.mt 04 |Page | 54 Amphetamines Increase mental alertness (“Uppers”) MDMA (methylenedioxymethamphetamine) = ecstasy Methamphetamine HCl = shabu Annabolic steroids Improves athletic performance by increasing muscle mass Cannabinoids Marijuana and hashish Tetrahydrocannabinol (THC) Psycoactive substance of marijuana Urinary metabolite: 11-nor-deltatetrahydrocannabinol (THC-COOH) Cocaine (Crack) Alkaloid salt Admin: Insufflation of IV or by inhalation/snorting Derived from coca plant (erythroxylon) Cardiac toxicity Prozac: inhibit the action of cocaine Urine metabolite: benzoylecgonine Opiates From opium poppy Heroin Morphine Codeine Methadone Major metabolites: N-acetylmorphine (heroin) and morphine Antagonist: Nalaxone (Narcan) Phencyclidine Hallucinogen (Angel dust or angel hair) Admin: Ingestion or inhalation Major metabolite: Phencyclidine HCl Sedative hypnotics Barbiturates (Secobarbital, pentobarbital, Phenobarbital) Benzodiazopines: Diazepam (Valium), Lorazepam (Ativan), Chlordiazepoxide (Librium) Major metabolite (barbiturates): Secobarbial Lysergic acid diethylamide (LSD, “Undulating vision” Lysergide) “Bad trip” – panic reactions Methaqualone (Quaalude) Pyramidal signs (Hypertonicity, hyperreflexia, myoclonus) Vitamins Vitamins Water soluble: B1, B2, B3, B5, B6, B9, B12, Biotin, C, Carnitine Fat soluble: A, D, E, K Vitamin A CN: Retinol Def: Night blindness Vitamin E CN: Tocopherol Def: Mild hemolytic anemia, RBC fragility Vitamin D2 CN: Ergocalciferol, Cholecalciferol (D2), 1,25-dihydroxycholecalciferol (D3) Vitamin D3 Def: Rickets (young), Osteomalacia (adult) Vitamin K CN: Phylloquinones, Menaquinones Def: Hemorrhage Vitamin B1 CN: Thiamine Def: Beriberi, Wernicke-Korsakoff syndrome Vitamin B2 CN: Riboflavin Def: Angular stomatitis, dermatitis, photophobia Vitamin B3 CN: Niacin/Niacinamide/Nicotinic acid/Nicotinamide Def: Pellagra (dermatitis, disorientation, weight loss) Vitamin B5 CN: Panthotenic acid Def: Depressed immune system, muscle weakness Vitamin B6 CN: Pyridoxine, Pyridoxal Def: Facial seborrhea Vitamin B9 CN: Folic acid, Pteroylglutamic acid lec.mt 04 |Page | 55 Def: Megaloblastic anemia Vitamin B12 CN: Cyanocobalamin Def: Megaloblastic anemia, neurologic abnormalities Vitamin C CN: Ascorbic acid Def: Scurvy Biotin Def: Dermatitis Carnitine Def: Muscle weakness, fatigue lec.mt 04 |Page | 56