Clinical Chemistry Reviewer by MTR

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 Preferred (Human: biohazard) materials Not for immunochem, dye-binding and bilirubin Matrix effect Improper product manufacturing Unpurified analyte Altered protein Precision study First step in method evaluation Nonlab. personnel 29% of errors (lab results) SD Dispersion of values from the mean CV Index of precision Relative magnitude of variability (%) Variance SD2 Measure of variability Inferential statistics Compare means or SD of 2 groups of data T-test Means of 2 groups of data F-test SD of 2 groups of data Cumulative Sum Graph V-mask (CUSUM) Earliest indication of systematic errors (trend) Youden/Twin Plot Compare results obtained from diff. lab Shewhart LeveyGraphic representation of the acceptable limits of variation Jennings Chart Trend Gradual loss of reliability Cause: Deterioration of reagents (Systematic error) Shift Values: one side or either side of the mean Cause: Improper calibration (Systematic error) Outliers Values: far from the main set of values Highly deviating values Random or systematic errors Kurtosis Degree of flatness or sharpness Precision Random error Accuracy Systematic error Random error Causes: (Imprecision; -Mislabeling Indeterminate) -Pipetting error -Improper mixing of sample and reagents -Voltage/Temperature fluctuation -Dirty optics Parameters: SD and CV Systematic error Causes: (Inaccuracy/Determinat -Improper calibration e) -Deterioration of reagents -Contaminated solution -Sample instability/unstable reagent blanks lec.mt 04 |Page | 1

Multirule Shewhart procedure Test method Reference method Analytical Run Physiologic Limit POCT Quality Assurance

Quality Patient Care Reference Range/ Interval Range/ Reference Values Wavelength Spectrophotometric meas. Photometric measurement LASER Visible region UV

IR Stray light Diffraction gratings Prisms Nickel sulfate Cutoff filter Bandpass Alumina silica glass cuvet Quartz/plastic cuvet Borosilicate glass cuvet Photodetector Barrier layer cell/

-Diminishing lamp power -Incorrect sample and reagent volume Parameter: Mean Control rules + Control chart Westgard: at least 40 samples Westgard: preferably 100 samples Control and patient specimens assayed, evaluated, and report together Referred to as absurd value Performed by nonlab personnel Tripod: Program development Assessment and monitoring Quality improvement Test request forms, clear instruction for patient prep., specimen handling… At least 120 individuals should be tested in each age and sex category Analytical Methods Distance bet 2 successive peaks (nm) Lower frequency = Longer wavelength (Ex. Red) Higher frequency = Shorter wavelength (Ex. Violet) Meas. light intensity in a narrower wavelength Meas. light intensity w/o consideration of wavelength Multiple wavelength (uses filter only) Light Amplification by Stimulated Emission of Radiation Light source for spectrophotometry Tungsten light bulb Mercury arc Deuterium lamp Mercury arc Xenon lamp Hydrogen lamp Merst glower Globar (Silicone carbide) Wavelength outside the band Most common cause of loss of linearity Most commonly used monochromator Cutting grooves Rotatable Prevents stray light Anti-stray light ½ peak transmittance Most commonly used cuvet UV Strong bases Converts transmitted light into photoelectric energy Simplest detector lec.mt 04 |Page | 2

photocell/ photovoltaic cell Phototube Photomultiplier tube Galvanometer/Ammete r Absorbance Double beam spectro.

Double-beam in space Double-beam in time Dydimium filter Holmium oxide filter Reagent blank Sample blank FEP

Cesium and Lithium Lithium AAS

Atomizer (nebulizer) Chopper Lanthanum/Strontium chloride Volumetric (Titrimetric) Turbidimetry Nephelometry Electrophoresis Iontophoresis Zone electrophoresis Endosmosis

No external voltage For filter photometers Contains anode and cathode Req external voltage Most common type Most sensitive UV and visible region Meter or read-out device A = abc (a = absorptivity; b = length of light (1cm); c = concentration) A = 2 – log%T Splits monochromatic light into two components: One beam  sample One beam  reference soln or blank (corrects for variation in light source intensity) 2 photodetectors (sample beam and reference beam) 1 photodetector Monochromatic light  sample cuvet and reference cuvet 600 nm 360 nm Color of reagents Optical interference (Hgb) Meas. light emitted by a single atom burned in a flame Principle: Excitation Lt. source and cuvette: Flame For excited ions (Na+, K+) Internal standards (FEP) Correct variations in flame Preferred internal std Potent antidepressant 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 Convert ions  atoms Modulate the light source Complex with phosphate Avoid calcium interference Unknown sample is made to react with a known solution in the presence of an indicator Light blocked Meas. abundant large particles (Proteins) Depend on specimen concentration and particle size Meas. amt of Ag-Ab complexes Scattered light Depends on wavelength and particle size Migration of charged particles in an electric field Migration of small charged ions Migration of charged macromolecules Movement of buffer ions and solvent relative to the fixed support lec.mt 04 |Page | 3

Cellulose acetate Agarose gel Polyacrylamide gel Electrophoretic mobility Isoelectric focusing Densitometry Capillary electrophoresis Southern blot Northern blot Western blot Chromatography Paper chromatography TLC Retention factor (Rf) value Gas chromatography

Gas Solid chromatography Gas Liquid chromatography Mass Spectrometry GC-MS MS/MS HPLC Hydrophilic gel Hydrophobic gel Ion exchange chromatography Partition chromatography Affinity chromatography Adsorption

Ex: gamma globulins Molecular size Electrical charge Charge and molecular size 20 fractions (ex. isoenzymes) Directly proportional to net charge Inversely proportional to molecular size & viscosity of the supporting medium Molecules migrate through a pH gradient pH = pI For isoenzymes: same size, different charge Scan & quantitate electrophoretic pattern Electro-osmotic flow DNA RNA Proteins Separation by specific differences in physical-chemical characteristics of the different constituents Fractionation of sugar and amino acid Sorbent: Whatman paper Screening: Drugs Relative distance of migration from the point of application Rf = Distance leading edge of component moves Total distance solvent front moves Separation of steroids, barbiturates, blood, alcohol, and lipids Volatile compounds Specimens  vaporized Mobile phase: Inert gases Differences in absorption at the solid phase surfaces Differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase Fragmentation and ionization Gold standard for drug testing Detect 20 inborn errors of metabolism from a single blood spot Most widely used liquid chromatography Fractionation of drugs, hormones, lipids, carbohydrates and proteins Gel filtration Separation of enzymes, antibodies and proteins Ex: Dextran and agarose Gel permeation Separation of triglyceride and fatty acid Ex: Sephadex Separation depends on the sign and ionic charge density Based on relative solubility in an organic solvent (nonpolar) and an aqueous solvent (polar) For lipoproteins, CHO and glycated hemoglobins Based on differences between the adsorption and desorption of lec.mt 04 |Page | 4

chromatography Fluorometry/Molecular Luminescence Spectro.

Quenching

Borosilicate glasswares Boron-free/Soft glasswares Corex (Corning) Vycor (Corning) Flint glass TD: To deliver TC: To contain Blowout Self-draining Transfer pipet

Graduated or measuring pipet

Micropipettes

Air displacement pipet Positive displacement pipet Dispenser/Dilutor pipet

solutes at the surfaces of a solid particle Det. amt. of lt. emitted by a molecule after excitation by electromagnetic 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 Major disadvantage of fluorometry pH and temperature changes, chemical contaminants, UVL changes Instrumentation For heating and sterilization Ex: Pyrex and Kimax High resistance to alkali Special alumina-silicate glass Strengthened chemically than thermally 6x stronger than borosilicate For high thermal, drastic heat and shock Can be heated to 900OC Soda-lime glass + Calcium, Silicon, Sodium oxides Easy to melt For making disposable glasswares Exact amount Does not disperse the exact volume w/ etched rings on top of pipet w/ o etched rings Drain by gravity 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 Serological: w/ graduations to the tip (blowout) Mohr: w/o graduations to the tip (self-draining) Bacteriologic Ball, Kolmer and Kahn Micropipettes: 400mg/dL Bilirubin: 25.2 mg/dL Interfere with: "TACGu” Total Protein Albumin Cholesterol Glucose Preferred position Patient should be seated/supine at least 20 mins before blood collection to prevent hemodilution or hemoconcentration Vasoconstriction  Reduced plasma volume Increased: “ECA” Enzymes Calcium Albumin Hemoconcentration Increased: “P(u)BLIC” Proteins BUN Lipids Iron Calcium Hemodilution Decreased: “TLC” Triglycerides Lipoproteins Cholesterol Increased: K+ (muscles) Decreased: Albumin (Fluid retention) Recommended: 1 minute application Hemoconcentration Anaerobiosis Increased: “C2LEA2K” Calcium Cholesterol Lactate Enzymes Ammonia Albumin K+ Increased: “TUNG2C3” Triglycerides Urea Nonesterified fatty acid Glucose GH Catecholamines Cortisol Cholesterol Increased: “THUG” Triglycerides Hypoglycemia (chronic alcoholism) lec.mt 04 |Page | 8

Ammonia Stress (anxiety)

Drugs

Diurnal variation

Sleeping patients Unconscious patients Venipuncture Tourniquet Needle After blood collection BP cuff as tourniquet Benzalkonium chloride (Zephiran) IV line on both arms IV fluid contamination

Renin blood level Basal state collection

Uric acid/Urates GGT Increases by 100-200μg/L/cigar Increased: “LAGIC” Lactate Albumin Glucose Insulin Cholesterol Medications affecting plasma volume can affect protein, BUN, iron, calcium Hepatotoxic drugs: increased liver function enzymes Diuretics: decreased sodium and potassium "CA3PI2TG” Cortisol ACTH ACP Aldosterone Prolactin Iron Insulin Thyroxine GH Specimen Collection and Handling Must be awakened before blood collection Ask nurse or relative Identification bracelet Median Cubital (1st)  Cephalic (2nd)  Basilic (3rd) Velcro or Seraket type 3-4 inches above the site Not exceed 1 minute Bevel up 15-30O angle Length: 1 or 1.5 inch (Butterfly needle: ½ to ¾ inch) Cotton  site Apply pressure for 3-5 minutes Inflate to 60 mmHg Disinfectant for ethanol testing Dilution – 1:750 Discontinue IV for 2 minutes Collect sample below the IV site Initial sample (5mL)  discard Increased: Glucose (10% contam. w/ 5% dextrose  increased bld glucose by 500 mg/dL) Chloride Potassium Sodium Decreased: Urea Creatinine Collected after a 3-day diet, from a peripheral vein Early morning blood collection lec.mt 04 |Page | 9

Lancet Incision (Skin puncture) 1.5-2.4mm Arterialized capillary blood Flea Indwelling umbilical artery 1000-3000 RCF for 10 mins Hemolysis

Refrigeration/Chilling (Low temp)

Photosensitive analytes

Oxalate Citrate EDTA

12 hours after the last ingestion of food 1.75mm: preferred length to avoid penetrating the bone 500 mg/dL  nonketotic hyperosmolar coma 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 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 FBS = 100-125 mg/dL FBS = Ferrocyanide (Yellow) (Colorless) Schiff’s base Measures beta-D-glucose (65%) Converts alpha-D-glucose (35%) to beta-D-glucose (65%) lec.mt 04 |Page | 15

NADH/NADPH Polarographic glucose oxidase Hexokinase method G-6-PD Interfering substances (Glucose oxidase) Hemolysis (>0.5 g/dL Hgb) Dextrostics OGTT IVGTT

Requirements for OGTT

Glucose load HbA1c

IDA and older RBCs RBC lifespan disorders Fructosamine (Glycosylated albumin/ plasma protein ketoamine) Galactosemia

Essential fructosuria Hereditary fructose intolerance Fructose-1,6biphosphate deficiency

Absorbance at 340nm Consumption of oxygen on an oxygen-sensing electrode O2 consumption α glucose concentration Most specific method Reference method Uses G-6-PD Most specific enzyme rgt for glucose testing False-decreased Bilirubin Uric acid Ascorbate Major interfering substance in hexokinase method (falsedecreased) Cellular strip Strip w/ glucose oxidase, peroxidase and chromogen Janney-Isaacson method (Single dose) = most common Exton Rose (Double dose) Drink the glucose load within 5 mins 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 Ambulatory Fasting: 8-14 hours Unrestricted diet of 150g CHO/day for 3 days Do not smoke or drink alcohol 75 g = adult (WHO std) 100 g = pregnant 1.75 g glucose/kg BW = children 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) High HbA1c Low HbA1c 2-3 weeks Useful for patients w/ hemolytic anemias and Hgb variants Not used in cases of low albumin Specimen: Serum Congenital deficiency of 1 of 3 enzymes in galactose metabolism Galactose-1-phosphate uridyl transferase (most common) Galactokinase Uridine diphosphate galactose-4-epimerase Autosomal recessive Fructokinase deficiency Defective fructose-1,6-biphosphate aldolase B activity Failure of hepatic glucose generation by gluconeogenic precursors such as lactate and glycerol lec.mt 04 |Page | 16

Glycogen Storage Disease Ia = Von Gierke II = Pompe III = Cori Forbes IV = Andersen V = McArdle VI = Hers VII = Tarui XII = Fanconi-Bickel CSF glucose < 0.5 C-peptide 5:1 to 15:1 D-xylose absorption test Gerhardt’s ferric chloride test Nitroprusside test Acetest tablets Ketostix KetoSite assay Normal Values (Carbohydrates)

Phospholipids Sphingomyelin Forms of phospholipids TLC + Densitometric quantitation Microviscosity Cholesterol

Autosomal recessive Defective glycogen metabolism Test: IVGTT (Type I GSD) Glucose-6-Phosphatase deficiency (most common worldwide) Alpha-1,4-glucosidase deficiency (most common in the Philippines) Debrancher enzyme deficiency Brancher enzyme deficiency Muscle phosphorylase deficiency Liver phosphorylase deficiency Phosphofructokinase deficiency Glucose transporter 2 deficiency Collect blood glucose at least 60 mins (to 2 hrs) before the lumbar puncture (Because of the lag in CSF glucose equilibrium time) Normal CSF : serum glucose ratio Formed during conversion of pro-insulin to insulin Normal C-peptide : insulin ratio Differentiate pancreatic insufficiency from malabsorption (low blood or urine xylose) Acetoacetate 10x more sensitive to acetoacetate than to acetone Acetoacetate and acetone Detects acetoacetate better than acetone Detects beta-hydroxybutyrate but not widely used RBS = anabolism Excessive tissue destruction Anabolism > catabolism Growth and repair processes Transports thyroxine and retinol (Vit. A) Landmark to confirm that the specimen is really CSF Maintains osmotic pressure Negative acute phase reactant Acute phase reactant Major inhibitor of protease activity 90% of alpha1-globulin band 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) Low pI (2.7) Negatively charged even in acid solution Acute phase reactant Binds and inactivates PSA Increased: Alzheimer’s disease, AMI, infection, malignancy, burns Acute phase reactant Binds free hemoglobin (alpha chain) 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]) Larges major nonimmunoglobulin protein Increased: Nephrotic syndrome (10x) Forms a complex w/ PSA Affinity w/ vitamin D and actin

Binds free heme HLA Filtered by glomeruli but reabsorbed lec.mt 04 |Page | 22

Transferrin/Siderophilin (beta) Complement (beta) Fibrinogen (bet. beta and gamma) CRP (gamma) Immunoglobulins (gamma) Myoglobin Troponins TnT (Tropomyosinbinding subunit) TnI (Inhibitory subunit or Actin-binding unit)

TnC Glomerular proteinuria Tubular proteinuria Overload proteinuria Postrenal proteinuria Microalbuminuria

CSF Oligoclonal banding

Serum Oligoclonal banding Alkaptonuria Homocystinuria

MSUD

Negative acute phase reactant Major component of beta2-globulin fraction Pseudoparaproteinemia in severe IDA Increased: Hemochromatosis (bronze-skin), IDA C3: major Acute phase reactant Between beta and gamma globulins General scavenger molecule Undetectable in healthy individuals hsCRP: warning test to persons at risk of CAD Synthesized by the plasma cells IgG>IgA>IgM>IgD>IgE Marker: Ischemic muscle cells, chest pain (angina), AMI Most important marker for AMI Specific for heart muscle Det. unstable angina (angina at rest) Only found in the myocardium 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 Binds calcium ions and regulate muscle contractions Most common and serious type Often called albuminuria Defective reabsorption Slightly increased albumin excretion Hemoglobinuria Myoglobinuria Bence-Jones proteinuria Urinary tract infection, bleeding, malignancy 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) 2 or more IgG bands in the gamma region: Multiple sclerosis Encephalitis Neurosyphilis Guillain-Barre syndrome Neoplastic disorders Leukemia Lymphoma Viral infections Ochronosis (tissue pigmentation) Impaired activity of cystathione beta-synthetase Elevated homocysteine and methionine in blood and urine Screen: Modified Guthrie test (Antagonist: L-methionine sulfoximine) Markedly reduced or absence of alpha-ketoacid decarboxylase 4 mg/dL of leucine is indicative of MSUD lec.mt 04 |Page | 23

PKU Normal Values (Proteins) Tests for GFR

Tests for Renal Blood Flow Tests Measuring Tubular Function

GFR Inulin clearance Creatinine clearance Urea clearance Cystatin C

BUN 2.14 Fluoride or citrate Thiosemicarbazide Ferric ions Diacetyl monoxime method Urease method Coupled urease Isotope dilution mass spectrometry NPN

Screen: Modified Guthrie test (Antagonist: 4-azaleucine) Diagnostic: Amino acid analysis (HPLC) Deficiency of tetrahydrobiopterin (BH4)  elevated blood phenylalanine Total protein = 6.5-8.3 g/dL Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL Kidney Function Tests Clearance: -Inulin clearance -Creatinine clearance -Urea clearance Phenolsulfonphthalein dye test Cystatin C BUN Creatinine Uric acid Excretion: -Para-amino hippurate test (Diodrast test) -Phenolsulfonphthalein dye test Concentration: -Specific gravity -Osmolality Decreases by 1.0 mL/min/year after age 20-30 years 150 L of glomerular filtrate is produced daily Reference method Best alternative method Measure of the completeness of a 24-hour urine collection Excretion: 1.2-1.5 g creatinine/day Demonstrate progression of renal disease or response to therapy Low MW protease inhibitor FilteredNot secretedCompletely reabsorbed (PCT) Indirect estimates of GFR Its presence in urine denotes damage to PCT Synthesized from Ornithine or Kreb’s Henseleit cycle First metabolite to elevate in kidney diseases Better indicator of nitrogen intake and state of hydration BUN  Urea (mg/dL) Inhibit urease Enhance color development (BUN mtd) Yellow diazine derivative Routinely used Urease: prepared from jack beans Urea ---(Urease)--> NH4 + Berthelot reagent (Measure ammonia) Glutamate dehydrogenase method UV enzymatic method Reference method For research purposes 45% Urea 20% Amino acid lec.mt 04 |Page | 24

Creatinine Enzymatic methods (Creatinine) Direct Jaffe method Interferences (Direct Jaffe)

Folin Wu Method Lloyd’s or Fuller’s Earth method Lloyd’s reagent Fuller’s earth reagent Jaffe reagent (Alk. picrate) Kinetic Jaffe method Azotemia Pre-renal azotemia

Renal azotemia

Post renal azotemia

Uremia

Uric acid Hyperuricemia

20% Uric acid 5% Creatinine 1-2% Creatine 0.2% Ammonia Derived from alpha-methyl guanidoacetic acid (creatine) Produced by 3 amino acids (methionine, arginine, lysine) Most commonly used to monitor renal function Creatinine Aminohydrolase – CK method Creatinase-Hydrogen Peroxide method – benzoquinonemine dye (red) Creatininase (a.k.a. creatinine aminohydrolase) Formation of red tautomer of creatinine picrate Falsely increased: Ascorbate Glucose Uric acid Alpha-keto acids (+) Red orange tautomer True measure of creatinine Sensitive and specific Uses adsorbent to remove interferences (UA, Hgb, Bili) Sodium aluminum silicate Aluminum magnesium silicate Satd. picric acid + 10% NaOH Popular, inexpensive, rapid and easy to perform Requires automated equipment Elevated urea and creatinine in blood Decreased GFR but normal renal function Dehydration, shock, CHF Increased: BUN Normal: Creatinine 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 Urinary tract obstruction Decreased GFR Nephrolithiasis, cancer or tumors of GUT Creatinine = normal or slightly increased Marked elevation of urea, accompanied by acidemia and electrolyte imbalance (K+ elevation) of renal failure Normocytic, normochromic anemia Uremic frost (dirty skin) Edema Foul breath Urine-like sweat From purine (adenine and guanine) catabolism Excretion: 1g/day -Gout -Increased nuclear metabolism (leukemia, lymphoma, MM, lec.mt 04 |Page | 25

Hypouricemia Methods (Uric acid) Phosphotungstic acid mtd NaCN

NaCO3 Lagphase Uricase method

Para-amino hippurate test Phenolsulfonphthalein dye test Concentration tests Specific gravity Osmolality

Direct methods (Osmolality) Incr. plasma osmolality Tubular failure Osmolal gap Osmolal gap: >12

polycythemia, hemolytic and megaloblastic anemia) – Tx: Allopurinol -Chronic renal disease -Lesch-Nyhan syndrome (HGPRT deficiency) Fanconi’s syndrome Wilson’s disease Hodgkin’s disease Stable for 3 days Potassium oxalate cannot be used Major interferences: Ascorbate and bilirubin Uric acid + Phosphotungstic acid ---(NaCN/NaCO3)--> Tungsten blue + Allantoin Folin Newton Brown Benedict Archibald Henry Caraway Incubation period after the addition of an alkali to inactivate nonuric acid reactants Simplest and most specific method Candidate reference method Uric acid (Absorbance at 293nm) ---[Uricase]--> Allantoin (No absorbance) Decrease in absorbance α uric acid concentration Measures renal plasma flow Reference method for tubular function Measures excretion of dye proportional to renal tubular mass 6 mg of PSP is administered IV Collecting tubules and loops of Henle Specimen: 1st morning urine Affected by solute number and mass SG >1.050: X-ray dye and mannitol 1.010 = SG of ultrafiltrate in Bowman’s space Total number solute particles present/kg of solvent (moles/kg solvent) Affectted only by number of solutes present Urine osmolality = due to urea Serum osmolality = due to sodium and chloride Det. by Colligative properties: Freezing point (incr. osm. = decr. FP) Vapor pressure (incr. osm. = decr. VP) Osmotic pressure (incr. osm. = incr. OP) Boiling point (incr. osm. = incr. BP) Freezing point osmometry = popular method Vapor pressure osmometry (Seebeck effect) Incr. vasopressin (H2O reabsorption)  decr. plasma osmolality Increased: BUN, creatinine, calcium Decreased: Phosphate Difference between measured and calculated osmolality Sensitive indicator of alcohol or drug overdose DKA lec.mt 04 |Page | 26

mOsm/kg Normal Values (Kidney Function Tests)

Liver Synthetic function Conjugation function Detoxification and Drug metabolism Excretory and Secretory functions Storage function Test measuring the Hepatic Synthetic Ability

Test measuring Conjugation/Excretion Function Test for Detoxification Function

Drug overdose Renal failure Creatinine Clearance: 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 [1:1 = Glomerular disease] [1.2:1 = loss of renal concentrating ability] [2 or 3 mg/dL Hemolytic B1 = increased B2 = normal UG = increased UB = negative Hepatocellular B1 = increased B2 = increased UG = increased UB = positive ALT = increased AST = increased Obstructive B1 = normal B2 = increased UG = decreased/negative UB = positive lec.mt 04 |Page | 29

Gilbert’s syndrome Crigler-Najjar syndrome

Dubin-Johnson syndrome & Rotor syndrome Lucey-Driscoll syndrome Methods (Bilirubin) Van den Berg reaction Evelyn and Malloy method

Jendrassik and Grof

Bilirubin Rosenthal White method Mac Donald method Ammonia

ALP = increased GGT = increased Cholesterol = increased Bilirubin transport deficit (uptake) B1 = increased B2 = decreased Conjugation deficit Type I = total UDPGT deficiency Type II = partial UDPGT deficiency B1 = increased B2 = decreased Danger: Kernicterus Bile is colorless Bilirubin excretion deficit Blockade of excretion into the canaliculi TB = increased B2 = increased Circulating inhibitor of bilirubin conjugation B1 = increased Free from hemolysis and lipemia Store in the dark Measured ASAP or w/in 2-3 hours Diazotization of bilirubin 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 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 Absorbs light maximally at 450nm Double collection method Collection: -After 5 mins (50% dye retention) -After 30 mins (0% dye retention) Single collection method Collection: -After 45 mins (+/- 5% dye retention) From deamination of amino acids Elevated levels are neurotoxic and often associated w/ encephalopathy and acetaminophen poisoning Diagnosis of hepatic failure and Reye’s syndrome In severe liver disorder: NH3  circulation  brain (conv. to glutamine)  increases pH  compromise the Kreb’s cycle  Coma due to lack of ATP for the brain lec.mt 04 |Page | 30

Methods (Ammonia)

Kjeldahl (Digestion) method Nesslerization of ammonia Berthelot reaction Normal Values (Liver Function Tests)

Enzyme concentration Substrate concentration Saturation kinetics Cofactors Coenzymes Activators

Metalloenzymes Inhibitors Competitive inhibitor Noncompetitive inhibitor

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 Specimen  PFF N2 ----------(hot conc. H2SO4 + CuSO4 + Hg + Selenium)----------> NH3 NH3 + K2Hg2I2 ----------(Gum Ghatti)----------> NH2Hg2I2 End color: Yellow (low to moderate N2) Orange brown (high N2) NH3 + Phenol + Hypochlorite -----(Na Nitroprusside)-----> Indophenol blue Total protein = 6.5-8.3 g/dL 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 Serum  Enzyme concentration =  reaction rate Reagent If enzyme > substrate,  substrate =  reaction rate When substrate concentration reaches a maximal value, higher concentration of substrate no longer results in increased rate of reaction Nonprotein entities Organic compound Ex. NADP  Coenzyme =  Velocity Inorganic ions Alters spatial configuration of the enzyme for proper substrate binding Ex. Ca2+ (#1 activator), Zn2+ (LDH), Cl- (AMS), Mg2+ (CK, ALP) Inorganic ion attached to a molecule Ex. Catalase, cytochrome oxidase Interferes with the enzymatic reactions Binds to the active site of an enzyme Reversible (Substrate > Inhibitor) Binds to the allosteric site (cofactor site) Irreversible lec.mt 04 |Page | 31

Uncompetitive inhibitor Isoenzymes Temperature 40-50’C 60-65’C Temperature coefficient (Q10) pH Storage Hemolysis Lactescence or milky specimen Enzyme nomenclature Enzyme classification

Oxidoreductases

Transferases

Hydrolases

Binds to the enzyme-substrate complex  Substrate = ES = Inhibition Same catalytic reactions but slightly different molecular structures Fractionation of isoenzymes 37’C = optimum temperature for enzyme activity Temperature = Reaction rate (movement of molecules) Denaturation of enzymes Inactivation of enzymes For every 10OC increase in temperature, there will be a two-fold increase in enzyme activity Most physiologic reactions occur in the pH range of 7-8 Enzymes: -20’C = for longer period of time Substrate and Coenzymes: 2-8’C LDH (LD4 & 5): Room temperature Mostly increases enzyme concentration Decreases enzyme concentration 1st digit: classification 2nd and 3rd digits: subclass 4th digit(s): serial number “OTHLIL” Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases Redox reaction Dehydrogenases: -Cytochrome oxidase -LDH -MDH -Isocitrate dehydrogenase -G-6-PD Transfer of a chemical group other than hydrogen from 1 substrate to another Kinases, Transaminases, Aminotransferases: -CK -GGT -AST -ALT -OCT Hydrolysis/splitting by addition of water Esterases: -ACP -ALP -CHS -LPS Peptidases: -Trypsin -Pepsin -LAP Glycosidases: lec.mt 04 |Page | 32

Lyases

Isomerases Ligases Active site Allosteric site Prosthetic group Holoenzyme Zymogen/proenzyme Emil Fisher’s/Lock and Key theory Kochland’s/Induced fit theory Enzyme kinetics Absolute specificity Group specificity Bond specificity Zero-order reaction First-order reaction Measurement of enzyme activity International Unit Katal Unit Nonkinetic assay Alkaline Phosphatase

Phenylalanine L-leucine Levamisole 3M urea Methods (ALP)

-AMS -Galactosidases Removal of groups w/o hydrolysis (product contains double bonds) Aldolase Decarboxylases: -Glutamate decarboxylase -Pyruvate decarboxylase -Tryptophan decarboxylase Intramolecular arrangements Glucose phosphate isomerase Ribose phosphate isomerase Joining of 2 substrate molecules Synthases Water-free cavity Where the substrate interacts Cavity other than the active site May bind regulatory molecules Coenzyme that is bound tightly to the enzyme Apoenzyme + Prosthetic group Inactive form of enzyme Shape of the key (substrate) must fit into the lock (enzyme) Based on the substrate binding to the active site of the enzyme Acceptable theory Enzymes catalyze reactions by lowering the activation energy level that the substrate must reach for the reaction to occur Enzyme combines w/ only 1 substrate and catalyzes only 1 reaction Enzymes combine w/ all the substrates in a chemical group Enzymes reacting w/ specific chemical bonds Reaction rate depends only on enzyme concentration Independent on substrate concentration Reaction rate is directly proportional to substrate concentration Independent on enzyme concentration Change in substrate concentration Change in product concentration Change in coenzyme concentration 1 micromole of substrate/minute 1 mole of substrate/second Absorbance is made at 10-second intervals for 100 seconds pH = 10.5 405nm Electrophoresis: (+) Liver  Bone (Regan)  Placenta  Intestine (-) Heat fractionation: (Δ Stable) Regan  Placenta  Intestine  Liver  Bone (Δ Labile) Inhibits Regan, placental and intestinal ALP Inhibits Nagao ALP Inhibits liver and bone ALP Inhibits bone ALP Low temperature = Increased ALP 1. Bowers and McComb (PNPP) – IFCC recommended lec.mt 04 |Page | 33

Increased ALP Acid Phosphatase Prostatic ACP RBC ACP Methods (ACP)

Aspartate Aminotransferase (AST/SGOT) Alanine Aminotransferase (ALT/SGPT) Methods (AST and ALT)

Increased Transaminases

Amylase

Methods (AMS)

Saccharogenic

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) Sprue Hyperparathyroidism Rickets (children) and osteomalacia (adults) pH = 5.5 405nm Sources: Prostate (major), RBC, platelets, bone Inhibited by L-tartrate ions Inhibited by cupric and formaldehyde ions 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) pH 7.5 340nm Sources: Cardiac tissue > Liver > Skeletal muscle > Kidney, pancreas, RBCs pH 7.5 340nm Major Source: Liver 1. Karmen method = Kinetic 2. Reitman and Frankel = Endpoint -Color developer: DNPH -Color intensifier: 0.4N NaOH 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 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 Samples w/ high activity of AMS should be diluted w/ NaCl to prev. inactivation Salivary AMS = inhibited by wheat germ lectin Substrate: Starch Reducing sugars produced Classic reference method (SU) lec.mt 04 |Page | 34

Amyloclastic Chromogenic Coupled-enzyme Lipase Methods (LPS)

Lactate dehydrogenase

Methods (LDH)

10-fold increase (LDH) 2-3x URL Creatine Kinase

Duchenne’s muscular dystrophy CK-MB Methods (CK) Adenylate kinase N-acetylcysteine Liver cells and RBC Cleland’s reagent and glutathione Electrophoresis CK relative index (CKI) Aldolase

5’ Nucleotidase

Degradation of starch Increase in color intensity Continuous-monitoring technique Late marker (AP) Most specific pancreatic marker Substrate: Olive oil/Triolein 1. Cherry Crandal (Reference method) 2. Tietz and Fiereck 3. Peroxidase coupling (most commonly used method) 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 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 Hepatic carcinoma and toxic hepatitis Viral hepatitis and cirrhosis Isoenzymes: CK-BB = most anodal, brain CK-MB = myocardium (20%) CK-MM = least anodal, skeletal and smooth muscles (Major, 94100%) Total CK: 50x URL (highest) Most specific indicator of myocardial damage (AMI) Not elevated in angina 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 Inside RBCs Interferes w/ CK assay Inhibited by adenosine monophosphate Activate CK Do not contain CK Partially restore lost activity of CK Reference method for CK CKI (%) = CK-MB/Total CK x 100 Isoenzymes: Aldolase A = Skeletal muscles Aldolase B = WBC, liver, kidney Aldolase C = brain tissue Marker for hepatobiliary diseases and infiltrative lesions of the liver Methods: 1. Dixon and Purdon 2. Campbell, Belfield and Goldberg lec.mt 04 |Page | 35

GGT

Methods (GGT)

Cholinesterase/ Pseudocholinesterase

Angiotensin-Converting Enzyme Ceruloplasmin Ornithine carbamoyl transferase G-6-PD Normal Values (Enzymes)

Located in the canaliculi of the hepatic cells Differentates the source of an elevated ALP level Sensitive indicator of occult alcoholism Increased: Obstructive jaundice Alcoholic hepatitis (most sensitive) Substrate: gamma-glutamyl-p-nitroanilide 1. Szass 2. Rosalki and Tarrow 3. Orlowski Monitor effects of relaxants (succinylcholine) after surgery Marker for organophosphate poisoning (Low CHS) Methods: 1. Ellman technic 2. Potentiometric A.k.a. peptidyldipeptidase A or Kininase II Converts angiotensin I  angiotensin II (lungs) Indicator of neuronal dysfunction (Alzheimer’s disease – CSF) Ferrooxidase enzyme For hepatobiliary diseases

Drug induced hemolytic anemia (primaquine, antimalarial drug) 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 Myoglobin Troponin T Troponin I CK-MB AST LD Rise 1-3 h 3-4 h 3-6 h 4-8 h 6-8 h 12-24 h Peak 5-12 h 10-24 h 12-18 h 12-24 h 24 h 48-72 h Normalize 18-30 h 7 d (10-14 5-10 d 48-72 h 5d 10-14 d d) Acute Pancreatitis Markers Amylase Lipase Rise 2-12 h 6h Peak 24 h 24 h Normalize 3-5 d 7d 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) lec.mt 04 |Page | 36

Vasopressin deficiency Volume and Osmotic regulation Electrolytes Myocardial rhythm and contractility Neuromuscular excitability Cofactors (enzyme)

ATPase ion pump Production and use of ATP from glucose Acid-base balance Replication of DNA and translation of mRNA Sodium Aldosterone Atrial natriuretic factor Hypernatremia

Hyponatremia

Thirst

Pseudohyponatremia (artifactual) Methods (Na+)

Potassium

7% solutes: (Increased in dehydration) -Proteins -Glucose -NPN -Lipids -Ions Excretion of 10-20L H2O everyday Sodium Potassium Chloride EC = Na+ > Cl- > HCO3- > Ca2+(5th) > iPO4 IC = K+ > Mg2+(4th) Potassium Calcium Magnesium Calcium Magnesium (CK) Zinc Chloride (AMS) Potassium Magnesium Magnesium Phosphate Bicarbonate Magnesium Major contributor of osmolality (92%, together w/ Chloride and Bicarbonate) 100 mg/dL glucose = 1.6 mmol/L sodium Sodium Potassium =  Magnesium  Sodium Excess water loss Decreased water intake Hyperaldosteronism (Conn’s disease) Hypothalamic disease (Chronic hypernatremia) Renal failure SIADH (increased water retention) Marked hemolysis (dilutional effect) 165 mEq/L Na+ = Severe water deficit Hyperlipidemia (turbidity) Hyperproteinemia 1. FEP 2. AAS 3. ISE = Glass aluminum silicate 4. Colorimetry = Albanese Lein Concentration in RBC is 105 mmol/L lec.mt 04 |Page | 37

Specimen Considerations (K+)

Hyperkalemia

Hypokalemia

pH and K+ Methods (K+)

Chloride Specimen Considerations (Cl-) Methods (Cl-)

Hyperchloremia

Hypochloremia

Reciprocal relationship with H+ 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 Decreased resting membrane potential  incr. contractility  lack of muscle excitability Decreased renal excretion (Dehydration, renal failure, Addison’s disease) Acidosis (DM) Muscle injury Spironolactone Increased resting membrane potential  arrhythmia Leads to hypomagnesemia Vomiting Diuretics Cushing’s syndrome Alkalosis Insulin overdose  pH by 0.1 =  K+ by 0.2-1.7 mmol/L Lithium heparin plasma = preferred 1. FEP 2. AAS 3. ISE = Valinomycin gel 4. Colorimetry = Lockhead and Purcell Chief counter ion of sodium in ECF Chloride methods measure bromide and iodide Cl- = HCO31. 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 Renal tubular acidosis Metabolic acidosis Diabetes insipidus (Dehydration) Prolonged diarrhea Prolonged vomiting (HCl) Aldosterone deficiency (Na+ = Cl- = K+) lec.mt 04 |Page | 38

Calcium

3 Forms of Calcium Vitamin D3 PTH Calcitonin Practical considerations (Ca2+) Hypercalcemia

Hypocalcemia Primary hypocalcemia Secondary hypocalcemia Methods (Ca2+)

Inorganic Phosphorus

3 Forms of Inorganic Phosphorus PTH Calcitonin Growth hormone

Metabolic alkalosis (HCO3- = Cl-) Marked hemolysis (dilutional effect) 99%  Bones 1%  ECF Absorbed in the duodenum Absorption is favored at an acidic pH 50% = Free/Ionized/Unbound/Active Calcium 40% = Protein-bound (Albumin) 10% = Complexed with anions  Ca2+ =  absorption (intestine) and reabsorption (kidney)  Ca2+ =  resorption (bone) and reabsorption (kidney)  Ca2+ =  urinary excretion (major net loss of calcium) Serum = specimen of choice  Albumin (1g/dL) =  Ca2+ (0.8 mg/dL) Acidosis (Ca2+: from Bones  Blood) Cancer Hyperthyroidism Milk-alkali syndrome Tetany Alkalosis (Ca2+: from Blood  Bones) Acute pancreatitis (Ca2+: binds to damage pancreatic tissues) Low PTH Parathyroid gland disease High PTH Renal failure ( excretion) 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 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.) 55% = Free 35% = Complexed with ions 10% = Protein-bound  PO4 =  Ca2+  PO4 =  Ca2+  PO4 (renal reabsorption) lec.mt 04 |Page | 39

Practical considerations Hyperphosphatemia Hypophosphatemia Methods (iPO4)

Magnesium

3 Forms of Magnesium PTH Aldosterone (& Thyroxine) Hypermagnesemia Hypomagnesemia Methods (Mg2+)

Bicarbonate Chloride shift Anion Gap Increased AG

Decreased AG

Cystic Fibrosis (Mucoviscidosis)

Fasting is required (Nonfasting:  PO4) Hypoparathyroidism Renal failure Hypervitaminosis D Alcohol abuse = most common cause Primary hyperparathyroidism Avitaminosis D (Rickets, Osteomalacia) Most accurate: unreduced phosphomolybdate formation (340nm) 1. Fiske Subbarow Method (Ammonium molybdate method) -Reducing agents: Pictol, Elon, Senidine, Ascorbic acid -(+) Phosphomolybdenum blue 53%  Bones 46%  Muscles and soft tissues 1%  Serum and RBC Vasodilator 55% = Free/Ionized/Physiologically active 30% = Protein-bound 10% = Complexed with ions Mg2+ =  Ca2+ =  PO4 Mg2+ =  K+ =  Na+ Addison’s disease Chronic renal failure Acute renal failure Chronic alcoholism 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) 90% of the total CO2 HCO3- diffuses out of the cell in exchange for Cl - to maintain ionic charge neutrality w/in the cell Difference between unmeasured anions and unmeasured cations QC for ISE Uremia/renal failure Ketoacidosis Lactic acidosis Methanol poisoning Ethanol poisoning Ethylene glycol poisoning Salicylate poisoning Hypoalbuminemia Hypercalcemia Hyperlipidemia Multiple myeloma Defective gene: Cystic fibrosis transmembranous conductance regulator (Chromosome 7) Miconeum ileus (Infants) lec.mt 04 |Page | 40

Pilocarpine Gibson & Cooke pilocarpine iontophoresis Iron

Methods (Iron)

Increased iron Decreased iron TIBC UIBC % Transferrin Saturation Transferrin Note

Normal Values (Electrolytes)

Foul-smelling stool URT infection  Na+ and ClSweat inducer Reference method (Sweat sodium and chloride) Prooxidant 3-5g = Total body iron Ferrous = Hgb Ferric = Transferrin and Ferritin 1. Colorimetric = HCl and Ferrozine -(+) Blue color 2. Anodic stripping voltammetry Hemochromatosis Viral hepatitis Non-IDA IDA Malnutrition Chronic infection UIBC + Serum Iron Increased: IDA, hepatitis, iron-supplemented pregnancy Decreased: Non-IDA, nephrosis TIBC – Serum iron Measure of reserve iron binding capacity of transferrin Index of iron storage Increased: Iron overdose, hemochromatosis, sideroblastic anemia Decreased: IDA (lowest), malignancy, chronic infection TIBC (μg/dL) x 0.70 = mg/dL Sodium 1/α Potassium Potassium 1/α Hydrogen ion Potassium α Magnesium Magnesium α Calcium Calcium 1/α Inorganic phosphate Chloride 1/α Bicarbonate 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: 40 y.o. = 10-250 μg/dL NB and Child = 100-200 μg/dL % Transferrin Saturation = 20-50%

Regulation of Acid-Base balance 20:1 4:1 Expanded HendersonHasselbalch equation Chloride-isohydric shift pCO2 pO2 Metabolic Acidosis

Metabolic Alkalosis

Respiratory Acidosis

Respiratory Alkalosis

Blood Gases and pH Lungs and Kidneys CO2 + H2O H2CO3 H2CO3 H+ + HCO3HCO3-: H2CO3 ratio HPO4: H2PO4 ratio pH = 6.1 + log [Total CO2 – (pCO2 x 0.03)] pCO2 x 0.03 Buffering effect of hemoglobin Index of efficiency of gas exchange Increased: Barbiturates, morphine, alcohol, heparin (12-15%) Reflects the availability of the gas in blood but not its content Excessive O2 supply  acidosis Causes: -Bicarbonate deficiency -DKA (normochloremic acidosis) -Renal failure -Diarrhea (HCO3-) Compensation: Hyperventilation Compensated:  HCO3- + pCO2 + pH 7.4 Causes: -CO2 excess (Hypoventilation) -COPD -Drug overdose (morphine, barbiturates, opiates) Compensation: Bicarbonate retention Compensated:  HCO3- +  pCO2 + pH 7.4 lec.mt 04 |Page | 42

Full compensation Partial compensation Buffer base Methods for Blood Gases and pH Factors affecting Blood gases & pH measurements

pH  normal range pH  near normal All forms of base that will titrate hydrogen ions Specimen: Arterial blood Blood gas analyzers: meas. pH, pCO2, pO2 For every 1OC above 37OC:  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 (Blood gases & pH)

1. Gasometer 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) Dissolved CO2 + H2CO3 + HCO3Continuous monitoring of pO2 Directly placed on the skin Min. requirement: -1 sample every 8 hours -3 levels of control (acidosis, normal, alkalosis) every 24 hours pH = 7.35-7.45 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 Hormone  blood circulation  specific receptor Hormone  interstitial space  adjacent cell Hormone  self-regulation Hormone  direct cell-to-cell contact Hormone  gut lec.mt 04 |Page | 43

Whole blood total CO2 Transcutaneous electrodes Blood gas QC Normal Values (Blood gases and pH)

Endocrine Paracrine Autocrine Juxtacrine Exocrine

Neurocrine Neuroendocrine Glycoproteins Polypeptides Steroids Amines Hypothalamus Pineal gland Pituitary gland Anterior Pituitary (Adenohypophysis) GH (Somatotropin) Dwarfism Acromegaly GH deficiency tests Tests for Acromegaly

FSH LH TSH (Thyrotropin)

ACTH (Corticotropin)

Prolactin

Panhypopituitarism Pituitary ischemia (Shechan’s) Posterior pituitary (Neurohypophysis)

Hormone  neurons  extracellular space Hormone  neurons  nerve endings FSH, hCG, TSH, LH ACTH, ADH, GH, angiotensin, calcitonin, CCK, gastrin, glucagons, insulin, MSH, oxytocin, PTH, PRL, somatostatin Precursor: cholesterol Aldosterone, cortisol, estrogen, progesterone, testosterone, vitamin D Derived from amino acids Catecholamines, T3, T4 Connected to the posterior pituitary by the infundibulum stalk Hypophyseal hormones: TRH, GnRH, GH-IH, GH-RH, PIF Melatonin: decreases pigmentation of the skin Master Gland Located in the sella turcica or Turkish saddle True endocrine gland Hormones: PRL, GH, FSH, LH, TSH, MSH, ACTH Most abundant of all pituitary hormones Structurally similar to PRL and HPL Markedly elevated during deep sleep Decreased GH Increased GH 1. Insulin tolerance test = Gold standard (Confirmatory test) 2. Arginine stimulation test = 2nd confirmatory test 1. Somatomedin C or insulin-like growth factor I (Screening) -Increased: Acromegaly -Decreased: GH deficiency 2. OGTT (Confirmatory) -75g glucose Spermatogenesis Helps Leydig cells to produce testosterone (male) Ovulation (female) Synthesis of androgens, estrogens, and progesterone Stimulates thyroid gland to produce T3 and T4 Increased: 1’ hypothyroidism, 2’ hyperthyroidism Decreased: 1’ hyperthyroidism, 2’ hypothyroidism, 3’ hypothyroidism 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 Initiation and maintenance of lactation Inhibited by Dopamine Highest: 4AM and 8AM, and 8PM and 10PM Increased: Menstrual irregularity, infertility, amenorrhea, galactorrhea From pituitary tumor (adenoma) or Ischemia Hemorrhage or shock in a pregnant female at the time of deliver Release but not produce oxytocin and vasopressin lec.mt 04 |Page | 44

Oxytocin ADH/AVP (Arginine vasopressin) Overnight water deprivation test (Conc. test) Neurogenic DI Nephrogenic DI SIADH

Thyroid Gland Follicle Follicular cells Parafollicular or C cells Thyroglobulin Thyroid hormone Biosynthesis

Protein-bound hormones Free hormones (FT3/FT4) Reverse T3 (rT3) I2 intake 295 mOsm/kg), decreased blood vol. Promotes factor VII and vWF release Diagnostic test for ADH True Diabetes Insipidus Failure of the pituitary gland to secrete ADH Failure of the kidneys to respond to normal or elevated ADH Syndrome of inappropriate ADH Sustained production of ADH Decreased urine volume Low plasma osmolality Low serum electrolytes Butterfly-shaped 2 lobes = connected by the isthmus Fundamental structural unit of the thyroid gland Secrete T3 and T4 Secrete calcitonin Preformed matrix containing tyrosyl groups Stored in the follicular colloid of the thyroid gland 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 Metabolically inactive Biologically inert Do not enter cells Storage sites Physiologically active Readily enters cells From removal of one iodine from T4 (product of T4 metabolism) Metabolically inactive Deficiency of hormone secretion 3,5,3’-Triiodothyronine Most active thyroid hormonal activity 75-80% is produced from the tissue deiodination of T4 Diagnosis of T3 thyrotoxicosis 3,5,3’5’-Tetraiodothyronine Principal secretory product All originated in the thyroid gland Transports 70-75 of TT4, and majority of T3 Transports 15-20% of TT4 No affinity for T3 Transports T3 and 10% of T4 TPO Tg TSHR Screening is recommended when a person reaches 35 yrs old and lec.mt 04 |Page | 45

Primary hyperthyroidism Secondary hyperthyroidism T3 Thyrotoxicosis (Plummer’s disease) Graves’ disease (Diffuse toxic goiter) Riedel’s thyroiditis Subclinical hyperthyroidism Subacute granulomatous/ Subacute nonsuppurative/ De Quervain’s thyroditis Hypothyroidism Primary hypothyroidism Hashimoto’s disease (Chronic autoimmune thyroiditis) Myxedema coma

Secondary hypothyroidism Tertiary hypothyroidism Congenital hypothyroidism (Cretinism) Subclinical hypothyroidism TRH stimulation test

Radioactive Iodine Uptake (RAIU) Thyroglobulin (Tg) assay

every 5 yrs thereafter  T3 and T4  TSH  T3 and T4  TSH  T3 N-T4  TSH 1’ Hyperthyroidism Most common cause of thyrotoxicosis (autoimmune) Women > Men Anti-TSH receptor Thyroid  woody or stony-hard mass No symptoms N-T3 and T4  TSH Hyperthyroidism Painful thyroiditis Neck pain, low-grade fever (-) anti-TPO,  ESR and Tg Treatment: Levothyroxine  T3 and T4  TSH Most common cause of 1’ hypothyroidism Thyroid is replaced by a nest of lymphoid tissue (T cells) Goiter (+) anti-TPO  TSH Severe form of 1’ hypothyroidism Peculiar nonpitting swelling of the skin Skin is infiltrated by mucopolysaccharides “Puffy” face, thin eyebrows  T3 and T4  TSH  T3 and T4  TSH  TRH Mental retardation (child) Screening: T4 Confirmatory: TSH N-T3 and T4  TSH Most specific and sensitive test for diagnosing thyroid disease Confirm borderline cases and euthyroid Graves’ disease : 1’ hypothyroidism : Hyperthyroidism Measure the ability of the thyroid gland to trap iodine Postoperative marker of thyroid cancer : Untreated and metastatic differentiated thyroid cancer, hyperthyroidism lec.mt 04 |Page | 46

rT3 Free Thyroxine Index (FT4I)

TT3, FT3, FT4

T3 Uptake test

TBG test

Fine-needle aspiration Recombinant Human TSH Tanned Erythrocyte Hemagglutination method Serum calcitonin test FT4 and TSH FT3 and FT4 Euthyroid sick syndrome Parathyroid gland PTH 1’ hyperparathyroidism

2’ hyperparathyroidism

3’ hyperparathyroidism

: Hypothyroidism, thyrotoxicosis factitia Assess borderline or conflicting laboratory results 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 FT4 test: differentiates drug induced TSH elevation and hypothyroidism TT3 or FT3: confirm hyperthyroidism Reference method (FT4): Equilibrium dialysis Measures the number of available binding sites of the thyroxine binding proteins (TBG)  TBG =  T3U  TBG =  T3U Confirm results of FT3 or FT4 or abnormalities in the relationship of TT4 and THBR test Estrogen: TBG Androgen: TBG Most accurate tool in the evaluation of thyroid nodules Test patients w/ thyroid cancers for the presence of residual or recurrent dis. Test for anti-Tg disorders Marker for familial medullary thyroid carcinoma Best indicators of thyroid status More specific indicators of thyroid function than meas. of total hormone Not affected by TBG Acutely ill but without thyroid disease  T3 and T4 N/ TSH  rT3 4 parathyroid glands Smalles endocrine gland Hypercalcemic hormone  Ca2+ (bone resorption and renal reabsorption) and Mg 2+  iPO4 Defective: Parathyroid gland Most common cause of hypercalcemia Parathyroid adenoma  PTH and iCa2+ Hypercalciuria Phosphaturia  Hypophosphatemia If goes undetected  severe demineralization (osteitis fibrosa cystica) In response to Ca2+ Hyperplasia of all 4 glands Causes: Vit. D deficiency and chronic renal failure  PTH  Ca2+ Occurs w/ 2’ hyperparathyroidism ( Ca2+) lec.mt 04 |Page | 47

Hypoparathyroidism Hyperparathyroidism Hypoparathyroidism Adrenal glands

Adrenal cortex CPPP ring 3 layers (Adrenal cortex) Cortisol

Porter-Silber method Zimmerman reaction

Pisano method Kober reaction Cushing’s syndrome (Hypercortisolism)

Screening tests (Cushing’s) Confirmatory tests (Cushing’s) Addison’s disease (1’ Hypocorticolism)

Autonomous function of hyperplastic PT glands or PT adenoma  PO4 Calcium phosphates precipitate in soft tissues Accidental injury of the PT glands (neck) during surgery Autoimmune parathyroid destruction  PTH =  Ca2+ Acidosis Alkalosis Pyramid-shaped Above the kidneys Adrenal cortex = outer (yellow) Adrenal medulla = inner (dark mahogany) Has prime effects on blood pressure Major site of steroid hormone production G cells: convert cholesterol  pregnenolone 17-carbon skeleton derived from cholesterol 1. Zona Glomerulosa = Mineralocorticoids (Aldosterone) 2. Zona Fasciculata = Glucocorticoids (Cortisol) 3. Zona Reticularis = Weak androgens (androstenedione, DHEA) 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 Meas. 17-OHCS Rgt: DNPH in H2SO4 + Alcohol (+) Yellow Meas. 17-KGS Rgt: m-dinitrobenzene (+) Reddish purple Oxidation procedure: Norymberski (Na+ bismuthate) For quantitating metanephrines and normetanephrines For estrogen Rgt: H2SO4 + hydroquinone (+) Reddish brown color Excessive production of cortisol and ACTH Overuse of corticosteroids Buffalo hump Hyperglycemia Hypertension Hypercholesterolemia  Lymphocytes 1. 24-hour urine free cortisol test 2. Overnight dexamethasone suppression tests = Most widely used (1mg) 3. Salivary cortisol test 1. Low-dose dexamethasone suppression test (0.5mg) 2. Midnight plasma cortisol 3. CRH stimulation test Primary adrenal insufficiency  Cortisol and aldosterone lec.mt 04 |Page | 48

 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 Differentiates: stimulation test) 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 Most sensitive and specific screening test for excess cortisol 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 Gold standard for 2’ and 3’ hypocorticolism test) Confirms borderline response to ACTH stimulation test Serum ACTH Differentiates: Cushing’s disease (ACTH) Cushing’s syndrome (0-ACTH) ACTH 17-OHCS and 17-KS Congenital Adrenal Enzyme deficiencies: Hyperplasia 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 lec.mt 04 |Page | 49

Bartter’s syndrome Gitelman’s syndrome Hypoaldosteronism Postural stimulation test Florinef Weak androgens

DHEA (Dehydroepiandrostero ne) Adrenal medulla

9:1 Norepinephrine

Epinephrine

Dopamine Pheochromocytoma

Clonidine test Neuroblastoma Methods (Catecholamines)

Bumetanide-sensitive chloride channel mutation  Aldosterone and Renin Thiazide-sensitive transporter mutation  Aldosterone Destruction of the adrenal glands Glucocorticoid deficiency 21-hydroxylase deficiency Test for aldosterone Synthetic mineralocorticoid 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) Principal adrenal androgen Converted to estrone Chromaffin cells: secrete catecholamines Precursor: L-tyrosine Norepinephrine/Epinephrine ---(Monoamine oxidase and Catechol0-methyl-transferase)---> Metanephrines and VMA Norepinephrine: Epinephrine ratio Primary amine  in CNS Metabolites: -3-methoxy-4-hydroxyphenylglycol (MHPG) = Major metabolite -VMA Secondary amine Most abundant medullary hormone “Flight or fight hormone” Metabolites: -Vanillylmandelic acid (VMA) = Major metabolite -Metanephrines -Normetanephrines -HVA Primary amine From the decarboxylation of 3,4-Dihydroxyphenylalanine (DOPA) Major metabolite: Homovanillic acid (HVA) Tumors of the adrenal medulla Catecholamines Classic “Spells”: tachycardia, headache, chest tightness, sweating, hypertension Differentiates: Pheochromocytoma (Catecholamines not suppressed) from Neurogenic hypertension (50% decreased in catecholamines) Norepinephrine (Children)  urinary HVA, VMA or both and dopamine Specimen: 24-hr urine and plasma 1. Chromatography: HPLC or GC-MS 2. RIA: sensitive screening test ->2000pg/mL = diagnostic for pheochromocytoma lec.mt 04 |Page | 50

Estrogens

Markers for Down Syndrome Karyotyping or FISH typing Progesterone Tests for menstrual cycle dysfunction and anovulation Tests for female infertility

Pancreas (Exocrine) Pancreas (Endocrine)

hCG Human placental lactogen (HPL)

Gastrin

Serotonin (5-hydroxytryptamine) 5-HIAA Somatostatin 1’ amenorrhea 2’ amenorrhea Cushing’s disease Cushing’s syndrome

Estrone = Postmenopausal women Estradiol = Premenopausal women (most potent, secreted by the ovary) Estriol = Pregnancy (placenta) AFP Unconjugated Estriol hCG Inhibin A Test for Down syndrome (amniotic fluid) Produced mainly by the corpus luteum Det. whether ovulation has occurred Luteal phase Estrogen Progesterone FSH LH hCG PRL FT4 TSH FSH LH Estradiol Progesterone Digestive enzymes (AMS, LPS) Acinus: functional secretory unit Hormones: Alpha cells (20-30%) = glucagon Beta cells (60-70%) = insulin Delta cells (2-8%) = somatostatin Produced by the syncytiotrophoblasts (placenta) Maintain progesterone production by the corpus luteum Stimulates development of mammary gland Increases maternal plasma glucose levels Diagnosis of intrauterine growth retardation Secreted by G cells (stomach) Stimulates parietal cells to secrete HCl Stimulus: Amino acid Zollinger-Ellison syndrome Pernicious anemia Synthesized by argentaffin cells (GIT) Metabolite: 5-HIAA Diagnostic marker for carcinoid syndrome Test: Ehrlich’s aldehyde test = (+) purple color A.k.a. GH-IH Inhibitor of GH, glucagon and insulin Menstruation having never occurred Absence of menses for 6 months Abnormal increased secretion of ACTH Chronic excessive production of cortisol by the adrenal cortex lec.mt 04 |Page | 51

Gynecomastia Hirsutism Mullerian agenesis Nonthyroidal illness Sipples syndrome (MEN II) Stein-Leventhal syndrome Thyroid stones Normal Values (Endocrinology)

Mixed function oxidase (MFO) system Intravenous route Liberation Absorption Distribution Metabolism Excretion Bioavailable fraction (f) Vd of a drug First-pass hepatic metabolism First order elimination Pharmacodynamics Pharmacokinetics Pharmacogenomics Therapeutic index Trough concentration Peak concentration Cardioactive Drugs Class I

-Large doses of glucocorticoids -Pituitary tumor (ACTH) = most common cause Development of breast tissue in males Excessive hair growth w/ a male distribution pattern in a female Most common endocrine disorder in women Congenital malformation or absence of the fallopian tubes, uterus or vagina N-FSH, LH and testosterone Illness that do not directly involve the thyroid gland Medullary carcinoma of the thyroid Pheochromocytoma Parathyroid adenoma Mild hirsutism w/ normal menses to excessive hirsutism w/ amenorrhea A.k.a. thyroid crisis Life-threatening Uncontrolled thyrotoxicosis T3: 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 Biochemical pathway responsible for the greatest portion of drug metabolism 100% bioavailability Drug  Release Drug  Blood (most: by passive diffusion) Drug  Tissues Drug  Chemical modification Drug  metabolites  excreted Fraction of the dose that reaches the blood Dilution of the drug after it has been distributed in the body Drugs  Liver  Decreased bioavailability Linear relationship bet. the amt. of drug eliminated per hour and the blood level of drug Relationship bet. drug concentration at the target site and response of the tissues Relationship bet. drug dose and drug blood level Study of genes that affect the performance of a drug in an individual Ratio bet. the minimum toxic and maximum therapeutic serum conc. Lowest concentration of a drug obtained in the dosing interval Drawn immediately (or 30 mins) before the next dose Highest concentration of a drug obtained in the dosing interval Drawn one hour after an orally administered dose (except digoxin) Rapid Na+ channel blockers (Procainamide, Lidocaine, Quinidine) lec.mt 04 |Page | 52

Class II Class III Class IV Digoxin Lidocaine (Xylocaine) Quinidine Procainamide (Pronestyl) Disopyramide Propanolol Amiodarone (Cordarone) Verapamil Antibiotics Aminoglycosides Vancomycin

Antiepileptic Drugs Phenobarbital Phenytoin (Dilantin) Valproic acid (Depakene) Carbamazepine (Tegretol) Ethosuximide (Zarontin) Gabapentin (Neurontin) Others (Antiepileptic) Psychoactive Drugs Lithium Tricyclic antidepressantas (TCA)

Fluoxetine (Prozac)

Beta receptor blockers (Propanolol) K+ channel blockers (Amiodarone) Ca2+ channel blockers (Verapamil) Tx: CHF Local anesthetic 1’ product of hepatic metabolism: MEGX (monoethylglycinexylidide) Common formulations: Quinidine sulfate and Quinidine gluconate Hepatic metabolite: NAPA (N-acetylprocainamide) Toxic effect: reversible lupus-like syndrome Substitute for quinidine Anticholinergic effects Tx: angina pectoris Iodine-containing drug Tx: angina, hypertension, supraventricular arrhythmias Tx: Gram (-) bacterial infections Nephrotoxic and ototoxic Tx: Gram (+) cocci and bacilli Toxic effects: “Red man syndrome” Nephrotoxic and ototoxic Long acting barbiturate Enhances bilirubin metabolism Inactive proform: Primidone Injectable proform: fosphenytoin Tx: petit mal and grand mal Tx: grand mal Drug of choice for controlling petit mal seizure Similar to neurotransmitter GABA Topiramate Lamotrigine (Lamictal) Felbamate Tx: Bipolar disorders (Manic depression) Imipramine Amitriptyline Doxepin Nortriptyline Tradazone Major metabolite: Desipramine 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) lec.mt 04 |Page | 53

(Acetylsalicylic acid) Method: Trinder assay Acetaminophen Hepatotoxic (Tylenol) 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) Immunosuppressant Cyclosporine s Tacrolimus (FK-506) Rapamycin (Sirolimus) Mycophenolate mofetil Lefluamide Chemotherapeutic Busulfan agents 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 Major metabolic pathway: Ethanol ------(Alcohol Dehydrogenase)------> Acetaldehyde Testing: Use benzalkonium chloride as antiseptic Methanol (Wood Cause blindness alcohol) 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) lec.mt 04 |Page | 54

Cyanide

Arsenic

Cadmium Lead

Mercury

Drugs of Abuse Opiates

Tranquilizers Barbiturates: Sedative Hypnotics Dopaminergic pathway stimulants Hallucinogens

Amphetamines Annabolic steroids Cannabinoids Tetrahydrocannabinol (THC) Cocaine (Crack)

Opiates

Binds to iron (ferric and ferrous) containing substances like hemoglobin and cytochrome oxidase “Odor of bitter almonds” Antidote: Sodium thiosulfate, amyl and sodium nitrite “Odor of garlic” “Metallic taste” Hair and nails: “Mees lines” Method: Reinsch test (Flat black) Significant environmental pollutant (+) GGT in urine sample 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) Amalgamate: mix or merge w/ other substances Specimen: -Whole blood (organic mercury) -Urine (inorganic mercury) Method: Reinsch test (Silvery gray) Morphine Codeine Heroin Methadone Diazepam (Valium) Oxazepam Phenobarbital Pentobarbital Amobarbital Cocaine Benzoylecgonine Amphetamine Phencyclidine Lysergic acid diethylamide Tetrahydrocannabinol Methaqualone Increase mental alertness (“Uppers”) MDMA (methylenedioxymethamphetamine) = ecstasy Methamphetamine HCl = shabu Improves athletic performance by increasing muscle mass Marijuana and hashish Psycoactive substance of marijuana Urinary metabolite: 11-nor-deltatetrahydrocannabinol (THC-COOH) 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 From opium poppy Heroin Morphine lec.mt 04 |Page | 55

Phencyclidine (Angel dust or angel hair) Sedative hypnotics

Lysergic acid diethylamide (LSD, Lysergide) Methaqualone (Quaalude) Vitamins Vitamin A Vitamin E Vitamin D2 Vitamin D3 Vitamin K Vitamin B1 Vitamin B2 Vitamin B3 Vitamin B5 Vitamin B6 Vitamin B9 Vitamin B12 Vitamin C Biotin Carnitine

Codeine Methadone Major metabolites: N-acetylmorphine (heroin) and morphine Antagonist: Nalaxone (Narcan) Hallucinogen Admin: Ingestion or inhalation Major metabolite: Phencyclidine HCl Barbiturates (Secobarbital, pentobarbital, Phenobarbital) Benzodiazopines: Diazepam (Valium), Lorazepam (Ativan), Chlordiazepoxide (Librium) Major metabolite (barbiturates): Secobarbial “Undulating vision” “Bad trip” – panic reactions Pyramidal signs (Hypertonicity, hyperreflexia, myoclonus) Vitamins Water soluble: B1, B2, B3, B5, B6, B9, B12, Biotin, C, Carnitine Fat soluble: A, D, E, K CN: Retinol Def: Night blindness CN: Tocopherol Def: Mild hemolytic anemia, RBC fragility CN: Ergocalciferol, Cholecalciferol (D2), 1,25dihydroxycholecalciferol (D3) Def: Rickets (young), Osteomalacia (adult) CN: Phylloquinones, Menaquinones Def: Hemorrhage CN: Thiamine Def: Beriberi, Wernicke-Korsakoff syndrome CN: Riboflavin Def: Angular stomatitis, dermatitis, photophobia CN: Niacin/Niacinamide/Nicotinic acid/Nicotinamide Def: Pellagra (dermatitis, disorientation, weight loss) CN: Panthotenic acid Def: Depressed immune system, muscle weakness CN: Pyridoxine, Pyridoxal Def: Facial seborrhea CN: Folic acid, Pteroylglutamic acid Def: Megaloblastic anemia CN: Cyanocobalamin Def: Megaloblastic anemia, neurologic abnormalities CN: Ascorbic acid Def: Scurvy Def: Dermatitis Def: Muscle weakness, fatigue

lec.mt 04 |Page | 56