Chemistry AQNA-LSRE3Q-Y2PCA AQ2M-5PVEDN-EP9AR AQ9Z-E54PBQ-YEPAL ASPN-SDGDLD-ZATA8 Instruments 1) Serum: liquid that remains after blood clots at bottom of tube, no clotting factors 2) Plasma: Liquid on top followed by buffy coat and RBCs, treated with anti-coagulant a) Contains water, proteins, lipids, ions 3) EDTA: not used for Na, K, Ca++. Gray top for glucose but not for BUN Instruments Interferences Principle Spectrometer: detects light waves to measure physicals characteristics of substance over a colored spectrum Spectrophotometer: measures colored substance that absorbs light at a specific wavelength Quantitatively measures light intensity using wavelengths. Polychromatic light, monochromatic Bilirubin in icteric samples interacts with blood components Nephelometry: particle [] proportional to amount of light blocked or scattered Used for low [], sensitive for small particles Turbidimetry: LIH samples interference at particular wavelengths Measures light intensity without wavelength. With wavelength requires filters, photometers, prisms, grading. Light not transmitted is absorbed. Light absorbed proportional to [] of analyte Transmittance ↑, absorbance ↓, [] ↓ Used for cloudy or turbid samples Better than turbidimetry but less precision Used for high [] scattering and measured at 180 degrees Used for serum, CSF, Ig measurements Light gets directed through turbid solution with suspended particles. Light gets transmitted, absorbed and scattered. No energy is absorbed when particles collide. Energy of photon before and after remains the same Measures light blocked Refractometry: Light enters two media (air/liquid) and bends Used for urine, plasma protein in blood measures how light is refracted when it passes a unknown compound. Amount of light refracted determines refractive index Fluorometry high intensity light source, sensitive to background noise, high specificity Absorbance of monochromatic light when excited followed by emission of longer wavelength at ground state Used for immunoassays or flow cytometry Similar to spectrophotometry but light turns into energy and energy is transferred Electrochemistry: Measurement of charge of potential difference at two different []s of same ions when they come into contact Galvanic cell: made of two half cells in a salt bridge spontaneously flow of electrons that go from lower electron affinity electrode Used for pH, blood gas analysis Find [] of solute in solution. Potentiometry: ion selective electrodes Potential between 2 electrodes is measured using voltmeter in which current flow is negligible aka system is equilibrium Potential difference is linearly proportional to [] Atomic Absorption Spectroscopy: Hollow cathode lamp and gas chamber with anode with inert gas which is a carrier molecule. Voltage is applied to cathode to ionize the gas so neutral matrix that carries the sample and voltage that applies to samples Atomic Absorption Uses UV- visible to detect, cooler flamer so atoms can emit or Determine [] of metal atoms/ions with hollow Spectroscopy: absorb radiation cathode lamp Measures one metal at a time Amount of light absorbed proportional to [] ions Flameless employs graphite furnace to hold sample in chamber. More sensitive Mass Spectroscopy: vaporized Used for drugs, aa composition, steroids and atoms ionized by ↑ specificity: two MS connected for further fragmentation knocking electrons off to make positive ions. Ions are After absorption, electrons move to excited state and then atomized in flame. Energy emitted once atoms return to ground state. Metals can absorb the radiation that is unique to the metal via lamp. Light not absorbed is measured Identify composition of chemical substance based on mass to charge ratio deflected by a magnetic field based on masses ICP spectroscopy (inductive coupled plasma): Argon to ionize samples and measures different metals at once Plasma is an ionization source that decomposes compared to flame or atomic absorption that measures one sample into its constituent elements and converts metal elements to ions Energy couples to argon using induction coil to form the plasma Ion Exchange chromatography Retains analyte based on ionic charges. Stationary phase surface displays functional groups that interact with analyte ions of opposite charge. Size exclusion/gel filtration: Polymer beads with controlled pore size. Molecules larger than pores do not enter beads and They travel quicklySmaller molecules enter beads. They are slowed down Gel permeation chromatography Determine relative molecular weight of polymer samples and distribution of MW Samples dissolved in solvent and separation occurs in column. Stationary phase: gel which fills up with column Eluent is mobile phase Affinity chromatography: high selectivity, resolution Separation method based on binding interactions between immobilized ligand and its binding partner. AB/Ag, enzyme/substrate, enzyme/inhibitor Protein purified and then desalted HPLC: High Performance Liquid Chromatography Separates compounds dissolved in a liquid sample and determines components in the sample Mobile phase: solvent which is delivered to stationary column (methanol, ethanol) by pump Mobile Solvent mixed with dissolved compound No need for gas phase compound, use with high MW compounds Stationary: polar or non-polar silica Gas Liquid chromatography: Separation of compounds that can be vaporized without decomposition Liquid mixed with solvent, injected into GC and is vaporized into gas phase. Inert carrier gas and sample heated into a long tube. Detector at end of tube Solutes carried by gas over stationary phase eluted and goes to detector in an order More volatile, faster elution due to less interaction with column ID components of liquid mixture and their relative [] and purify components based on retention time, peak size Thin layer chromatography Screening of drugs in urine Stationary phase is silica gel over glass support. Elute TLC with organic polar solvent= mobile Monitor chemical reactions and purifications More polar compounds climb slower and less polar ones will fly upwards Direct antigen on plate. Another protein like albumin added to block binding sites. Enzyme linked Ab added. Unbound ones washed off and bound ones to the Ag left. Add substrate for signal Immunoassays: Enzyme Linked Immunosorbant Assay ELISA – Direct/Indirect Indirect Ag added on plate. An Ab with no enzyme that sees Ag is added. Then enzyme-Ab added….. Quantify Ag between 2 layers of Ab. ELISA – Sandwich Can only measure proteins, subject to interferences such as hook effect (Ag excess so dilute sample) and mouse antibodies give false + High Sensitive, specificity No limit to [Ab] Greater dynamic range ELISA – Competitive -competition between unlabeled Ag (unknown sample) with labeled Ag for antibody. Reference Ag pre-coated. Higher the sample Ag [], weaker the signal -Free labeled Ag not bound to Ab ([] of Ab sites is limited) -More sample Ag less Ag-ref Ab bound so weaker signal -Binding of labeled Ag to Ab allows distinction of bound and free Homogeneous Immunoassay Chemiluminescence / Luminometry Measure [] of free labeled antigen or complex by signal interference. EMIT More sample Ag, less enzyme labeled Ag to Ab, ↑ free labeled Ag which reacts with substrate. More product means higher the analyte in sample No need to separate bound and free No drug antibody binds to antigen no enzyme activity Measure chemi and bioluminescent (enzymecatalyzed) reactions Higher sensitivity, wide range, inexpensive Signal proportional to [] of analyte in serum Current proportional to pO2 in sample Amperometry- anodes immersed in buffer and membrane (permeable to gas) separates electrode and buffer from blood sample Measures current produced via oxidation/reduction of substance at electrode at fixed potential Measures pO2 w/ platinum cathode + Ag/AgCI anode No O2 diffusing in buffer no current b/c polarization Used in blood gas analyzer O2 diffuses in buffer from sample reduced at cathode and current flows Albumin has high negative charge and travels fast Anode (+), cathode (-) Agarose gel gives proteins = negative charge Electrophoresis Silver nitrate used for CSF Oil red O and fat red for lipoproteins Analytical Chemistry Principles Terms: Glycosuria: glucose in urine Glomerulonephritis: kidney not filtering, bubbles in urine Diabetes insipidus: can’t retain water 1) Osmometry: molecular mass depending on colligative properties changing when solute dissolves in pure solvent, measures hydration levels in serum or urine a) Freezing point depression – FP ↓ as solute ↑ i) Serum is cooled and FP is recorded ii) If FP lower than the reference temp, then higher [] of particles b) Vapor pressure depression – liquid to gas as temp ↑ i) VP of solvent ↓ as dissolved solute ↑ c) Boiling point ↑ -not used d) Hemolysis doesn’t interfere e) Heparinized plasma/urine ok but EDTA is not 2) Osmolal gap (mOsm/kg) = measured FP – calculated from solute [] Osmolal Gap (mOsm/kg) What is it Osmotic activity per kg of solvent (water) Serum molecules= Na, Cl, glucose, urea Osmolarity (mOsm/L) Range 10-12 mOsm/kg ↑ Gap -toxins -dehydration -hypernatremia -glycosuria -lactic acidosis -ADH not working Osmotic activity per liter of whole solution 3) Anion Gap – estimated of unmeasured electrolytes (Ca, Mg, K, sulfate, proteins) a) Na- (CI + HCO3-) 7-16 mmol/L b) Increased gap (common) i) MUDPILES – abnormal accumulation of acids (metabolic acidosis) ii) Low Mg, Ca iii) Assess instrument errors c) Decreased gap (uncommon) i) Increased cations, hypoalbuminemia d) MUDPILES i) Methanol – metabolized to formaldehyde acid so pH ↓ ii) Uremia - ↓GFR, pH ↓, decreased acid excretion iii) Diabetes – ketones ↑ iv) Propylene glycol - lactate v) Isoniazid – lactate to lactic acid vi) Lactate acidosis – hypoxia, sepsis, tumors, drugs vii) Ethylene glycol – increases lactic acid viii) Salicylates - makes lactate 4) Both anion gap and osmolality assess unmeasured cations ↓ Gap -Diabetes insipidus -fluid intake ↑ -glomerulonephritis Quality Assurance and Control 1) In-laboratory Quality factors: pre and post analytical phase a) Pre is accession/processor, patient ID, test order b) Post-test: cycle begins and ends with doctors and lab as partners 2) Accuracy closeness of measurements to target a) Analytical error: i) Random: variability upon repeated measurement (1) Imprecision, Always present ii) Systematic: directional bias, not random (1) May or may not be present (2) Recovery, method, comparison 3) Precision same value for repeated measurements a) Can’t have accuracy without precision. b) Low accuracy, high precision: systematic error c) Dispersed: random error 4) Statistical parameters i) Mean ii) Standard deviation (s or sigma) (1) Measures of dispersion around mean (2) Greater the SD, more spread out the observations (3) Levy Jennings Chart for QC values a. How many SD from the mean, sees trends shifting b. Labs use +/- 2sd as criteria for acceptable ranges 5) Interfering substances a) Hemolysis Hgb release absorbs light and cell membrane destroyed i) High peroxidase activity affects bilirubin and inhibited by free Hgb b) Lipemia (turbidity) excess of fat or lipids in blood i) Volume displacement (1) Pseudohyponatremia (Na<135) (2) Chylomicrons or hyperproteinemia ii) Light scattering increases absorbances c) Bilirubin (icterus) d) Fibrin interfere with accuracy 6) Random Error a) Widening of distribution of control results poor precision, Related to instrument problems 7) Systematic Error a) Change in position of distribution of control results via abrupt shift due to maintenance, reagent change, i) Abrupt shift in the mean due to maintenance, reagent change, calibration Long-term SD increase Instrument aging More calibrations New reagent lots Control material variation Long-term mean change Control material deterioration Reagent stability or new lots Calibrator stability or new lots Pull frozen surveys and re-run (if analyte stable) 8) Westgard Multi-rule (1981): uses 5 controls to judge run acceptability a) Levy Jennings Chart uses single set of control limits such as +/- 2SD b) Decision criteria chosen to maximize efficiency i) Decreased false rejection of assays ii) Increased detection of true error Term Positive predictive Value/sensitivity Definition Proportion of positive tests that represents disease Negative predictive value/ specificity Detects absence of disease Reference range Defines healthy population and 5% of healthy population is abnormal -reflects total population variability Reported directly by method without dilution Results above AMR are diluted Reportable Range Coefficient of Variation Comparing SD at different [] level Lower the CV, higher reproducibility is Calculation 𝑇𝑃 ; 𝑓𝑜𝑟 𝑃𝑉 (𝐹𝑁 𝑡𝑜 𝐹𝑃) 𝑇𝑃 + 𝐹𝑁 𝑇𝑁 ; 𝑓𝑜𝑟 𝑃𝑉 (𝐹𝑃 𝑡𝑜 𝐹𝑁) 𝐹𝑃 + 𝑇𝑁 Electrolytes, Acid-Base Balance and Kidneys Terms AVP/ADH (vasopressin) – antidiuretic hormone. Retains water so urine has low solutes and ↑ volume Diabetes Insipidus (DI) – can’t retain water (urine output ↑, serum osmo ↑) Polyuria - large amount of urine Polydipsia - constant thirst SIADH - too much AVP makes body retain too much water Diuretics – makes you urinate more Pyelonephritis – kidney inflammation due to bacterial infection Nephrotic syndrome- high protein in urine due to kidney disorder Diabetes mellitus- high blood sugar, not enough insulin Heparin: green top, anticoagulant by inhibiting thrombin formation; plasma, not serum Renin: part of renin angiotensin aldosterone system, renin is released from kidneys in response to low blood pressure/vol (in attempt to retain water and constrict vessels to raise pressure back) Respiration: supply cells with oxygen for metabolic processes and remove CO 1. Osmolality [] of dissolved particles in cell (relative to ECF) a. independent of temperature and pressure b. Na is primary contributor to serum osmolality c. BV regulated by renin-catecholamines d. Favors cations moving inside cell and anions go out e. 2*Na + (Glucose/18) + (BUN/2.8) Serum Osmolality Hypertonic (cell shrinks) Hypotonic (cell swells) ECF solutes ↑ so water leaves cell, water deficit ICF solutes ↑ so water comes in, excess water Higher osmolality makes AVP ↑, more thirst, high BV, polydipsa Hypernatremia Hyperglycemia Renal: tubular disease, diabetes insipidus (polyuria) Sweating, diarrhea, vomiting, burns Lower osmolality AVP ↓, ↓ BV Factors ↑ Serum osmolality Water loss DI Na overflow hyperglycemia Urine Osmolality ↓ in DI and polydipsia, solutes coming out ↑ in inappropriate ADH and hypovolemia (decreased blood volume) Hyponatremia Forced ingestion or IV fluid Requires renal failure, suppression of thirst, diuretics Catecholamine secretion, Angiotensin II ↑, Ald ↑ Urine Osmolality solutes↑ Syndrome inappropriate ADH ↑ ibuprofen use Factors ↓ SIADH Renal failure Diuretic use Adrenal insufficiency Fluid Volume (excess) DI ↓ Inverse relationship: DI ↑ serum osmolality, ↓ urine osmolality SIADH: ↓ serum osmolality, ↑ urine osmolality 2) Electrolyte measurement: Na, K, CI (measured by ISEs), total CO2 3) Calcium measurement: a) PTH breaks down bone to release Ca and reabsorbs in kidneys b) Vit D liver INACTIVE but converted into 1,25-dihydroxychlecalcierol in liver (ACTIVE form) c) Calcitonin inhibits Vit D and PTH activity d) When uncapped O2 ↑, CO2↓ so pH increases (low pH increases binding so iCa ↓) e) When CO2 ↑, pH decreases (H+ replaces Ca so more iCa) f) If pH = 7.20 -7.60, needs to be corrected to 7.40 g) Actual blood is reported if whole blood is used Calcium Forms Free ionized Ca (45%) Active -When ↓ iCa, PTH ↑ -Vit D enhances Ca absorption and in kidneys -calcitonin ↑ when Ca ↓ -heparin binding to Ca+2 lowers iCa Protein bound (45%) Complex (10%) Bound to albumin (pH dependent) ↑ binding, ↑ pH Bound to anions 4) Bicarbonate converted to CO2 species a) tCO2 HCO3- + H2CO3 (carbonic acid, bound CO2, dCO2) b) 90% of measured tCO2 = HCO3c) Uncapped tubes CO2 ↓ 6 mmol/L/hr 5) Phosphorus 6) Things to remember a) Increases in HCO3 (bases) and ↓ in CO2 alkaline Analyte Mechanism of Action Hyper Na+ ↑/↓ thirst due to plasma osmol Hypernatremia -DI (lack of AVP) diluted urine Hyperaldosteronism (blood pressure ↑ and lowers K) Renal tubular disease, cushing’s syndrome Hyponatremia Direct OR indirect Diuretics hypoaldosteronism, ↑ K measurement of [] -diarrhea, vomiting, burns Overhydration, SIADH, nephrotic Pseudohyponatremia syndrome, Addison’s dz drawing above low Na IV line Hyperkalemia Hypoaldosteronism (adrenal insufficiency) metabolic acidosis (H displaces K) ↑ RBC lysis Decreased renal excretion Hypokalemia Hyperaldosteronism, excess insulin lower K, diuretics Hypomagnesemia Symptoms: weakness, low HR Rapid uptake of ingested K into cells *K is ICF. Glucose having party ECF. K went out to see party. Insulin stops the party so K back into cells ↓K Pseudo hyperkalemia: hemolysis ↑K 136-145 mmol/L 85% reabsorbed ECF cation Kidney K+ Excites muscles, contraction 3.4-5.0 mmol/L ADH ↑, K ↓ ADH ↓, K ↑ Kidney ICF cation Cl98-107 mmol/L Kidney Involved in osmotic pressure, Hyperchloremia blood volume, ionic neutrality -dehydration -Excess loss of HCO3- (ClECF anion compensates for HCO3- in RBCs so metabolic acidosis) diarrhea Bicarbonate Buffer excess H+ in blood HCO3-/ (CO2) ECF 22-29 mmol/L Increased tCO2, ↑HCO3Metabolic alkalosis Emphysema Severe vomiting Hypo Notes No direct regulatory mechanism Treatment: insulin + glucose Hypochloremia Vomiting (HCl ↓), diuretics, burns, aldosterone deficiency Diabetic ketoacidosis Pyelonephritis (salt losing renal disease) Metabolic alkalosis (HCO3- ↑) Follows Na Cl changes proportionally with Na Decreased tCO2, ↓HCO3Metabolic acidosis Diabetic ketoacidosis Salicylate toxicity 90% CO2 in form of HCO3Total CO2: dissolved CO2, H2CO3, HCO3- Sweat Cl: ↑ in cystic fibrosis Ca2+ tCa: 8.610.3 mg/dL 5th abundant inorganic Muscle contraction, membrane transport, enzyme reactions, coagulation iCa: 4.6-5.3 mg/dL Calcitonin inhibits bone reabsorption Parathyroid, kidney, and pH dependent Vit D Mg2+ 4th most major cation (53% in bone) 1.7-2.4 mg/dL Blocks Ca channel Kidney PTH enhances reabsorption Hypercalcemia Caused by hyper-PTH when Ca is ↓, excess Vit D, low calcitonin, cancer, multiple myeloma Symptoms: nephrolithiasis (kidney stones), DI Hypermagnesemia renal failure so ↓ excretion, Dehydration, bone destruction Symptoms: cardio, neuromuscular, GI, lethargy, metabolic ICF Inorganic Phosphorus PO4- Nucleic acids, 80% in bone Heparinized whole blood or blood gas syringes EDTA, oxalate, citrate: NO (False ↓, chelated) Uncapped serum ↑pH, lowers pCO2, bound Ca ↑, resulting in ↓iCa B-hydroxyquinoline is added to remove Mg++ Symptoms: paresthesia, seizures, heart block Tetany: Ca++ first, then Mg or K (spasms) Hypomagnesemia -excretion by pyelonephritis, glomerulonephritis -Hyper-PTH, hyper-Ca, hyper-Na, -Digoxin, gentamicin, diabetes mellitus, alcohol Pregnancy and lactation Hemolysis can interfere LIH affects it Serum or heparinized plasma Treat by replacement Inverse relationship with Ca (when Ca ↑, PO4 ↓) Hyperphosphatemia ↑ tissue turnover (infection), hypoPTH (↓ Ca, PO4 retained) -renal failure, excess Vit D Not regulated, waste Monitor for critically ill Patients Tourniquet >30sec, fist clenching, hypoxia, exercise will ↑ Delayed processing will ↑ 2.5-4.5 mg/dL Kidney Lactate Hypocalcemia Hypo-PTH, high calcitonin, Vit D ↓, massive transfusion Hypoalbuminemia (affects Tot Ca), PseudohypoPTH -heparin binding to Ca+2 lowers iCa Hypophosphatemia Hemolysis increases PO4Diabetic ketoacidosis (DKA), sepsis, hyper-PTH ↓PO4 and ↑ Ca Serum or heparinized plasma Citrate, oxalate, EDTA =NO Aerobic glycolysis and anerobic Green top OK if iced Gray top tube recommended (inhibits glycolysis) Blood gases Terms Buffer system- resists change in pH; weak acid or weak base and its salt 1) Whole blood in pre-heparinized syringes required (measured in mm Hg) a) Parameters affected by rate of respiration b) Must be anerobic collection c) In ice to prevent glycolysis 2) Acid-Base metabolism – 4 buffers in whole blood a) Bicarbonate-carbonic: minimizes pH changes in plasma, RBCs (most important), low Cl i) Metabolic processes optimal at pH ~7.40 ii) Henderson-Hasselbalch equation as tCO2 is transported iii) Normal HCO3-/H2CO3 ratio is 20:1 b) Hgb: minimizes pH and most intracellular buffer i) When O2 ↓, Hgb binds to H+ and transported to lungs oxygenated Hgb releases H+ ii) CO2 inhaled c) Plasma proteins and Phosphate and albumin 3) Arterial blood vs venous and vs cord blood a) Hypercapnia (↑ pCO2) AND hypocapnia (↓ pCO2) b) Arterial pH range = 7.35-7.45 c) Dissolved CO2 = H2CO3 + dCO2 in blood d) tCO2= 22-26 mmol/L (45/2 e) pCO2: 35-45 mm Hg (90/2) f) pO2: 85-105 mm Hg (I like my oxygen at 100 but 90 will do) g) Exposure of specimen to air: pCO2 decreases, pH increases, pO2 increases h) Prolonged testing glycolysis pCO2↑, pH↓, pO2↓ Arterial Venous Cord blood Measurement <7.35 Glass electrode Acidemia pH >7.35 Glass electrode Alkalemia pH Higher than venous Lower than arterial Clark electrode O2 Measure for Higher Severing Haus pCO2 hypercapnia electrode Red (Hgb +O2) dark Color 4) Calculated is HCO3- and Base excess a) Base excess: pos values = metabolic alkalosis / neg values = metabolic acidosis. 10) Disorders a) Metabolic: HCO3- [] ……kidneys, non-respiratory b) Respiratory: dissolved CO2 [] ……. lungs, respiratory 11) Mixed acid-base disturbances a) Normal: HCO3-= 24 and pCO2=40 b) Fall in HCO3-: 24-12=12 c) Anticipated Fall in pCO2: 40-15=25 d) Estimated is pCO2 is 15 so ↓ e) Respiratory alkalosis due to respiratory stimulation by ASA f) Metabolic acidosis due to oxidative phosphorylation by ASA and ↑ anion gap 12) Oxygen and Gas Exchange a) Tissue oxygenation requires available O2, ventilation, gas exchange between air and blood i) Fe2+ (ferrous) and binds to O2oxyhemoglobin ii) H+Hgb= deoxyhemoglobin iii) metHgb= hemoglobin with Fe3+ (ferric), does not bind O2 iv) COHb=carboxyHb-CO displaces O2 b) Release of O2 from Hgb to tissue by releasing H+ [] and pCO2 levels in tissue i) Saturation of Hgb with oxygen = 95% ii) When below 95%, not enough O2 or ↓, CO2 ↑ in carboxy-hgb iii) pO2 levels drop during hypoxia, pulmonary diffusion ↓, obstruction 13) Oxygen Hemoglobin Binding a) When temperature decreases in lungs i) ↑↑ O2 binding to Hgb (shift left/up) ii) pCO2 ↓, pH ↑ b) When temperature increases in tissue i) ↓↓ O2 binding to Hgb and release to tissues (shift right/down) ii) pCO2 ↑, pH ↓ 14) Oxygen saturation (functional) is proportion of oxygenated Hgb a) Estimates lung gas exchange 15) Fractional O2 Hgb saturation is proportion of all oxygenated species deliverable to tissue a) COHb and MetHb don’t bind so saturation less than 100% 16) BMP a) Na, K, CI, CO2, glucose, BUN, creatinine, calcium 17) Case Study 1 pH HCO3- pCO2 Metabolic acidosis Normal (↓) Metabolic alkalosis Normal (↑) Respiratory acidosis Causes Renal failure (can’t excrete acid) Diabetic ketoacidosis Severe diarrhea (base loss) Salicylate poisioning (muscle twitch, K ↑) Acid excreted; base retained Diuretics Severe vomiting Loss of HCl Compensation Respiratory compensation Hyperventilation to ↑ pH, CO2 ↓ Reabsorption of HCO3- CO2 ↑ means more acid Obstruction, pneumonia Metabolic compensation bicarbonate retention. Hyperventilation, Hypoxia, CHF, seizures Renal compensatory mechanism excretes bicarbonate, pH ↓ Hypoventilation ↑ CO2, then pH normal Normal (↑) Respiratory alkalosis Normal (↓) Kidney/ Non-protein Nitrogens Terms Azotemia: Urea, Creatinine, Uric acid, Ammonia ↑ in plasma in renal failure Anuria: no urine Oliguria: low urine output Rickets: childhood disease, softening and weakening of bones 1) Protein breakdown in body produces urea and ammonia 2) Glomerular Filtration a) Filters small solutes, cell free, low protein (MW cutoff such as albumin) b) Glomerular filtration rate (GFR): 125 ml filtrate per minute c) If ADH is present water ↑, urine []ed 3) Water Balance/ renin-angiotensin-aldosterone axis a) When osmol ↑, blood volume ↓ i) ADH/AVP secreted by pituitary gland ii) Hypothalamus increases thirst iii) Renin and angiotensin retain water iv) Vasoconstriction of arteries b) When osmol ↓, blood volume ↑, ↑ pressure i) AVP not secreted and kidney excretes diluted urine ii) ↑ aldosterone, Na and water reabsorbed, K out, ↑ volume 4) Non-protein Nitrogen a) Urea, Creatinine, Uric acid, Ammonia – high levels lead to azotemia if renal impairment b) Creatinine clearance (ml/min) evaluates GFR i) Timed collection – how much blood passes glomeruli each minute ii) More sensitive than BUN or creatinine c) eGFR more sensitive than creatinine clearance i) time collection not needed ii) Assess kidney damage 5) Liver converts drug to more polar metabolites and Kidney excretes them 6) Erythropoietin hormone by kidneys stimulated during hypoxia to produce more RBC by bone marrow a) Epo ↓ anemia, renal disease 7) Vitamin D hormone a) D2 (diet) and D3 (sunlight) and forms into active 1,25-dihydroxycholecalciferol b) Causes increase of Ca and phosphorus by ↑ absorption c) Deficiency causes rickets, osteopenia, osteoporosis Non-protein N Urea What is it? Urea: N-compound, total amount of urea, reflects protein metabolism Clinical Significance ↑ serum urea: renal failure, glomerular nephritis, obstruction, ↑ catabolism 2.5-8 mg/dL Excreted by kidney in urine Liver BUN 7.0-22 mg/dL Creatinine Male: 0.9-1.3 mg/dL Female: 0.6-1.1 Differs per age, sex Cystatin C -Represents nitrogen in urea ↑ in renal impairment, protein diet -Rises more rapidly than serum ↑ dehydration, oliguria, anuria creatinine -Reflects kidney function, affected by diet muscle energy storage Renal disease/failure: ↑ Creatinine in blood, ↓ in urine Excreted by Kidney ↓ creatinine: good Urease hydrolyzes urea into ammonia measured Do not use gray top BUN to Urea conversion: BUN X 2.14 = Urea Indicator of GFR but not as sensitive Jaffe Reaction: creatinine reacts w/ pierie acid in alkaline solution red complex Interferences: glucose, acetoacetate and ascorbic acid No color= bilirubin interference Marker for GFR Small proteins made by all cells Not affected by muscle mass, gender, age, diet Uric Acid By-product of purine catabolism Liver Elimination regulated by kidneys, GI tract Deamination of aa and converted to urea Ammonia ↓ serum urea: liver disease, vomiting, malnutrition Notes ↑ serum urea: prerenal, renal, postrenal 11-32 umol/L Excreted by kidneys ↑ in gout. Renal failure, preclampsia, leukemia, chemotherapy Uricase-peroxidase coupled reaction ↓ in liver disease, reabsorption disorder ↑ means toxic to CNS, liver failure and Reye Syndrome (fatty liver and brain swelling), cirrhosis, viral hepatis Disorders can’t convert to urea so urea ↓? Green top, venous Ice Renal Dz Azotemia Definition Increases blood nitrogenous wastes Uremia Renal failure, same as azotemia Oliguria <500 mL/day urine Anuria <100 mL/day Glomerulonephritis Inflammation of glomerulus Nephrotic Syndrome Glomerular disease Acute kidney injury (AKI) Pre-renal: circulation failure Renal (intrinsic): glomerulonephritis Post-renal: urinary tract obstruction Reduced GFR and impaired urine [] Chronic kidney disease Renal Calculi (stones) ↑ [] of insoluble constituents Symptoms/Causes Kidney defect, obstruction, ↓ creatinine Hematuria or ↑↑ RBC casts proteinuria in urine (albumin, nephrotic syndrome) ↑↑↑ high protein, Few casts hypoproteinemia(albumin ↓ in serum), hyperlipidemia Serum creatinine ↑ or Metabolic acidosis: oliguria, shock, obstruction, oliguria, anuria, ↑ ECF volume Diabetes mellitus, hypertension, hypocalcemia, anemia Low urine and reabsorption Bacterial urease: increases pH 7) 24-hour urine a) [] dependent on water b) 24 hr collection normalizes (Void at start time) c) High potential for inaccurate collection d) Reference analyte to creatinine a) Kidney: urea, uA, BUN, creatinine, glucose, calcium, Mg, phosphate, lactate b) Liver: ammonia, carbs, fat, ketones, vit A, enzymes Treat: hemodialysis or transplant Calcium oxalate or calcium phosphate(80% common) Liver Terms Hepatocytes – functional liver cells Kupffer c ells – macrophages Cholestasis = obstruction of bile flow Albumin- transports bilirubin and 60% of plasma protein Hypophosphatasia: insufficient bone calcification (↓ALP) 1) Hepatic Artery 20-30% of Oxygen-rich blood 2) Portal Vein 70-80% of Blood rich in digested nutrients 3) 5 Major Liver Functions a) Excretion/secretion - bili, cholesterol, drug, hormone excretion makes clotting proteins w/K b) Metabolic - fats, proteins & carbs, along with regulation of glycogen storage c) Detoxification - filters blood, binds toxins to inactivate, chemically modify the drug for excretion d) Storage - glycogen, vitamins, iron, and blood for daily bodily maintenance and function e) Immunity- Kupffer cells and IgA secretion; phagocytosis of bacteria and other substances 4) Bilirubin: pigment in bile derived from Hgb breakdown of old RBCs a) Heme oxygenase catalyzes porphyrin ring green biliverdin converts to bilirubin (hydrophobic) b) Iron=recycled! Transported by transferrin to liver or BM for storage Liver pathophysiology Cause Jaundice High bilirubin (>3.0 mg/dL) High bilirubin Bilirubin buildup Icterus Kernicterus Signs yellow discoloration Notes Skin, eyes Yellow colored Serum, plasma yellow staining of Newborns histology Brain damage 5) Liver makes clotting factors that is vitamin K dependent and ↓ synthesis prolong PT & aPTT 6) GI absorbs substances and liver detoxifies before circulation 7) Hepatic Signs a) Gilbert syndrome (inherited) – metabolism defect i) Reduced UDPGT to convert unconjugated to conjugated b) Crigler-Najjar syndrome (congenital)- metabolism defect i) Deficiency of UDPGT c) Dublin-Johnson syndrome (congenital)- transport defect i) Defective transportation of conjugated bilirubin to bile canaliculi ii) Liver has black pigmentation iii) If obstructive liver disease occurs not excreted in urine, breakdown by general protein catabolism Urine is normal d) Neonatal jaundice i) Low levels of UDPGT, Kernicterus may occur ii) Treatment: UV light, exchange transfusion e) Intra-Cholestasis i) Hepatocyte injury like cirrhosis Jaundice Type Prehepatic Causes ↑ RBCs ↑ bilirubin hemolysis rate exceeds bilirubin conjugation Signs Hyperbilirubinemia, hemolytic anemia Liver function is fine Hepatic Post-hepatic Malfunction of liver cells No conjugation of bilirubin so secretion ↓ Obstruction blocks bile to intestines extrahepatic cholestasis hepatitis 1) Gilbert syndrome (common) 2) Crigler-Najjar disease (rare, serious) 3) Dubin-Johnson syndrome 4) Neonatal jaundice (acquired) 5) Intrahepatic cholestasis Gallstones, tumor, edema 1) 2) 3) 4) 5) Test Results Unconjugated bili ↑ Conjugated bilirubin N Urobilinogen ↑ Bilirubin: 0, no backup Serum Fe ↑ 1) Indirect/unconjugated bili: ↑ 2) Indirect/unconjugated bili: ↑↑↑ 3) Indirect bili: ↑↑↑; urine normal if there is obstruction, Direct bili: 0 or ↑ 4) Indirect/Unconjugated bili: ↑↑ 5) Conjugated bili: ↑ 1) Total serum bilirubin: ↑↑↑ 2) Unconjugated bili is N 3) Plasma conjugated bili: ↑↑ 4) Bilirubin ↑ 5) Urobilinogen: ↓ so stool is pale Liver function is fine [↑ urobilinogen: hepatitis) Disease Causes Signs/ symptom Reye Syndrome Aspirin therapy and viral infection Only in kids -Swelling in the liver and brain, Fatty liver, encephalopathy Cirrhosis (chronic) Scar tissues by Chronic alcoholism, hepatitis Jaundice, Edema, Bleeding Drug-induced -Ethanol -Acetaminophen immune-mediated injury against liver cells Cholestasis = obstruction of bile flow Liver inflammation Hep B and C Virus, drugs, radiation, bacteria, toxins or parasitic liver scarring jaundice dark urine Alcoholic Injury Decreased Fatty Liver Increased AST, ALT, GGT (slight) Alcoholic hepatitis albumin AST, ALT, GGT, ALP, bili and PT(prothrombin) (moderate) Alcoholic cirrhosis albumin AST, ALT, GGT, ALP total bili PT Bilirubin specimen serum/plasma Patient condition Tube handling -Light sensitive ↓ bili -Time sensitive -hemolysis ↓ bilirubin rxn Urine -Light sensitive -Air sensitive -Ascorbic interference 8) How to assess Liver enzymes in Serum markers for cholestasis, ALP, GGT, tbili, albumin, ammonia, AFP a) Urobilinogen collective term for stercobilinogen, meso-bilinogen and urobilinogen b) Porphyrin causes photosensitivity in skin and excess neuropsychiatric symptoms Fasting (prevent lipemia which ↑ bili) Liver Analysis Total bilirubin Range Babies: 2-6 mg/dL and ↑ Adults: 0.2-1.0 Measured by Measured Notes 1) dbili 0.0-0.2 mg/dL Measured Polar, soluble, free 2) Unconjug/indirect bili 3) Delta bilirubin 0.2-0.6 mg/dL Calculated Non-polar, bound to albumin in plasma Direct bilirubin bound to albumin Alcohol, drugs, hepatitis High urine= defective liver functions (cirrhosis, Hep B or C) Urine bilirubin Urobilinogen Colorless in urine Brown in stool Dipstick Urine sample Bilirubin testing accelerator needed (Jendrassik-Grof), LIH interference, avoid light No accelerator needed accelerator needed Not seen in urine (too big), Use serum Conjugated bili ↑ Ehrlich’s reaction. Red product low = biliary obstruction or carcinomas of the liver Liver Proteins/Tests Plasma Total Protein 6.0-8.0 g/dL Function protein degradation, cytokines Pre-albumin -Nutritional status indicator -Transports thyroid -hormones Albumin (plasma protein) Determines liver dz severity and transporter -osmotic pressure Normal=acute Low=decreased 3.5-5.0 g/dL Hyper Hyperproteinemia: dehydration, tourniquet time ↑ ↑ with steroid therapy, alcoholism, renal failure Hypo Hypoproteinemia: ↓ synthesis, burns, liver dz Dehydration, tourniquet time ↑ (no clinical significance) ↓ synthesis, ↓ osmotic pressure, malnutrition, nephrotic syndrome, burns Notes ↓ in liver disorders, poor nutrition Highest [] of all plasma proteins Aspartate Aminotransferase (AST) 5-30 U/L Cardiac muscles, liver, RBCs Alanine Aminotransferase (ALT) 6-37 U/L Alkaline phosphatase (ALP) Bone, biliary tree, placenta (source of ALP) 5’nucleotidase (5NT) Gamma glutamyl transferase (GGT) If AST>ALT, alcoholic hepatitis, cirrhosis, liver cancer convert proteins into energy for the liver cells Hepatic damage, hepatitis, cirrhosis, jaundice, mononucleosis, MI Hemolysis can affect If AST and ALT ↑ AML Less specific to liver If ALT>AST, viral and drug Liver disease, cirrhosis, hepatitis, Hep C, obstruction carcinoma Metabolize amino acids More specific than AST pH=10, Mg activation Bile obstruction Differentiates biliary dz vs bone disease ALP>ALT, AST for bone dz, biliary dz ↑ as bone fractures heals Paget Dz (bone), growing kids, 3rd trimester ↑ 5NT and ↑ALP = liver dz Normal 5NT and ↑ ALP= bone dz Biliary obstruction Not found in muscle or bone ↑ GGT due to drug Assessed with ALP toxicity, biliary obstruction, alcohol Hemolysis can affect Hypophosphatasia Hydrolyzes 5’-esters No bone source like ALP Lactate Dehydrogenase (LD/LDH) Indicates cell injury, cellular ↑ MI, liver disease, muscle respiration trauma, renal infarct, -non-specific hemolytic, pernicious anemia In AML, ↑ over 48 hrs and normal 7-10 days Glucose and galactose Ability to metabolize carbs Hemolysis can affect Not increased in pregnancy or childhood Normal with bone dz and pregnancy Hemolysis no interference LIH Prolonged contact of serum to cells -LD convers pyruvate to lactate while oxidizing NADH to NAD VI. Non-Liver enzymes for testing PT (prothrombin time) Hepatitis Signs Test result Notes High in liver dz Long PT monitor dz progression not assess bleed risk AST ALT ↑ ↑ Biliary obstruction ALP ↑ 5’NT ↑ GGT BIli ↑ ↑ ↑ ↑ Hepatitis Markers- Liver dysfunctions Terms Perinatally: childbirth Parenterally: intravenous or intramuscular (needles) 1) Immunoglobulins proteins that serve as Abs, made in plasma cells as immune response Globulins Increased Decreased IgM Cannot cross placenta Infections, Waldesntrom Inherited diseases (pentamer) Prior to onset of symptoms, macroglobulinemia, declines in 3-6 months, acute rheumatoid arthritis, Anti-A, Anti-B abs hepatis, mononucleosis IgG (80% Crosses placenta Viruses, bacteria, toxins, Kidney damage and infections in serum) After IgM and lifelong HIV Late stage response IgA Increases after birth in mucus, Attack before Abs attack Immune disorders tears, saliva, semen internal tissues IgD On B cells and binds to Connective tissue antigens disorders IgE Attaches to basophils and Allergies, parasitic mast cells infections IgM anti-HAV IgG anti-HAV HbcAg Anti-Hbc HBsAg anti-HBs Hepatitis B e antigen anti- HCV HAV primary marker for infection past infection HBV indicator of active viral replication indicates a past or current hepatitis B infection. current hepatitis B infection indicating recovery and immunity from hepatitis B virus infection Present in acute or chronic infection HCV looks for antibodies to the hepatitis C virus in blood (core antigen) (Hepatitis B core antibody) (hepatitis B surface antigen) enveloped can not differentiate current or past infection Hep type A B Gene material RNA DNA Mode of transmission fecal-oral Parenterally Perinatally Sexually Parenterally Sexually Chronic Vaccine infection No Yes Yes Yes Incubation period 2-6 weeks 8-26 weeks Notes C RNA D E Yes No 2-15 weeks chronic hepatitis major RNA Parenterally Sexually Yes Yes 3-10 weeks RNA fecal-oral Rare No 3-6 wks Only develops if coinfected with Hep B self-limiting disease (resolves itself) Liver produces: Glucose synthesis Function Glycogenolysis Gluconeogenesis Both maintain stable glucose levels Lipid synthesis -FA breakdown -Lipoprotein production to transport lipids -Made in Rough ER of liver Protein Synthesis Liver function Testing Most common acute and chronic hepatitis Components Two sources: - Glycogenolysis uses stored glycogen - Gluconeogenesis using pyruvate, lactate, and amino acids FA -> acetyl COA -> Trig, phospholipid or cholesterol 1. APR- Acute Phase Reactant (inflammation markers) 2. Albumin (osmotic pressure and transport) 3. Clotting factors 4. Amino acids Detected in all body fluids Past or current contact with blood can’t tell past, current Notes Glycogenesis- stores glucose as glycogen Cholesterol mainly produced in the liver and not obtained from diet Fetal liver site of hematopoiesis during late pregnancy Pancreas Terms Hypovolemia- body looses too much blood or fluid Steatorrhea- oily stool Lipiduria- lipids in urine, oval fat bodies 1) Pancreatic activity controlled by a) Secretin -regulates bicarbonate b) cholecystokinin- stimulates pancreas to release pancreatic enzyme 2) Secretes hormones a) Insulin – β-cells – lowers glucose levels b) Glucagon – α-cells – raises glucose levels c) Somatostatin – δ-cells – down-regulates others d) Gastrin – stimulates stomach acid production 3) Enzymes a) Lipase – triglycerides to monoglycerides i) Present in pancreas, stomach, intestine ii) Pancreas content 9000x other tissues b) Proteases –proteins to amino acids i) Trypsin and chymotrypsin ii) Elastase c) Nucleases – nucleic acids to nucleotide Disease What is it? Causes Signs Lab Pancreatitis Inflammation from Biliary obstruction Hypovolemia Amylase rise w/in 5-8 autodigestion by Alcoholism Hypocalcemia hours. Elevated 3-5 days activated enzymes hyperlipidemia Steatorrhea HyperMalabsorption (↓ B12 Lipase- rise w/in 4-8 PTH/hypercalcemia pernicious anemia) hours. Elevated 8-14 days and ↓ over time Cystic altered mucus Dilation of ducts Intestinal obstruction Genetic testing: sweat Fibrosis secretion in lungs and cyst filled mucus Malabsorption chloride testing pancreas Failure to thrive in kids Abnormal Na+ and Cl- transport across membranes Pancreatic Exocrine Function tests What is it? Method Notes Fecal Fat measures the amount of fat in the stool Stool Fat Stain determines the presence or (qualitative) absence of excess fat Fecal Elastase Determine pancreatic insufficiency (does not make enough enzyme) Pancreas Tests Amylase (AMS) 28-100 U/L Lipase (preferred) Oil Red O or Sudan III – microscopic assessment Immunological measurement What is it -starch to glucose and maltose Requires calcium co-factor -Present in saliva, pancreas -Breaks down carbs Increased Acute pancreatitis, cholecystitis, opiates, GI issues Breakdown of fats to fatty acid and glycerol Pancreatitis, obstruction, cholecystitis, glomerular filtration ↓, ulcers ~38 U/L More specific and sensitive Carcinoembryonic CEA to monitor antigen (CEA) colon cancer Pancreatic Tumors Insulinoma Gravimetric assay Decreased Obstruction of salivary glands (mumps) Urine Amy ↑ longer than serum Low= pancreatic insufficiency malabsorption or diarrhea Error -Opiates falsely Serum or ↑ levels heparin -Triglycerides plasma suppresses AMS Common enzyme excreted in urine, Hemolysis b/c Hgb inhibits LPS Turbidimetry Monitors colon cancer Effects Hyperinsulinism Severe hypoglycemia Glucagonoma Hyperglycemia VIPoma Severe diarrhea ZollingerGastric HCl Ellison hypersecretion syndrome Watery diarrhea Adenocarcinoma clay-colored stools 72 hr stool collection CA 19-9 is a non-specific marker to monitor Stomach 1) Secretions HCI, pepsinogen to pepsin, intrinsic factor binds B12 2) Small intestine functionSite of digestion and nutrient absorption a) Carbohydrates absorbed as monosaccharides b) Protein absorbed as aa and dipeptides c) Lipid absorbed as fatty acids, cholesterol 3) Large intestine function (colon) a) Water absorption, forms solid stool Stomach disorders Description NSAIDS dec. mucus production, allows acid erosion H. pylori Tolerates acid, attached to mucosal cells Causes ulcers Cyto-toxins Inflammatory response Zollinger-Ellison Excess acid + diarrhea syndrome Stomach cancer Often w/ H. pylori Intestinal Disease Maldigestion Malabsorption Function ↓ breakdown of food, can’t absorb ↓ absorption of nutrients Celiac Disease Antibodies against gliadin, a protein of gluten in wheat, barley, rye Destruction↓ absorptive surface, malabsorption Lactose hydrolysis, bacterial metabolism sulfur gas, acids Osmotic influx, bloating, cramps Lactose intolerance Colon cancer Bowel Dz (IBD) 1. Ulcerative colitis 2. Crohn’s disease Results from maldigestion Loose stools, greasy, odor, anemia -Anti-tissue transglutaminase (TTG)–IgA (tTg-IgA) (sensitive and specific) -Anti-gliadin – IgA or IgG Breath test Bacteria form H2 from unabsorbed lactose CEA used to monitor disease Autoimmune with genetic component Limited to colon and mucosal lining abdominal pain and diarrhea -use pANCA testing In small bowel but may affect all of abdominal pain and diarrhea GI tract malabsorption -use ASCA testing Test H. pylori test antibody ELISA antigen (stool) Urea breath test (UBT) Xylose Absorption Test non-metabolizable carbohydrate Colon cancer testing Ova and parasite test Leukocytes test Stool osmolality or electrolytes Stool α1 -antitrypsin What is it Does not distinguish current vs prior infection indicates active infection Drink liquid that has labeled urea Bacteria will convert urea to CO2 Notes preferred <25% urine excretion = malabsorption -Use urine -Blood sample if renal disease 1. Fecal occult blood test (FOBT) 2. fecal immunochemical test 3. Carcinoembryonic antigen-CEA 1. Blood in stool (3 samples) 2. Blood in lower intestine 3. Monitoring only, not dx Does not detect worms, only eggs are passed through the stool infection or inflammation Low gap – secretory diarrhea High gap- malabsorption or ingesting unabsorbable items protein-losing enteropathy (Loss of serum proteins from the digestive track) Stool sample Liquid stool sample A1A is resistant to digestive enzymes Cause: IBD, GI obstruction, neoplasm, Celiacs Lipids 1) Lipid content differs due to sex hormones and aging (trig ↑ with age) a) Men= lower HDL, higher cholesterol, higher trig b) Women=higher HDL, lower cholesterol, lower trig Components What is it Notes Fatty acids Hydrolyzed by lipids Binds to albumin and is -used for energy storage free in plasma -endogenous (unesterified) Triglycerides 95% stored in fat and released Transported by (made from -exogenous and endogenous chylomicrons and carbs, has FA, VLDL glycerol)) Glycerol recycled into trig Phospholipids (in liver) Cholesterol (in liver) 2 FA + 1 phospholipids to glycerol Surface of lipid layers and lipoproteins 4 rings + 1 C-H side chain -Not a fuel source -↑ fat absorption in intestine, makes Vit D -precursor to steroid hormones -exogenous & endogenous Notes ↑ double bonds= ↓ MP Trans FA=risk CHD, high LDL, low HDL Hydrophobic Glycerol contamination can increase trig Hydrophilic and hydrophobic (amphipathic) LDL is primary carrier to cells HDL carries out of cells Amphipathic Released into circulation as lipoproteins 2) Lipoprotein Features (water insoluble lipids and soluble proteins) a) Outer layer apolipoprotein (water soluble)structure, binding site b) Transports hydrophobic molecules c) Lipoprotein metabolism i) Insoluble fats need to be soluble for intestinal absorption FA cleaved off by lipase ii) Lipid absorption, exogenous, endogenous pathway, reverse cholesterol transport 3) Exogenous pathway a) GI tract absorbs fat + cholesterol (packaged as chylomicrons) b) Secreted into circulation and tissue binds lipoprotein lipase c) Chylomicrons are cleaved off Trigs and taken up by liver d) Trigs release FA, cholesterol, aa e) Some lipids HDL 4) Endogenous pathway a) Hepatic derived lipids to cells for energy b) FA and cholesterol in liver VLDLtravel to peripheral cells lipolysis c) VLDL remnant taken up by liver or become LDL 5) Reverse cholesterol Transport pathway a) Uptake of cholesterol by HDL and esterification of HDL b) Returns excess cholesterol transported to liver for excretion prevents atherosclerosis (choronary heart disease) Lipid Panel Range (mg/dL) Total Cholesterol good<200-239>bad LDL (bad) (60-70%) good<100-129>bad HDL (20-30%) Bad<40-59>good Indicators of heart dz Low levels: alcoholic cirrohosis 12 hour fasting serum Non-fasting: lipemic, fat droplets, ↑ trig, chylomicrons on top layer w/ refrigeration Triglyceride Good<150-199>bad Lipoprotein class Chylomicrons Where made Intestine Function Transporting dietary trigs and cholesterol Turbid appearance and Apo B-48 absorbed by intestinal epithelia chylomicrons float on top if plasma stored in fridge Enter circulation and Uptake by hepatic cells (normal metabolism) 86% trig Milky turbid sample Liver Major triglyceride transporter to cells for energy needs or store as fat Biggest but least dense, Exogenous VLDL Features HDL (good) smallest and most dense Major cholesterol carrier liver and intestine apo B-100 apo-E apo-C pre-beta Turbidity but not milky (due to small size) -Methsclerotic plaque 50% cholesterol apo B-100 Best predicator of cardiovascular disease Direct OR Indirect-remove chylomicrons, VLDL, LDL Direct Unloads trigs, transports cholesterol to cells Best indicator for cardio dz Recycled chylomicrons and VLDL 50% protein Transports excess cholesterol from tissues and deliver back to liver. Removed using bile salts Testing: 55% trig Normal metabolism: VLDLs secreted into blood by liver LDL (lethal) Marker bad beta Apo A-1 Good alpha 6) Factors that affect cholesterol measurement a) Thyroxine i) Hypothyroid: hyper-cholesterol ii) Hyperthyroid: hypo-cholesterol b) Hormones i) Post-menopausal: ↑ LDL ii) Pregnancy c) Stress, nephrotic syndrome d) Turbidity ↓ absorption e) HDL: high trig levels (400 mg/dL) can interfere f) LDL = total cholesterol – [HDL +trig/5], not valid if trig is >400 7) Hypo and Hyperlipoproteinemia – acquired or inherited a) Most common is atherosclerosis Dyslipidemias What is it Cardiovascular disease Plaque in heart, myocardial infarction Peripheral vascular -plaque formation, paresthesia disease (PVD) Blocks blood flow, injures cells, inflammation, WBC Cerebrovascular disease stroke Hyperlipoproteinemia -Hypercholesterolemia: genetic defect, predisposed to ↑↑ LDL levels -Hypertriglyceridemia: lipase deficiency so can’t hydrolyze trig and VLDL to cells as FA hypolipoproteinemia -Abetalipoproteinemia: everything low -Hypobetalipoproteinemia: low total cholesterol and normal to low trig -Hypoalphalipoproteinemia: ↑↑↑ trigs and low HDL Positive Cardiac Risk Factor >45 yrs male, >55 years female Family history of CHD Currently smoking hypertension HDL low Diabetes Mellitus (↑ cholesterol) Metabolic syndrome Hyperlipoproteinemia Type I Type IIa Type IIb Type III Type IV Type V Notes Treated by statin: blocks cholesterol synthesis and removes LDL Diseased state: trig stays high an chylomicrons not cleared Negative Cardiac Risk Factor HDL high LDL low Increased Lipoprotein ↑↑ chylomicrons ↑ LDL ↑ LDL and VLDL ↑ IDL ↑ VLDL ↑ VLDL w/ ↑ chylomicrons Cholesterol level Normal to ↑ ↑↑ ↑↑ ↑↑ Normal to ↑ ↑ to ↑↑ Triglyceride level ↑↑↑ Normal ↑↑ ↑↑↑ ↑↑ ↑↑↑ Cardiac 1) Symptoms of heart disease a) Syncope = abnormal heart rhythm b) Edema= lowered cardiac function c) Cyanosis = pulmonary insufficiency d) Dyspnea = reduced function e) Angina pectoris = CAD Cardiac diseases What is it Congenital heart failure Abnormal heart or vessel formation (CHF) -excess fluid in lung but kidney compensates by retaining liquid Acute coronary Caused by atherosclerosis (thick artery wall) syndrome (ACS) Hypertension High blood pressure of 140/90 Infectious agents dz -rheumatic heart dz autoimmune response -streptococcus infection -endocarditis staph and strep -pericarditisinflammation of heart membrane Notes Rubella infection Plaque formation, thrombosis Obesity, inactivity 2) Blood test biomarkers sensitive to myocardial infarction a) Collect multiple samples. AST and LDH is non-specific for heart, not used 3) Troponin a) Rises at 4-10 hours after onset, Peaks at 12-48 hours, elevated for 4-10 days b) Complex of 3 proteins: TnC (binds Ca, not useful), TnI (inhibition of ATPase), TnT (tropomyosin-binding) Cardiac Markers Troponin Very specific & sensitive for MI Myoglobin Where found skeletal AND cardiac muscle Function Notes Binds to actin in muscle and replaces Ca+ binding (Inhibits contraction) Released into the blood upon injury to the heart, muscle *Found in all muscle types skeletal AND cardiac muscle Heme protein that binds oxygen-binding protein 30-90 ng/mL non-specific Creatine Kinase (CK) 15-60 U/L hsC-reactive protein 0.3-8.6 mg/dL B-type (brain) natriuretic peptide (BNP) <100 pg/mL hsCRP Homocysteine Acute-Rise and fall Chronic-Elevated but stable Released into blood when heart attack or severe muscle damage Notes Enzyme activity Not found in the healthy population Positive values indicate myocardial injury but not acute ↑↑ AMI marker and necrosis Slowest to return to baseline -absence rules out AMI but ↑ -Most rapidly rising marker (30 isn’t diagnostic min) -Fastest to return to baseline in 24 hrs ↑↑ in renal disease intracellular storage site for oxygen Transfers PO4 from creatine to ADP for muscle contraction and serves as heart’s primary energy reserve ↑↑ associated with cardiac issues, skeletal issues, CNS Healthy population: CKMM as major isoenzyme CK-BB: not detectable Rises and returns to normal in 2-3 days 100% sensitivity by 6-8 hours CK-MB>CK when heart is damaged (AMI) Troponin tested with CK-MB Liver Used as predictor and assessment for CAD Released during injury or inflammation Heart Promote excretion of Na and water and ↑ GFR and ↓ reabsorption of Na Secreted in response to ventricular pressure and when ↑ fluid in CHF Concurrent skeletal muscle damage may mask cardiac damage Correlation of increased CRP to CAD due to atherosclerosis Regulates cardiac homeostasis Causes dilation of blood vessels, reduces fluid load Released by ventricular walls during hypertension and ↑ volume ↑↑ is risk factor Skeletal, heart brain CK-MB (myocardial band) <6% of total CK Marker for CHF Lowers blood pressure Marker for chronic inflammation Amino acid associated with B6, B12 and folic acid ↑↑ risk factor BNP antagonist to reninangiotensin-aldosterone system (RAAS) which ↑ blood pressure by vasoconstriction and ↑Na Patient must be free of other inflammatory processes Pre-proBNP cleaved to BNP and NT pro-BNP: BNP C-terminal is active N-terminal is inactive If BNP used as medication, monitor NT pro-BNP for monitoring Tumor Markers 1) Small proteins, abnormally high []s in blood, tissues, body fluids 2) Placental Tumors a) Phantom hCG- low levels or falsely elevated residual tumor or interference b) Urine hCG- interference antibodies too big 3) Glycoprotein tumor markers a) Mucins: CA 15-3, CA 27.29 b) Blood group antigens: CA 19-9, CA 72-4 4) Protein tumor markers a) Immunoglobulins Multiple myeloma markers b) Thyroglobulin (TG)thyroid cancer, falsely low due to anti-TG abs c) Chromogranin ANeuroblastoma, pheochromocytoma, carcinoid 5) Tumor marker assays a) Sandwich ELISA assays stable i) But beware of hook affect high []ed samples that registers negative which have excess of antibody that binds itself and doesn’t interact with the assay ii) Poorly standardized meaning you must establish new baseline for each assay to compare. If a sample has been tested previously in an assay, then run the new sample with both methods and report both. Subsequent samples get the new assay 6) Markers used to confirm diagnosis Tumor Hormones ACTH Calcitonin (not procalcitonin) Parathyroid hormone Prolactin hCG (human chorionic gonadotropin) Tumor markers Prostate-specific Antigen (PSA) >2.5 ng/mL (biopsy) <10% : cancerous >25%: benign hCG AFP (alpha fetal protein) CEA (carcinoembryonic antigen) Ca 15-3, Ca 27.29 CA-125 CA 19-9 CA 72-4 Where Function Anterior pituitary thyroid ↑ glucose Insulin antagonist Lowers Ca levels -marker of sepsis Pituitary gland During pregnancy and after birth for breast milk or ↓ sex drive in men Used to diagnose trophoblastic (placenta) cancers -correlates well with tumor[] Placenta in pregnancy Found Prostate, breast tissue, but only small [] in serum Notes Bound PSA=stable, inactive Free PSA =immunoreactive (detectable) Placenta/testicular Liver and germ ↑ in amniotic fluid cell (testicular -↑ AFP, ↑ burden and ovarian) -liver cancer marker All except liver, prostate, testicular Fetal GI tract Lung, breast, pancreatic, ovaries Disease Notes Prolactinoma Cushing’s disease, lung carcinoma Medullary thyroid carcinoma Parathyroids or ectopic Pituitary or ectopic Pancreatic, biliary, testicular cancers Measure FSH, LH, TSH or whole hCG molecule Bacterial infection Hyper-PTH Seen in Free PSA: Total PSA Ratio ↓ in cancer Notes Broader ranges and not race specific Many false positives Trophoblastic cancer liver dz (cirrhosis, hepatitis), cancer -does not correlate well ↓ in maternal serum during pregnancy (Down syndrome) Elevated in liver dz, IBD, pancreatitis Not good for screening Elevated in other cancers Not good in screening Ovarian cancer Blood group Ag Pancreatic, hepatic, -not expressed in 5gastric 10% population Ovarian, breast Used to monitor only Need type A or A/B blood or not good to use Nutrition and Trace Elements/metals 1) Ferrous is Fe+2 (soluble) and Ferric is Fe+3 (insoluble) a) Absorbed as Fe+2 and recycled, free form used as transport b) Ferrous bound to transferrin (excess, rarely saturated) c) Overabundance of free Fe is toxic 2) 65% of Fe is in Hgb – O2 transport a) Transported by haptoglobin and transferrin. Stored as ferritin. Chaperoned by hemosiderin 3) Copper a) Enzyme co-factor that bounds to ceruloplasmin and circulates b) ↓ copper: microcytic hypochromic anemia and neutropenia c) ↑ copper: Wilson Dz (rings around eyes) i) ↓ ceruloplasmin and serum Cu + ↑↑ urine Cu Fe related Tests Function Notes Notes Notes Serum Fe TIBC (binding -reflects transferrin Salts added to serum to Trsf sat = Fe/TIBC capacity) levels saturate binding sites (15-60%) -amount of on transferrin 175-400 mcg/dL transferrin bound to -unbound Fe Trsf= 0.8 x TIBC Fe precipitated -supernatant measured RST, heparinized Unsaturated IBC Ferritin -measures unbound Fe only UIBC + serum Fe= total Fe Falsely ↑ when inflammation Normal Fe during cirrhosis but truly low due to malnutrition Proportional to stored Fe Levels Disease Normal Storage Fe Depletion (No anemia) IDA Anemia of chronic Disease (inflammation) Thalassemia Hemochromatosis Sideroblastic Anemia Fe (ug/dL) Transferrin Sat (%) 20-55 250-425 N N ↓ ↓ ↓ ↓ ↑ ↑ ↓ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ N ↑ ↑ ↑ 65-175 (M) 50-170 (F) N TIBC Ferritin 20-250 (M) 10-120 (F) ↓ Diseases Notes IDA Stages 1. Iron Depletion (no -Low Fe so storage used anemia) -Ferritin maintains serum Fe 2. Iron-deficient -depleted stores erythropoiesis (no anemia) 3. IDA (hypochromic anemia) Chronic Dz of Anemia (ACD) Levels Hematology Ferritin ↓ Normal RBC morphology Fe ↓, ferritin ↓, transferrin RBC microcytic, ↑RDW saturation ↓ TIBC ↑ Fe ↓, ferritin ↓, transferrin ↓,Low Hgb, MCV, MCH, TIBC ↑ MCHC; ↑ RDW Microcytic ↓ Fe interferes with bacterial growth and erythropoiesis Ineffective erythropoiesis Cytokines induce hepcidin Which prevents Fe release in GI tract ↓ absorption Iron Overload/ Hereditary -Fe deposited in pancreas, hemochromatosis heart, livercirrhosis or diabetes, CHF Fe ↑, transf sat >50%, TIBC ↓ to normal 4) Porphyrins and porphyria - Ring structure containing Fe in ferrous or ferric a) Key components in heme biosynthetic pathway b) If abnormal, can lead to build up of intermediates and excreted (not good) c) Coloration of bruising i) Heme biliverdin bilirubin ii) ↓ heme doesn’t negatively regulate biosynthetic pathway symptoms d) Collect urine and stool. If negative, then acute porphyria unlikely e) Heme synthesis- Takes place in mitochondria and cytosol 5) Fat soluble vitamins (stored) a) Vitamin A carotene, retinol, retinal (vision and epithelial cells) b) Vitamin D cholecalciferol (absorbs Ca) c) Vitamin E antioxidant (free radical scavenger) d) Vitamin K phylloquinones, menaquinones (makes clotting factors) i) Not secreted in urine readily so deficiency develops slowly (↓ intake, or absorption) 6) Water soluble vitamins (not stored so secreted) a) Vitamin B Thymine, convert carbs to energy, muscle contraction, ATP i) Found in breads and cereal ii) Measure in whole blood, not plasma b) Vitamin B3 Niacin, required precursor NAD and NADP i) ↓ B3 means dermatitis, diarrhea and dementia c) Vitamin B6 Pyridoxine, co-factor for ALT and AST d) Vitamin C Ascorbic acid, antioxidant, maintains metals in reduced form i) ↓ C means scurvy, reopening of wounds, bleeding gums e) Vitamin B12 Cobalamin, needed for thymidine synthase and folate i) Absorption required low pH in stomach and intrinsic factor ii) Transported in blood via transcobalamin to liver iii) Reference: 180-900 pg/mL iv) Tested with methylmalonic acid: ↑ MMA reflects B12 deficit v) Decreased gastric acidity, ↓ intrinsic factor, malabsorption, megaloblastic anemia f) Folate dietary intake only i) Needed for RBC production ii) Tested on whole blood, deficiency rare Carbohydrates and Diabetes Terms Addison’s Dz: 1) Monosaccharides glucose, fructose, galactose (reducing agents) 2) Disaccharides sucrose (non-reducing), lactose (milk sugar) a) Glycolysis can also produce lactate under oxygen deficit b) Liver dz ↑ lactate. Hypoxia and metabolic disorder c) Avoid tourniquetfalsely raise blood lactate levels Metabolic Pathways Function Notes Glycolysis (anaerobic) Makes pyruvate Tricarboxylic acid cycle Utilizes oxygen as part of cellular Acetyl CoA reacts with carbon molecule (aerobic)/Kreb’s cycle respiration ATP, NADH, FADH2, CO2 Glycogenesis Stores glucose as glycogen Glucose glycogen Glycogenolysis Breaks glycogen to glucose during Glycogen glucose fasting Gluconeogenesis (nonGenerates glucose from pyruvate, Noncarbs glucose carb source) lactate and aa 3) Specimen collection a) Glucose ↓ by 10 mg/dL per hour in whole blood b) Refrigerated stable c) Arterial and capillary values are higher d) Normal CSF values are two-thirds of plasma glucose levels 4) Glucose Reference Ranges a) 70-100 mg/DL (fasting 8 hrs) b) Below 20-30 mg/dL is life threatening c) Hypoglycemia: <50-60 AND Hyperglycemia: >100 5) Ketone measurement a) Acetoacetate created from fatty acid oxidation NADH ↑ reduces to BHB b) BHB measured. If treated, should ↓ BHB Hormone & glucose levels Insulin Glucagon ACTH Growth hormone Cortisol Human placental lactogen Epinephrine T3 & T4 Source Action Beta cells Alpha cells of pancreas Anterior pituitary Anterior pituitary Adrenal cortex Placenta ↓ glucose, uptakes glucose in cells ↑ glucose, glycogenolysis ↑ glucose, insulin antagonist ↑ glucose, insulin antagonist, acromegaly = hyperglycemia ↑ glucose, gluconeogenesis, addison’s dz ↑ glucose, insulin antagonist Adrenal medulla Thyroid gland ↑ glucose, glycogenolysis, hyperglycemia, inhibits insulin ↑ glucose, glycogenolysis Hormones control glucose Function Insulin (by pancreas) Facilitates uptake of glucose into cells -Inhibits glycogenolysis C-peptide (by pancreas) Reflects pancreatic secretion of insulin Glucagon (by pancreas) Acts on liver to ↑ blood glucose -glycogenolysis, gluconeogenesis, lipolysis ↑ glucose absorption Thyroxine(T4) Somatostatin Measurement -hypoglycemia -Diabetes Mellitus (DM2) ONLY hormone to ↓ glucose -Hypoglycemia assessment Endogenous insulin: C-peptide ↑ Exogenous insulin: normal/↓ C-peptide *better to use Secreted when ↓ glucose to increase glucose levels Inhibits insulin, glucagon, GH and results in ↑ glucose Test Interpretation to Diagnose Diabetes Mellitus Stage Fasting glucose plasma Normal Diabetes Mellitus (body not using sugar well so high) Pre-Diabetes Affect Released when glucose ↑ <100 mg/dL >126 mg/dL TEST Oral glucose tolerance test (OGTT) 2 hr PG <140 mg/dL >200 mg/dL plus 2 hr PG >200 mg/dL unexpected weight loss, polyuria, polydipsia Impaired glucose tolerance Casual glucose plasma Impaired fasting glucose if 101-125 mg/dL GTT: how well body uses sugar, baseline test is 75g glucose given and monitor after 2 hours Hgb A A1c <5.5% >6.5% 5.6-6.4% Glucose Disease Hypoglycemia Causes ↓ Glucose liver disease, Addison’s dz Type I Diabetes mellitus (DM) Insulin deficiency Excess ketones Type 2 Diabetes Mellitus Diabetic Ketoacidosis (DKA) Gestational Diabetes Insulin resistance due to sedentary life, obesity Blood sugar ↑↑ and ketones ↑ During pregnancy, insulin resistance, hormones interfere w/ insulin Microalbuminuria Minimal glomerular damage Minute amounts of albumin -early renal damage DM Pathogenesis Acute Chronic Autoantibodies Symptoms Early: chills, sweating Late: speech ↓, coma, CNS damage Insulin ↑ Ketoacidosis, glycosuria, polyuria, polydipsia, polyphagia (↑ hunger) ↑ glucose so insulin treatment ineffective Common in type I DM Fruity breathy odor Fetus: hypoglycemia b/c fetal not resistant More than URL diabetic nephropathy Diagnosis Not as prone to ketoacidosis Fasting and interval tests: ↑ glucose after one hour and ↓ decreases 30-300 mg/dL in preferred sample DM Type I DM Type II B-cell destruction, genetic, viral Insulin resistance, B-cell dysfunction Glucose deficit, ketoacidosis Hyperosmolar hyperglycemic state Stroke, damage to tissues, infections Islet cell Abs, GAD65, Antiinsulin Abs 6) Hgb A more dominant, HbA1C = glucose a) Proportional to average blood glucose b/c RBC permeable to glucose molecules b) Glucose + Hgb glycosylated hemoglobin c) Results glucose level of 1-3 months d) Variants Hgb S or C can alter A1C levels e) Advantages: less variability, no need to fast, chronic implications f) Disadvantages: anemia ↓ RBC lifespan, G6PD deficiency, injection of EPO ↑ span g) Measured by HPLC 7) Fructosamine/Glycated proteins ketoamine products a) Any blood protein can by glycated – measured by HPLC b) Useful for Hgboapathies, hemolytic anemia, GDM c) Measures levels for 2-3 weeks Endocrinology-Pancreas, thyroid, adrenal glands Terms Addison’s Dz: adrenal insufficiency, no cortex so ALD and cortisol ↓ Conn’s syndrome: benign tumor so ALD ↑↑↑ causing hypernatremia and hypokalemia 1) PANCREAS- collection of glands, organs, and cells that secrete hormones or non-hormones a) Cells produced in one site have specific regulatory effects on activity of other target cells at a distance Gland types Features Examples Endocrine No ducts, remain as blocks in Hypothalamus, pituitary, adrenal cortex tissue so secretes hormone in Ex: pancreas releases insulin due to presence blood stream of another hormone Exocrine Has ducts, activity at distant Sweat and salivary glands, pancreas secrete or same site, non-hormone juice to lumen Hormones Classification Proteins/polypeptides Steroid-cholesterol derived Features Response Mins to hours to days Water soluble so no carrier protein triggers protein kinase intracellular signals with cells Day to weeks to months Lipid soluble so transport protein Active intracellularly so binds to nuclear receptors and modulates gene txn Aromatic aa derivatives from tyrosine T3 and T4 (thyroid Transported by protein for gene txn hormones) Intracellular activity Catecholamines (epinephrine, Water soluble, neurotransmitters norepinephrine) Affects intracellular cascades via kinase activity 2) Endocrine axis concept hypothalamus, pituitary gland, endocrine gland a) Signals have positive and negative feedback b) Ex: hypothalamus releases factors that stimulates pituitary gland to produce trophic hormones such as TSH i) TSH hormones stimulate thyroid to make T3, T4 ii) Hormones bind to receptors and effects target tissue so T4 goes back inhibiting production of TSH iii) Hormones act in negative feedback loop c) Levels of Endocrine Disease i) Tertiary disease (rare) hypothalamus Slow response Fast response ii) Secondary (less common) pituitary iii) Primary (more common) endocrine gland (thyroid, adrenal, gonads) d) Hypofunction is when primary function ↓ i) Upstream have ↑ed levels of hormones released levels to make something e) Hyperfunction is when primary function ↑ i) everything upstream ↓ due to negative feedback Pituitary Gland Type Anterior pituitary gland Or adenophypophysial Hormones found Direct effectors Growth hormone (GH)/somatotropin Prolactin (Prl) Males: 5-21 ng/ml Females: 6-30 ng/ml Features release GHRH, GHIH by hypothalamus ↑: Gigantism, acromegaly ↓: pituitary dwarfism, mental issues Inhibited by somatostatin -deiodinase T4 into T3 Increased in pregnancy, nipples -when (hypothyroidism) TRH ↑, Prl induced -constant dopamine inhibition -suppressed GnRH in hypothalamus -prevents secretion of FSH, LH Indirectly trophic hormones Adrenocorticotropic pigmentation of Cushing’s and stimulates cholesterol hormone (ACTH) Follicle stimulating hormone (FSH) Luteinizing hormone (LH) Thyroid stimulating hormone (thyrotropic, TSH) Growth hormone Posterior pituitary gland Or neurohypophysial Made in hypothalamus but stored in this gland Cyclic nonapeptide (9 ass’s) Acts on kidneys to increase water reabsorption Hypothalamic osmoreceptors and cardia/carotid baroreceptors Vasoconstrictor Deficiency leads to diabetes insipidus Arginine Vasopressin (AVP, antidiuretic hormone, ADH) Oxytocin Involved in lactation and labor Important in creating bonds between mother and baby Pitocin is synthetic hormone used to induce labor 1) Insulin-Like Growth Factors synthesized by liver in response to GH a) IGF-1 and IGF-BP3: blood levels correlated with GH secretion 2) Growth hormone disorders/Testing a) GH stimulates glucose but High glucose level inhibits GH b) Insulin lowers blood sugar levels and uptakes glucose c) Done on fasted individual Normal subjects -GH has negative feedback to inhibit GH. Oral test ↑ glucose so no need to stimulate Hypersecretion GH <1 ng/mL Hyposecretion -GH doesn’t suppress and GH ↑ False positive: liver disease, uremia Insulin tolerance, hypoglycemia If GH doesn’t increase, then confirms deficiency of GH 3) Prolactin disorders – action is to influence lactation Abnormality of -When PRL ↑, dopamine ↓ Hyper-Prl -PRL ↑ when TRH ↑ (>150-200 ng/mL) APG, proportion to tumor size -false negative values when high dilute -suppressed GnRH leading to ↓ FSH and LH ovulation disturbed Prolactinoma Amenorrhea and galactorrhea (f) Lactation in men -52 yo male with 6 mo history of headaches, left sided weakness and left hearing loss -Serum prolactin 7.3 ng/ml (4.1-18.4) is low (without dilution) -MRI shows massive tumor -Dilution of sample (1:1000) yielded prolactin 122,260 ng/ml 4) THYROID- Vascularized with Precursor colloid thyroglobulin, takes up iodine and creating T3 and T4 a) Parafollicular C cells minoritySecrete calcitonin b) Hypothalamic-Pituitary-Thyroid Axis i) Hypothalamus thyrotropin (TRH) which acts on pituitary gland ii) Pituitary gland ↑ TSH and acts on thyroid gland iii) Thyroid T3 and T4 iv) Negative feedback TRH and TSH inhibited v) T4 is thyroxine and T3 is triiodothyronine (active) 5) Thyroid hormone synthesis organification a) T4 is the primary secreted hormone by the thyroid i) Transported bound to protein: (1) Thyroxine-binding globulin (TBG) is 60-75% (2) Albumin is 15-30% (3) Transthyretin (prealbumin, TBPA) is 10% ii) Only free hormones are biologically active (1) 99% of T3 and T4 is bound (2) T3 is the active form and binding is reversible b) Peripheral thyroxine metabolism i) T3 accounts for most activity in peripheral tissue ii) When T4 is converted to T3 by deiodination form biologically inactive (1) This is called rT3 reverse (inactive) iii) T3 is more active but only in proper confirmation 6) Thyroid- Important in fetal and neonatal CNS development Hyperthyroidism Grave’s Disease (autoimmune, 60-80%) Antibodies stimulate TSH receptor (TSI, TSIg, TRAb) triggers neonatal hyperthyroidism due to IgG ↑ ↓ TSH and thyroid hyperactivity Thyroid nodules Single or multinodular, benign Thyroiditis Inflammation due to anti-thyroid antibodies destroying thyroid Initial stages of hashimoto ↓ TSH, ↑ T3 and T4 High levels of hCG triggers TSH Choriocarcinoma caused by cross-rxn Increased in iodine ingestion Common in females Thyroid cancer Pregnancy Tumor disrupts hormones Hypothyroidism Hashimoto’s thyroiditis-later stages 90% have antibodies to thyroid peroxidase (antiTPO) and thyroglobulin (anti-Tg) Constant attack of thyroid by antibodies Decreases T3 and T4 levels, TSH ↑ Thyroidectomy or radio ablation treatment Congenital: 1:4000 births so newborn screening Secondary or tertiary : <1% cases, TSH or TRH failure rare Thyroid hormone resistance is rare autosomal dominant Lab evidence shows increased TSH with normal T4 but no symptoms Common in developed countries and females. Most common worldwide due to iodine deficiency -Calcitonin used as marker -thyroglobulin used for monitoring epithelial cancers 1st trimester: TSH ↓ TBG ↑ due to estrogen 2nd & 3rd trimester: FT4 and FT3 ↓ Total T4 and T3 ↑ 7) Why measure free hormones? a) In pregnancy binding protein ↑ so bound T4 ↑ but free T4 is normal Thyroid Tests TSH 0.27-4.20 mcIU/mL What is it? -primary test, reflects hypothalamic response to FRT4 and T3 levels -Total T3: 90-200 ng/dL Free T4 (thyroxine) Total T4 4.8-10.4 mcg/dL Free T3 Increased Hypothyroidism -insufficient T4, ↑ TSH Gland not responsive to lower TSH Hyperthyroidism No antibodies so hormones increasing Decreased Hyperthyroidism -FRT4 ↑↑, TSH ↓ ↑ T4 in pregnancy due to estradiol hypothyroidism Bound T4 + free PTH T3 uptake indirect measurement of TBG Controls Ca levels Anti-TPO Indicates inflammation, improper immune system Hyper-PTH vit D ↓, adenoma Causes Hashimoto, pregnancy and babies Hypo-PTH hypothyroidism 8) Anti-Thyroglobulin (anti-TG) a) Tumor marker for papillary of follicular thyroid cancer (treated by ablation) b) False readings if there are anti-thyroglobulin antibodies so always include an competitive assay to determine their presence c) Liquid chromatography or MS which has no interference from antibodies 9) Thyroid disorders a) Hyperthyroidism example: increase T4 and T3, low TSH, increase free T4 i) Thyroid hormones increasing because no antibodies b) Grave’s disease: low TSH but has antibodies present so TSI positive c) Subclinical hypothyroidism is just normal T4 10) Non-thyroidal Illness (NTI) a) Abnormal results but no thyroid issues b) Thyroid assessment should not occur during acute illness Excess Hypo-thyroid Grave’s dz (primary) Thyroid stimulating Ig stimulates thyroid receptors Hashimoto (initial) -autoimmune hCG in women Deficiency hyper-thyroid Hashimoto (late stage) Tests TSI and TRab Anti-TPO and anti-TG High hCG triggers TSH Sheehan’s syndrome (2ndary) 11) ADRENAL GLANDS a) Cortex produces steroid hormones b) Medulla produces catecholamine hormones c) HPA axis: Stress hypothalamus release CRHACTH stimulatedcortisol/steroid hormones 12) Steroids = derived from cholesterol 3 classes of steroids What is it Notes Glucocorticoids (cortisol) -maintains blood -Negative feedback on ACTH and CRH pressure -cortisol high in mornings and low at night -anti-inflammation -stress ↑CRH -cause glucose ↑, protein catabolism Mineralocorticoids 60% bound -low K inhibits aldosterone (aldosterone) RAAS -BNP inhibits aldosterone secretion -promotes Na reabsorption and K secretion Conn’s Dz: ↑ ALD, Na ↑, K↓, hypertension Addison’s dz: ↓ ALD, ↓NA and Cl, ↓ cortisol ↓, low Hgb, ↑ACTH when primary, ACTH ↓ when secondary Sex hormones 1) Androgens: testosterone Made by testes, adrenals, ↑ during puberty, tumors, masculinization in ovaries females 17-ketosteroids ↓ in hypogonadism (metabolites) 2) Estrogens: estradiol Made by ovaries ↑ during fetal development, 3rd trimester, puberty in grils ↓ means pregnancy complication 3) dehydroepiandrosterone Makes sex hormones Peaks at puberty and declines w/ age (DHEA) 13) Aldosterone Regulation a) Renin-angiotensin aldosterone system (RAAS) i) Renin converts angiotensinogen to angiotensin I converted to angiotensin II stimulates aldosterone ii) Angiotensin makes blood vessels narrow and aldosterone tells kidneys to retain fluid Adrenal Gland Testing Cortisol 6-25 mcg/dL (AM) 3-16 (PM) What is it? primary Increase Cushing’s syndrome: ↑ cortisol, ↓ plasma protein Decrease Addison’s dz: ↓ cortisol Dexamethasone suppressed cortisol Testing 4% free but rarely measured Adrenocorticotropic hormone (ACTH) 7-50 pg/mL secondary Cushing’s syndrome excess cortisol ↑ ACTH Met alkalosis ↓↓ ACTH (no hyperpigmentation in 2ndary), ↓CRH (rare) primary: Cortisol ↓, ACTH ↑ Addisonian crises Inject synthetic ACTH ↑ of 10 mcg of cortisol = normal Treatment: prednisone Corticotropin releasing hormone (CRH) RAAS Aldosterone CRH goes up when cortisol ↓ renin secreted when blood pressure ↓ ↑ in upright position ↓ in Conn’s but Ald is ↑ Maintains Na, K in blood ↑ in upright position ↑↑, low renin: adrenal issues ↑↑, high renin: kidney issue Na+ ↑ Conn’s syndrome Low Na, ↑K Addison’s Dz ↑ means infertility in females ↓ means infertility in males Testosterone Catecholamine Epinephrine, norepinephrine, dopamine VMA=metabolites Metabolic acidosis Plasma or urine Reproductive Endocrinology 1) Hypothalamic-Pituitary Gonadal Axis a) Hypothalamus makes GnRH. Pituitary makes FSH and LH b) Ovaries make estradiol and progesterone. Testes make testosterone 2) Menstrual Cycle a) Day zero = date after period b) Day 14: endometrial lining forms again i) LH peaks at ovulation and FSH slight increase only, estradiol builds up, progesterone increases after ovulation c) Luteal phase is after egg is ovulated and lining is completed i) If no placenta to increase progesterone or estradiol, then levels drop ii) FSH return back to normal Phase Description Focus Follicular Follicular proliferation, endometrium thickens E2 inhibits FSH so FSH ↓ Ovulatory (mid-cycle) Day 14 (ovulation), follicular rupture E2 stimulates FSH, LH Ovum captured by fallopian tube -LH peaks and stimulates P4 Luteal Ruptured follicle becomes corpus luteum NO fertilization: P4 peaks and drops LH and FSH neg feedback E2 peaks then drops off Fertilization occurs: hCG synthesis maintains corpus luteum P4 maintains endometrium 3) hCG (alpha=FSH, LH and Beta=TSH) a) Non-placenta hCG tumors increase with age (5-14 mIU/mL) and FSH > 20 so not pregnant i) In pregnancies, FSH ↓ so hint 4) Menopause Depletion of ovarian follicles, ↓ estradiol a) ↑ FSH and LH (no negative feedback) Hormone Origin in Men/Function Origin in Female/Function Gonadotropin releasing hormone (GnRH) Hypothalamus, stimulates FSH, LH -released in pulsatile fashion -activationsexual maturity By pituitary -spermatogenesis and inhibin secretion By pituitary -libido and testosterone production same FSH LH (luteinizing) Estradiol (E2) Progesterone (P4) hCG (human chorionic gonadotropin) Testosterone Inhibin By pituitary -testosterone and sperm production By Testes -sex characteristics, spermatogenesis Active form = DHT By testes, inhibits FSH and LH By pituitary -follicular maturation and E2 synthesis By pituitary -follicular rupture and P4 synthesis By ovaries and placenta -endometrial proliferation and ↑ HDL -Pos feedback in ovulation -Negative feedback in follicular By ovaries -endometrial maintenance, breast development By placenta Fertilizationmaintains P4 -by testes Tests LH (age, sex, cycle) Function -follicular rupture -P4 synthesis -testosterone production FSH (age, sex, cycle) -follicular maturation -E2 synthesis -spermatogenesis Estradiol (age, cycle dependent) By ovaries and placenta -endometrial proliferation -Pos feedback in ovulation Negative feedback in follicular -sex characteristics (mass) spermatogenesis Active form = DHT Bound to SHBG, albumin (weakly bound), free -endometrial maintenance -breast development ↑ HDL Made in luteal phase but decreases -detects pregnancy -germ cell tumor -serum and urine Bound to testosterone and estradiol ↑ affinity, ↓ capacity -When bound, not available for tissue ↑↑↑↑ indicates ectopic pregnancy Testosterone 260-1000 ng/dL Progesterone (age, pregnancy, cycle dependent) hCG 25 mIU/ML Sex-hormone binding globulin (SHBG) Notes E2 stimulates LH at day 12-13 during ovulation (+ feedback) and LH peaks LH stimulates P4 and egg is captured Luteal phase: FSH ↑ Notes Inhibited by E2 b/c E2 needed for maintenance so no need for FSH during follicular phase ↓ FSH ↓ in menopause Made in luteal phase but decreases Inhibits LH and FSH during luteal phase Disorders of Female reproduction Pseudohermaphrodites Precocious puberty Primary amenorrhea Secondary amenorrhea Polycystic ovary syndrome (PCOS) hyperandrogenism Disorders of Male Reproduction Hypogonadotropic hypogonadism Hypergonadotropic hypogonadism Androgen Insensitivity Syndrome What is it? Gonadal sex different than genital sex -normal chromosomes Dependent or independent of GnRH -↑LH or tumors lack of menses by age 16 -underdeveloped ovaries -insensitive androgens -↑↑↑ FSH: non-ovarian response Loss of normal menses -pregnancy, PCOS, stress Irregular or absent ovulation, presence of cysts in ovaries, hyperandrogenism male hair pattern, deep voice, muscle mass, testosterone ↑, DHEAS normal, obese Insulin resistance LH ↑↑, FSH normal to low What is it? Pituitary not responding to GnRH Hyperprolactinemia (PRL suppressed by dopamine so if PRL ↑, will suppress GnRH) ↓ testosterone (<200), ↓ FH/LH Gonadal dysfunction, no response to male steroids ↓ Testosterone, with ↑↑ FSH X-linked, no male development, no upper female geneital tract N to ↑ testosterone, high LH Bone Metabolism (Parathyroid) 1) Calcium levels controlled by endocrine system a) PTH, Vitamin D and Calcitonin a) Bone stores calcium and stimulates osteoclasts (break down bone) 2) Bone stimulates osteoclasts which break down bone a) ↑ serum Ca b) Kidney ↑ reabsorption of Ca AND ↓ reabsorption of phosphate c) Induces 1-alpha-hydroxylase which forms Vit D 25 d) Intestine ↑ calcium absorption Bone Tests Notes Lab PTH Measure PTH intact ↑ PTH when low free iCa ↓ PTH when high Ca Calcitonin Made by thyroid cells Vit D <20 ng/ml = deficient 30-80 ng/mL = normal >80 is toxic Calcium Hormone more than vitamin Vit D status = Vit D OH Renal Dz= 1,25-diOH ↑ PTH, Vit D suppressed Raises Ca levels Lab ↑↑ PTH, ↓ PO4 Intraoperative PTH (ioPTH) Baseline done first. >50% PTH decline=success ↑PTH due to ↑ Ca, Vit D ↓ or renal dz Hypocalcemia with ↓ PTH PTH tested during surgery to verify removal of hyperplastic glands Secondary hyperPTH Hypoparathyroidism Rickets (kids) Paget’s Disease Tumor marker for thyroid cancer Measures D3 (UV exposure) and D2 (plant origin) Hypercalcemia: PTH increases Ca, Uncapped serum ↑pH, Vit D↑, low calcitonin lowers pCO2, bound Ca Hypocalcemia: high calcitonin, Vit ↑, resulting in ↓iCa D ↓, transfusion Disorders of PTH What is it Hyperparathyroidism Hyperplasia of one or more glands Osteoporosis Notes EDTA, single Ab Loss of bone mass Bone mineral density (BMD) Decreased mineralization of bone opposed to loss of bone -bowed legs ↑ osteoclasts bone formation Vit D ↓, high ALP Notes PO4 forms salts with insoluble Ca so plasma Ca ↓, iCa ↑ Psuedo-hypo-PTH: hypocalcemia with ↑↑PTH meaning defect Osteomalacia in adults 3) Case study a) Hypercalcemia and Vit D from liver is high but D from kidneys is normal along with PTH b) PTH promotes 1,25 Vit D from kidneys c) What is causing the liver Vit D and Ca? i) Supplement taking high dose 4) Bone turnover markers a) Osteocalcin and ALP for bone formation i) Binds calcium to bone b) Pyridinium and telopeptide for osteoclast activity i) Urine or serum test Toxicology and therapeutic: Drug Monitoring (TDM) 1) Measure blood drug [] a) Adequate and effective [] b) Avoid toxicity c) Drug screens lack specificity but are sensitive 2) Therapeutic (and toxic) response to drug is directly related to [] at a specific receptor a) Blood [] of the drug reflects [] at the target receptor but can’t measure the receptor b) Therapeutic range for each drug: desired effects between point A and B i) Point A: [] in which minimum effective [] takes place ii) Point B: minimum [] for toxic effect 3) Pharmacokinetics- what the body does to a drug a) Absorption, distribution, metabolism, elimination i) Drugs measured directly or bound drugs ii) Free drug found in liver, tissues, site of action, blood 4) Pharmacodynamics-what a drug does to the body a) Effects, mechanism of action 5) Absorption a) Intravenous administration Complete absorption, already in circulation b) Intramuscular or subcutaneousSlower, but complete c) Oral i) Stomachintestineabsorbed by body ii) Bioavailability that reaches blood varies iii) Inefficient routes iv) Vascularized area drug gets their quickly 6) Distribution a) Accumulates in central blood supply and delivered to interstitial fluids OR it goes directly to tissues and organs b) Greater [] being delivered than eliminated c) Drug levels should be drawn after distribution is complete i) Drug distributed from blood to tissue overrepresentation of amount at tissues b/c it’s in transit 7) Protein binding of drugs a) Albumin binds acid and neutral drugs b) When measuring total drugs correlate that with protein [] c) Bound drug measurement total drug low and albumin low and free drug normal d) Also consider competitive inhibition so you have excess of free drug e) Free DPH/phenytoin = high because VPA displaced DPH i) When VPA was withheld, free DPH = normal (1) Competitive inhibition f) Elimination i) Hepatic Metabolism -clearance may be decreased (1) Renal excretion- ↓eGFR also ↓ clearance 8) Metabolism a) Detoxify drug or increase polarity for urinary excretion b) Also form active drug or a toxin c) Hepatic cytochrome (CYP) system – enzymatic activity by liver d) Genetic variability e) Xenobiotics: not natural to animal life f) Phase I: yields a polar, water-soluble, active metabolite, can be substrates for phase II i) Cytochrome reaction with drug g) Phase II: yields large polar metabolite with hydrophilic groups to form water-soluble inactive compounds for excretion i) Drug=enzyme substrate (1) 1st order: enzyme >> drug (dose dependent) so increase dose (exponential) (a) Most drugs use this (b) More drug there is, more it is excreted (2) 0 order: enzyme < drug (fixed amount) and enzymes no longer saturated (a) Phenytoin exhibits first order at low []s and high [] switches to 0 order (b) Alcohol too (3) When a patient receiving regular dose. Then increasing it even little bit can change drug [] 9) Peak and Trough a) Peak = highest [] at site of action b) Trough = lowest [] and just before the next dose c) Used for narrow range drugs where it becomes toxic d) And eventually a steady peak and trough is established e) Make sure you don’t draw before 5 half-lives 10) Case study a) Person takes valium and Tylenol with codeine after surgery and antifungal b) Codeine gets secreted in urine via cytochrome c) Valium is partially metabolized so bioactive and antifungal inhibits cytochrome activity d) Inhibited by antifungal and creating bioavailable sedatives so valium and codeine ↑↑ 11) Therapeutic Index = Toxic [] divided by therapeutic [] a) High positive value for over-the-counter analgesic, sedatives and antibiotics b) Low number = lithium, anticonvulsants, antibiotics, digoxin, immunosuppressants 12) Sample collection a) Steady state requires after 5 half-lives. Just before next dose at trough b) Peak for antibiotics to make sure they are reaching high [] c) Do not use serum separator tubes with gel barriers b/c can interact with drugs and give false low value Cardiac drugs Digoxin Procainamide Lidocaine Quinidine Description Natural product Low therapeutic index (0.82 ng/ml) -JBND, long distribution -weakly protein bound Metabolized by liver PA and NAPA both active Notes Measured Immunoassay -half life based on genetic factors Immunoassay, HPLC 13) Anti-convulsant: liver metabolism so any liver defects leads to accumulation a) PB and DPH is given for same purpose, but DPH has lower half life and more side effects Anti-convulsant Carbamazepine Phenobarbital (PB) Phenytoin (DPH) Description Range: 4-12 mcg/ml JBND, trough levels Metabolized by liver and metabolite of primidone PA and NAPA both active Low solubility so absorption rates in oral variable -JBND Notes DPB, PB, VPA ↑ rate of CBZ clearance ↓ clearance with VPA and aspirin Bound or free 80% protein bound Induced by alcohol, PB and CBZ -protein binding competition by aspirin and VPA 90% protein bound but free measured 40-60% bound to plasma proteins Valproic Acid (VPA) Protein bound and competed with DPH -JBND 93% protein bound When competition for binding ↑, free VPA ↑ Primidone Metabolized to PB so monitor both (same thing) 20% protein bound 14) Bronchodilators Bronchodilators theophylline Description First order kinetics of elimination Caffeine Treats neonatal apnea Less dosing due to longer half life Notes Both are used for neonatal apnea but short half life so less common to use Measured 50% protein bound 15) Antibiotics Antibiotics Aminoglycosides (Gentamycin, tobramycin, amikacin) Aminoglycosides (Carbenicillin, Ticarcillin, Piperacillin) Vancomycin Description Bactericidal against aerobic gram ves. Competitive inhibitors of protein synthesis Covalent reaction, blocks antigenicity and antibiotic reaction Notes When administered directly in blood distribute fast to extracellular fluid but do not cross cell membrane or bind to plasma proteins Glomerular filtration issues accumulation Measured by IA Glycopeptide, effective against gram +ves esp MRSA Given by IV Inhibits cell wall synthesis Crystal degradation if patients have renal disease 16) Immunosuppressants a) All are isolated from fungi, molds and block T cell proliferation Immunosuppressants Cyclosporine A Tacrolimus Description WHOLE BLOOD sample -draw trough levels Less nephrotoxic than cyclosporine. Collect whole blood after 5 days Measured IA, high protein bound and []ed in RBCs IA, high binding to FK-506 proteins within cells so plasma [] is 8% of whole blood IA Sirolimus Collect whole blood after 3 days Everolimus Mycophenolic Acid Water-soluble Given as prodrug hydrolyzed in liver to MPA LC-MS/MS 17) Psychiatric drugs Psychoactive Drugs Description Measured Lithium Treats manic phase of bipolar disorder -JBND, very low therapeutic index -renal excretion SSRI and antidepressants ISE but slight Na interference Not mentioned Rarely need monitoring 18) Clinically toxicology a) Acetaminophen: in Tylenol i) Overdose hepatic failure ii) Urine spot screening can detect routine use iii) Serum quantification needed b) Salicylates i) Aspirin- acetylsalicylic acid (ASA) rapid hydrolysis to salicylate ii) Salicylic acid keratolytic gels iii) Oil of wintergreen enhanced toxicity due to CNS penetration iv) Enhances anaerobic glycolysis v) Accumulation leads to metabolic acidosis c) Ethanol d) Iron i) Corrosive, shock, acidosis ii) Transferrin saturation >100% indicates free iron toxic iii) Chelation used as treatment which binds up free iron and excreted e) COHgb and metHgb by CO-Oximeter i) CO is colorless, odorless and tasteless. Can bind to Hgb and exclude O2 due to higher affinity (a) Can also bind to megHgb and cytochrome a3 (2) Directly released from hgb or indirectly using blood gas analyzers (3) Treatment is to administer oxygen (4) COHgb is stable ii) Methemoglobin: form of Hgb that’s been oxidized (Fe2+ Fe 3+) iii) Doesn’t bind to O2 iv) Treated with ascorbic acid to reduce level in blood v) Unstable so transported in nice and measured in short time 19) Volatiles are measured by flame ionization GC. Easily vaporized when not stable Volatiles Methanol Isopropanol Acetone Ethanol Ethylene glycol Description Not a significant CNS depressant -rubbing alcohol (70%),depresses CNS activity but longer half time Weak CNS depressant than EtOH Most abused drug Based on whole blood. CNS depressant. Metabolized by liver ADH to acetaldehyde Zero order elimination Mild CNS depressant and induced by ADH Causes calcium oxalate from metabolism 20) Toxic metals like lead use whole blood and measured using ICP-MS a) Inhibits Hgb biosynthetic pathway and PBG Drugs of Abuse Amphetamines Barbiturates Benzodiazepines Cannabinoids Cocaine Methadone Opiates Phencyclidine (PCP) Description -Methamphetamine metabolized to amphetamine -low therapeutic index -CNS depression, mild sedation, inhibits GABA and AMPA Diazepam and nordiazepam -low addiction potential, effective Blood THC peaks in minutes and decreases in 2 hours Lipid soluble compound but what’s being measured id soluble carboxylated THC -snorted as hydrochloride -used as anesthetic and excreted as benzoylecgonine Positives have to be confirmed Measurement IA on urine IA on urine Used as pain relief, euphoria, sedation -abused as heroin which is hydrolyzed to morphine -morphine excreted as glucuronide Surgical anesthetic, hallucinations -often smoked with THC or tobacco Poorly detected by IA IA on urine IA on urine IA targets BEG IA on urine Extra: 1) Alpha 1 globulins 2) Alpha 2 globulins 3) Beta globuin: carrier proteins for iron (transferrin) and lipoproteins a) Increased in elevated beta lipoprotein and IDA 4) Gamma globulin: increase in inflammation Cirrhosis, hepatitis a) Decreased in acquired immunodeficiency 5) Alpha 1-antitrypsin (AAT)- increase in acute phase and pregnancy a) decrease in emphysema in neonates 6) AFP- increase in amniotic fluid and maternal serum in neural defects (spina bifida) a) liver cancer marker b) decrease in maternal serum during pregnancy associated with down syndrome 7) Haptoglobin: binds free hemoglobin, a) increase in acute phase and nephrotic syndrome b) decrease in transfusion rxn, hemolysis and liver disease 8) Ceruloplasmin transports copper a) increased in acute phase and pregnancy b) decrease in wilson’s disease 9) albumin attracted anode because of positive attitude 10) ACP (acid phosphatase)- seen in PSA, increase in bone cancer, useful for rape cases, in seminal fluid 11) Cholinesterase-destroys ecetylycholine after nerve impulse transmission. Decreases in muscular 12) Role of fibrinogen: 13) Role of Lp-PLA2
