MLS Review - CC - Electrolytes & Anion Gap

o MEDICAL LABORATORY SCIENCE REVIEW CLINICAL CHEMISTRY: ELECTROLYTES & ANION GAP OUTLINE • Electrolytes o 2 Classifications of Electrolytes o Regulation of Electrolytes o Functions of Electrolytes o Clinically Significant Electrolytes o Concentration of Cations and Anions in Extracellular and Intracellular Water • o Sodium o o o o o o o o Potassium Chloride Bicarbonate Magnesium Calcium Phosphate Lactate Reference Values of Electrolytes Anion Gap o Renal and skin excretion of electrolytes ▪ Excess electrolytes in the plasma can be excreted through kidneys (urine) and skin (sweat) ▪ Deficient electrolytes, the kidneys can retain and reabsorb electrolytes to conserve the level of electrolytes present in the body ▪ Rule of thumb: any thing that is excess in plasma is removed through the kidneys or any thing inadequate in plasma is conserved by kidneys Hormonal activity ▪ Hormones can regulate level of electrolytes in blood by controlling retention and excretion process of electrolytes FUNCTIONS OF ELECTROLYTES ELECTROLYTES • • • • Ions capable of carrying an electric charge (+ or - charge) Participate in various metabolic activities Contribute to normal physiologic activities in the body Essential in metabolic activities of cells and tissues • • • 2 CLASSIFICATIONS OF ELECTROLYTES ACCORDING TO THE CHARGE IT CARRIES • • Anions o Ions that carry negative charge o Move toward the anode (positively charged field) o Eg. Chloride (Cl-), Bicarbonate (HCO3-), Phosphate (PO4-) Cations o Ions that carry positive charge o Move toward a cathode (negative field) o Eg. sodium (Na+), potassium (K+), magnesium (Mg2+), calcium (Ca2+) ACCORDING TO ITS LOCATION/DISTRIBUTION • • • Electrolytes are present where water is located o Electrolytes are dissolved and present in water Intracellular - found inside/within cell (cytoplasm/cytosol) o Cytosol - water component of the cell o E.g.: K+, PO4 Extracellular - found outside cell, plasma or interstitial fluid o Extracellular intravascular - inside the blood vessel/ within the plasma o Extracellular extravascular - outside blood vessel/ within the interstitial fluid between tissues o E.g.: Na+, Cl- • • • • CONCENTRATION OF CATIONS AND ANIONS IN EXTRACELLULAR AND INTRACELLULAR WATER Concentration of Water Cation Extracellular Intracellular (mmol/L) (mmol/L) Na+ 136 - 145 15 K+ 3.5 - 1.5 150 Ca2+ 2.15 - 2.15 1 Mg2+ 0.63-1 13.5 • • • • • REGULATION OF ELECTROLYTES • Concentration of electrolytes in the blood is regulated by the following factors: o Diet ▪ Most electrolytes are mainly derived from exogenous sources (food, fluid intake) o Intestinal absorption of electrolytes ▪ GIT plays an important role in regulating electrolytes through absorption process Volume and osmotic regulation (Na+, Cl-, K+) o Plasma volume control o Movement of water in and out of the cell Myocardial rhythm & contractility (K+, Mg2+, Ca2+) o Heart activity (movement and contractions) Neuromuscular Excitability (K+, Mg2+, Ca2+) o Nerve transmission and synapse regulation Cofactors in enzyme activation (Mg2+, Ca2+, Zn2+) o Act as activators to enhance enzyme activity Regulation of adenosine triphosphatase (ATPase) ion pumps (Mg2+) o For active transport of ions in cell membranes Acid-base balance (K+, Cl-, HCO3-) o For pH regulation & equilibrium Production & use of ATP from glucose (Mg2+, PO4-) o For energy during glycolysis Concentration of Water Anion Extracellular Intracellular (mmol/L) (mmol/L) HCO323 - 29 10 Cl98 - 107 1 HPO42- 0.78 - 1.42 50 SO420.5 10 Na+ - major extracellular cation K+ - major intracellular cation Cl- - major extracellular anion PO4 - major intracellular anion Electrolytes are involved in plasma osmolality (POSM) o Plasma osmolality - amount of solute per kilogram of solvent in the plasma o Concentration of solute (substance) vs. solvent (plasma) SODIUM (Na+) DESCRIPTION & REGULATION • • • • Aka Natrium Most abundant cation in the extracellular fluid Major extracellular cation Na+-K+-ATPase ion pump moves 3 Na+ ions out of the cell in exchange for 2 K+ ions o Regulates sodium in and out of the cell o Allows movement of electrolytes through active transport o Important to maintain balance of positive charge in and out the cell o Electroneutrality - balance of charge 1 • • Plasma concentration depends in renal regulation o Intake of water in response to thirst ▪ ↑ Na = ↑ POSM = activation of thirst center in the hypothalamus of the brain = urge to drink water • Thirst center - can lower Na levels ▪ Intake of water = ↓ Na • More solvent is taken in ▪ Sodium is one of the major contributors of plasma osmolality - affected by sodium intake/secretion o Excretion of water as affected by AVP → H2O reabsorption in the kidneys ▪ Water loss = ↓ plasma vol. = ↑ Na ▪ ↑ AVP = ↑ water retention = ↓ Na ▪ AVP activation = water reabsorption/conservation (to normalize/lower plasma sodium) ▪ AVP - Arginine vasopressin hormone • Renewed name of antidiuretic hormone (ADH) o The blood volume status, which affects Na excretion through: ▪ Blood volume affect excretion/retention of Na ▪ Aldosterone • Promotes increased Na reabsorption in kidneys • Conserves Na by increasing K excretion • Na & K have inverse relationship in the nephrons in the kidneys ▪ Angiotensin II • Stimulate adrenal gland to increase aldosterone secretion to conserve sodium • Activated during Na deficiency • Active form of Angiotensin ▪ Atrial Natriuretic Peptide (ANP) • Antagonist of aldosterone • Promote increased urinary excretion of Na • Produced by the heart • Activates when there is excess Na in the body The plasma sodium concentration depends greatly on balance of renal regulation. o Reference values: 135-145 mmol/L o Threshold critical value: 160 mmol/L & 120 mmol/L ▪ >160 mmol/L = severe hypernatremia ▪ <120 mmol/L = severe hyponatremia • Can lead to severe sequelae • Requires addition of Na source on the IV line • IV line administration of electrolytes is the most optimal way of increasing electrolytes in emergency cases o CSF Sodium: 135 - 150 mmol/L ▪ Na can pass through the blood brain barrier (BBB) CLINICAL APPLICATIONS • • Hyponatremia (<135 mmol/L) - ↓ plasma Na levels o Cause of Hyponatremia (↓ Na+) ▪ Increased Sodium Loss • Hypoadrenalism (↓ aldosterone) o Hormonal deficiency • Potassium (K+) deficiency o When K is low, K should be conserved and reabsorbed while excreting sodium • Diuretic use (Thiazide) • Ketonuria (Na+ lost with ketones) • Salt-losing nephropathy • Prolonged/severe vomiting or diarrhea o Continuous loss of electrolytes ▪ Increased Water Retention • Renal failure (dilution of Na) • Nephrotic syndrome (↓ COP-PV, ↑ AVP) o ↑ AVP = ↑ water retention = ↓ Na • Congestive Heart Failure (CHF) • Hepatic cirrhosis ▪ Water Imbalance • Syndrome of Inappropriate Antidiuretic Hormone (SIADH) (↑ AVP, ↑ H2O retention) • Pseudohyponatremia o Classification of Hyponatremia by Osmolality ▪ With Low Osmolality • Increased sodium loss (critical ↓ Na) • Increased water retention (dilutional effect) ▪ With Normal Osmolality (Normal osmolality due to compensation of other substances) • Increased non-sodium cations • excess (mental disorder medication for) • Increased gamma globulins • Severe hyperkalemia • Severe hypermagnesemia • Severe hypercalcemia • Pseudohyponatremia • Hyperlipidemia • Hyperproteinemia • Pseudohyperkalemia ▪ With High Osmolality (Na did not influence osmolality but other substances affect POSM) • Hyperglycemia (glucose is major contributor of osmolality) • Mannitol infusion Hypernatremia (>145 mmol/L) - ↑ plasma Na levels o Cause of Hypernatremia (↑ Na+) ▪ Excess Water Loss • Diabetes insipidus (↓ AVP) o ↓ AVP = ↓ Water retention • Renal tubular disorder (↓ urine conc.) • Prolonged diarrhea • Profuse sweating • Severe burns ▪ Decreased Water Intake • Old/infants/mental impairment ▪ Increased Sodium Intake or Retention • Cushing syndrome (↑ Na reabsorption) • Hyperaldosteronism (↓ AVP) • Hypertonic salt solution 2 DETERMINATION OF SODIUM • • Specimen o Serum - red top o Plasma - green top (lithium heparin, ammonium heparin, and lithium oxalate) o False ↑ with marked/significantly increased hemolysis ▪ Sodium is present inside the cell ▪ Hgb, lipids, bilirubin are interfering agents that are released during hemolysis Methods o Flame Emission Spectrophotometry (FES): ▪ Yellow after excitation ▪ Preferred over AAS o Atomic Absorption Spectrometer (AAS) ▪ Not commonly used for Na ▪ Used for ions not easily excited o Ion-selective electrode (ISE) ▪ Uses Glass ion-exchange membrane ▪ It is considered as the reference method and internal standard use due to its rapidness o Colorimetric/chemical method: Albanese-Lein ▪ Cupric sulfate and NaOH ▪ Uses magnesium uranyl acetate ▪ End product: yellow complex • • • CLINICAL APPLICATION • POTASSIUM (K+) DESCRIPTION AND REGULATION • • • • • Aka Kalium Major Intracellular Cation o Most abundant inside the cell Functions: o Regulation of neuromuscular excitability ▪ ↑ K+ → ↑ cell excitability → ↓ RMF (muscle weakness) ▪ ↓ K+ → ↓ cell excitability → ↑ RMF (arrythmia or paralysis) ▪ RMF - resting membrane potential • Inversely proportional with cell excitation ▪ Part of synapse for neuromuscular transmission o Contraction of heart ▪ K Controls majority of heart activity o ICF volume ▪ ICF - volume of cytoplasm o H concentration ▪ K can buffer the excess H ions in plasma ▪ When there I increased H concentration in plasma, K will move out of the cell to allow H ions to enter the cell causing increase of pH of plasma (↑ H = ↓ pH) ▪ Exchange of ions in and out of the cell Aldosterone: inversely proportional to the regulation of sodium and potassium o Conserves potassium by excreting sodium o ↑ K excretion in urine in exchange for Na reabsorption Na+, K+ -ATPase pump o For every 3 Na ions move in, 2 K ions move out of cell through active transport mechanism and vice versa o ↓ function = ↓ cellular entry of K → ↑ plasma K (hyperkalemia) → hypoxia, digoxin overdose, hypomagnesemia, propanolol (β-blocker) ▪ Slow entry of K in cell → ↑ plasma K o ↑ function = ↑ cellular entry of K → ↓ plasma K (hypokalemia) → insulin, epinephrine (↑ K) ▪ Rapid entry of K in cell → ↓ plasma K ↑ K with exercise, hyperosmolality (DM), cellular breakdown o Patients are discouraged to do arm exercise (rapid flexing, fist clenching) ▪ K is released from muscle fibers during physical activities → ↑ plasma K o Prolonged tourniquet application is not allowed (↑ K) o Hemolysis will increase plasma K Reference values: 3.5-5.2 mmol/L o Ref value is low as most of the K is intracellular Threshold critical values: o 6.5 mmol/L: hyperkalemia o 2.5 mmol/L: hypokalemia • Hypokalemia o Aka hypopotassemia o Causes of Hypokalemia (↓ K+) ▪ GI Loss • Vomiting, Diarrhea • Gastric suction • Intestinal tumor, malabsorption • Cancer therapy, laxatives ▪ Cellular Shift (↑ K uptake in cell) • Alkalosis (plasma) • Insulin overdose ▪ Renal Loss • Diuretics, nephritis, CHF • RTA (↓ H, ↑ K excretion) • Cushing’s syndrome (↑ Na, ↓ K reabsportion) • Hyperaldosteronism • Hypomagnesemia (↑ aldosterone) ▪ Decreased K Intake Hyperkalemia o Aka hyperpotassemia o Causes of Hyperkalemia (↑ K+) ▪ Decreased Renal Excretion • Renal failure • Hypoaldosteronism (↓ Na) • Addison’s disease (↓ Na, ↑ K reabsorption) ▪ Increased Intake • Oral/IV - K+ replacement therapy ▪ Cellular Shift • Acidosis (plasma ↓ H, ↑ K) • Muscle/cellular injury • Chemotherapy/ leukemia • Hemolysis ▪ Artifactual • Hemolysis, Thrombocytosis • Prolonged tourniquet DETERMINATION OF POTASSIUM • Specimen o Serum, plasma (heparin) ▪ False ↑ with hemolysis o 24-hour urine ▪ K can be excreted in the urine 3 • Methods o FES: violet after excitation o AAS (not commonly performed) o ISE: valinomycin membrane ▪ Reference method for K determination • Very rapid, specific, and sensitive ▪ Valinomycin - antibiotic with high affinity with K • Increases specificity for K o Colorimetric/chemical Method: Lockhead & Purcell (turbidimetric using sodium tetraphenylboron for ppt) CLINICAL APPLICATIONS • • CHLORIDE (Cl-) DESCRIPTION AND REGULATION • • • • • Major Extracellular Anion Predominant in plasma Similar with sodium o Na and Cl have direct proportional relationship o Cl can serve as rate limiting component for Na reabsorption in the nephrons o Na can also promote Cl reabsorption o Na excretion can cause Cl excretion Involve in maintaining osmolality blood volume and electric neutrality (chloride shift) o Level of Cl in plasma can regulate the negative charge in and out of the cell o Chloride and bicarbonate are involved in maintaining the negative charge balance in and out of the cell o When HCO3 is generated from carbonic acid formation/dissociation as negative electrolyte, H ion is also produced → HCO3 is released from the cell → the negative charge in the cell will decrease → Cl in the plasma will enter the cell to compensate to the movement of HCO3 outside the cell o Chloride shift - compensation of Cl to the loss of negative charge inside the cell by entering the cell to maintain electroneutrality o H ions are also produced in carbonic acid dissociation → excess H ions move out of the cell → H ions bind with reduced hgb form of RBCs (deoxyhemoglobin) → deoxyhemoglobin will buffer the excess H ions produced Rate limiting component in Na+ reabsorption o When Na is reabsorbed, Cl is passively reabsorbed o When Na is excreted, Cl is passively excreted o Na & Cl in plasma, exist as salt o Na & Cl can be excreted through skin via perspiration/sweat Hyperchloremia (↑ Cl-) o Cause of Hyperchloremia ▪ Excess Loss of HCO3• GI Losses • Renal Tubular Acidosis (RTA) • Metabolic acidosis Hypochloremia (↓ Cl-) o Cause of Hypochloremia ▪ Excess Loss of Cl • Prolonged vomiting • Diabetic ketoacidosis • Aldosterone deficiency • Salt-losing pyelonephritis DETERMINATION OF CHLORIDE • • Specimen o Serum, plasma (lithium heparin/green top) ▪ False ↓ with marked hemolysis (dilutional effect) o 24-hour urine Methods o ISE: ion exchange membrane ▪ Membrane should contain tri-noctylpropylammonium chloride decanol ▪ Reference method for Cl determination o Amperometric-coulometric: Cotlove Chloridometer ▪ Reaction: Ag2 + 2Cl → AgCl2 ▪ Used in sweat which is useful for diagnosis of cystic fibrosis (CF; aka mucoviscidosis) • CF - disorder of exocrine gland ▪ Px are given sweat-inducing drugs (Pilocarpine) ▪ When sweat is collected after Pilocarpine administration, px should immediately rehydrate o Schales and Schales ▪ Titration with mercuric nitrate ▪ Indicator S-diphenylcarbazone ▪ Reaction: Hg++ + Cl → HgCl2 Excess Hg++ + diphenylcarbazone → violet complex ▪ Gibson-Cooke if used in sweat chloride analysis o Colorimetric: ▪ Reaction: reddish color BICARBONATE (HCO3-) REGULATION AND CLINICAL APPLICATIONS • • • Reference values: 98-107 mmol/L • • 2nd Most Abundant anion in the ECF o 2nd to chloride Bicarbonate diffuses out of the cell once it is generated in exchange for chloride (chloride shift) Accounts for more than 80% of total CO2 with HCO3 Major component of the buffering system of the blood o Bicarbonate-carbonic acid buffer system (buffers plasma pH) o Buffers excess H ions by combining with excess acid to produce carbonic acid 4 • • • • • Along with chloride, HCO3 maintains negative ionic charge neutrality of the plasma and the cell (electroneutrality) o Bicarbonate exits, chloride enters the cells Normally, HCO3 is reabsorbed in the kidneys o 85% is reabsorbed in proximal convoluted tubule o 15% is reabsorbed in distal convoluted tubule HCO3 is an alaklaine agen ↑ HCO3 o Metabolic Alkalosis ▪ Severe vomiting ▪ Hypokalemia ▪ Hypoventilation ▪ Excessive alkali intake ↓ HCO3 o Metabolic Acidosis ▪ Hyperventilation CLINICAL APPLICATIONS • DETERMINATION OF BICARBONATE • • Specimen: o Serum, plasma (heparin) o False ↓ if tube is left uncapped ▪ CO2 can be released, decreasing HCO3 o If left uncapped, loss of 6 mmol/L HCO3 per hour Methods o ISE: PCO2 electrode o Enzyme method ▪ Reaction: ▪ Measures decreased absorbance of oxidized NAD at 340 nm MAGNESIUM (Mg+) PHYSIOLOGY AND REGULATION • • • • • • • • Divalent cation Second Major intracellular cation o 2nd to potassium Involved in neuromuscular conduction, enzyme phosphorylation and protein anabolism Mainly derived from exogenous sources (diet) Distribution of Mg in the body: o Bone: 53% o Muscle and other organs and soft tissues: 46% o Serum and RBC: <1% Forms in serum o Protein-bound: 33% o Free or ionized: about 61% ▪ Physiologically active form of Mg in serum o Complexed with PO4- and citrate: 5-6% Parathyroid Hormone (PTH) o Produced in the parathyroid gland o Promotes increased renal reabsorption of Mg o Promotes increased intestinal absorption of Mg ▪ Mg can be derived from diet Aldosterone and thyroxine (T4) o Promotes increased renal excretion of Mg & K o Promotes sodium absorption • Hypomagnesemia o Cause of Hypomagnesemia (↓ Mg+) ▪ Reduced Intake • Poor diet/starvation • Prolonged Mg-deficient IV • Chronic alcoholism ▪ Decreased Absorption • Malabsorption syndrome • Pancreatitis, diarrhea • Vomiting, laxative use, etc. • Neonatal (due to surgery) • Primary-selective malabsorption • Congenital (transport defect in SI) ▪ Others • Excess lactation • Pregnancy o Cause of Hypomagnesemia (↓ Mg+) - Increased Excretion ▪ Renal • Tubular disorder • Pyelonephritis • Glomerulonephritis ▪ Endocrine • Hyperparathyroidism (↑ Ca ↓ Mg) • Hyperaldosteronism (↑ Na ↓ Mg) • Hyperthyroidism (↑ Mg excretion) • Hypercalcemia (↑ Ca ↓ Mg) • Diabetic ketoacidosis (glycosuria) ▪ Drug Induced • Diuretics (Furosemide, Thiazide) • Antibiotics (Gentamicin) • Cyclosporin (Immunosuppressant) • Digitales and Digoxin (Glycosides) Hypermagnesemia o Causes of Hypermagnesemia (↑ Mg2+) ▪ ↓ Excretion, acute/chronic renal failure (GFR < 30 mL/min) ▪ Hypoaldosteronism (↓ Na ↑ Mg reabsorption) ▪ ↑ increased intake (medications and therapy) ▪ Dehydration (pseudohypermagnesemia) ▪ Bone carcinoma and bone metastases (due to ↑ in bone loss) DETERMINATION OF MAGNESIUM • • Specimen: o Serum, plasma (lithium heparin) o 24 hour urine o Hemolysis cause false elevation Methods o Calgamite Method (colorimetric) ▪ Mg2+ + calmagite → reddish-violet (532 nm) ▪ Mg is directly proportional to absorbance o Formazen Dye Method ▪ Mg2+ + Dye → colored Complex (660 nm) ▪ Mg is directly proportional to absorbance o Methylthymol blue Method ▪ Mg2+ + Chromogen → Colored complex ▪ Mg is directly proportional to absorbance 5 o • Titan yellow ▪ Serum TCA filtrate + titan yellow → red compound • TCA - trichloroacetic acid ▪ Serum undergoes deproteinization process to precipitate and remove proteins o Flourometric method ▪ Mg2+ + 8-hydroxy-5-quinoline sulfonic acid → fluorescence (380-410 nm) Reference values: 0.63 - 1.0 mmol/L or 1.26 - 2.10 mEq/L o Lower than sodium and other electrolytes 1-α-hydroxylase ! CALCIUM (Ca+2) REGULATION AND DISTRIBUTION • • • • • • • • • • A divalent cation 5th most common element in the body For muscle contraction to produce movement Evaluated with phosphorus for bone metabolism o Part of bone mineralization Major inorganic constituents of the bone For blood coagulation o Ca cause activation of enzymes & coagulation factors Absorbed in upper small intestine in the presence of vit D o Vit. D must be in active form to be utilized ▪ UV rays activates Vit. D ▪ Prolonged UV exposure cause disease o Without vit D, Ca will just be part of the excreta Distribution of Ca in the body o Bone and teeth: 99% o Blood and ECF: 1% Calcium in the blood is distributed as: o Ionized Ca+2 (45% of total calcium) ▪ Unbound or freeform in the plasma ▪ Physiological active form of calcium o Protein-bound Ca+2 (40% of total calcium) ▪ Present/attached with albumin (transporter) o Complex Ca+2 (15% of total calcium) ▪ Bound to anions (HCO3-, PO4, Lactate) Factors affecting Ca+2 level in blood: o Bone resorption ▪ Bone destruction ▪ Initiated by osteoclasts (bone-destroying cell) ▪ Calcium levels in the bone are released and increases Ca in blood ▪ Promoted by parathyroid hormone (PTH) • PTH mobilizes calcium from bones to blood • PTH levels is directly proportional to blood Ca o Bone deposition ▪ Bone mineralization ▪ Blood calcium levels decreases due to Ca deposition to bone ▪ Calcium in the blood enters the bone to promote calcification of the bone ▪ Promoted by calcitonin • Calcitonin inhibits PTH activity & vit D action o Intestinal absorption ▪ Calcium is absorbed in small intestine from diet ▪ Promoted by activated vitamin D • Promotes intestinal absorption • Activated Vit. D also increase bone resorption • Vit. D derived from diet but in inactive form • Inactive vit. D is activated by UV rays • • • • Hypercalcemia suppress PTH release Hypocalcemia promote secretion of PTH o Renal 1-α-hydroxylase - important for vit. D activation Vit. D - aka cholecalciferol o 1,25 dihydroxycholecalciferol - active form of vit. D When UV rays pass through the skin, it hastily activates vit. D to promote calcium absorption o However, excessive UV exposure can be harmful which can cause skin disorders ▪ Best time to be exposed in early morning o Melanin serves as a protection against UV rays o Melanin-poor race (Caucasians) are more prone to the harmful effects of UV rays (extremely low melanin) o Melanin-rich race (Africans) can’t activate vit D due to inability of UV to penetrate their skin affecting activation of vit D making prone susceptible to bone diseases o Asians have enough melanin levels to allow UV rays to promote vit. D. activation and to protect from the harmful effects of UV rays CLINICAL APPLICATIONS • • Hypocalcemia o Cause of Hypocalcemia ( ↓ Ca+) ▪ Primary hypoparathyroidism: PTH ↑ excretion of Ca ▪ Hypo/hypermagnesemia: ↓ PTH qty. & activity, Vit. D resistance ▪ Hypoalbuminemia: chronic liver disease, nephrotic syndrome, malnutrition ▪ Acute Pancreatitis: ↑ lipase ▪ Vit. D deficiency: ↓ absorption ▪ Renal Disease ▪ Rhabdomyolysis: ↑ PO4 release from cells which binds calcium Hypercalcemia o Cause of Hypercalcemia (↑ Ca+) ▪ Primary hyperparathyroidism ▪ Malignancy ▪ Multiple Myeloma ▪ ↑ Vitamin D ▪ Thiazide diuretics: ↑ Ca2+ reabsorption ▪ Prolonged immobilization: ↑ resorption 6 DETERMINATION OF CALCIUM • • • Specimen o Serum, plasma (lithium heparin) o 24 hour urine o Hemolysis cause false elevation of Ca Methods o Atomic Absorption Spectophometry (AAS) ▪ Reference method for Ca & Mg determination o ISE (liquid membrane electrode) o Ortho-cresolphthalein complexone (CPC) o Arsenzo III dye o Clark and Collip: redox titration method ▪ Precipitation of Ca+2 as Calcium Oxalate (CaC2O4) ▪ CaC2O4 + H2SO4 → oxalic acid (H2C2O4) ▪ H2C2O titrated with KMNO4 → pink color ▪ Ca is directly proportional to the color o Ferro and Ham: precipitation with chloranilic acid ▪ Ca+2 + Sodium chloranilate → Ca chloranilate ▪ Ca Chloranilate + EDTA → chloranilic acid ▪ Ca is directly proportional to the product o EDTA titration methods ▪ Bacarra ▪ Dower ▪ Zobel ▪ EDTA - chelating agent for Ca chelation Reference values: 8.6-10 mg/dL • • CLINICAL APPLICATIONS • • • • PHYSIOLOGY AND REGULATION • • • • • • Major Intracellular Anion o Counterpart of chloride as major anion Omnipresent in distribution o Present in various locations o Majority is found in the bones (85%) o 15% is present in extracellular environment (ECF) Two forms: o Organic PO4 = Intracellular PO4 ▪ Principal anion within the cell o Inorganic PO4 = Extracellular PO4 (plasma, serum, ECF) ▪ Ion part of blood buffers ▪ Measured in laboratory tests Component of phospholipids, nucleic acids, creatine phosphate, and ATP Levels of phosphate is inversely related to calcium Growth hormone (GH) decreases renal excretion of PO4 Phosphate in diet is absorbed maximally at the jejunum in the small intestine Hypophosphatemia o Cause of Hypophosphatemia (↓ PO4) ▪ Hyperparathyroidism (↑ renal excretion) ▪ Vit. D Deficiency/ antacid use (↓ intestinal abs) Hyperphosphatemia o Cause of Hyperphosphatemia (↑ PO4) ▪ Hypoparathyroidism ▪ Lymphoblastic leukemia, intensive exercise, neoplastic disorders ▪ Hypervitaminosis DETERMINATION OF PHOSPHATE PHOSPHATE (PO42-) • If Ca is absorbed, PO4 is excreted with promotion by PTH If PO4 is absorbed, Ca is excreted Specimen o Serum, plasma (lithium heparin) o 24-hour urine o Hemolysis cause false increase of PO4 Methods o Ammonium phosphomolybdate complex (340 nm) o Fiske-Subbarow Method (600 nm) ▪ Reaction: • Phosphorus + Ammonium molybdate → Ammonium phosphomolybdate (purple) • Ammonium phosphomolybdate + aminonaphtholsulfonic acid → Molybdenum blue (reduced ammonium phosphomolybdate) o Reducing Agent: Pictol (Amino Napthol Sulfonic Acid) o Other Reducing Agents: ▪ Elon (methyl amino phenol) ▪ Ascorbic acid ▪ Senidine (N-Phenyl-P Phenylenediamine Hydrochloride) LACTATE DESCRIPTION AND REGULATION • • • An anion biproduct of anerobic metabolism Indicator of severity of oxygen deprivation (hypoxia) o ↓ tissue O2 = ↑ plasma lactate o Hypoxemia - low blood O2 o Hypoxia - low tissue O2 Increases when O2 supply is reduced in tissues 7 • • CLINICAL APPLICATIONS • • Increased Lactate o Hypoxic Conditions (Type A) ▪ Lactate Acidosis • Shock • MI • Severe congestive heart failure • Pulmonary edema, severe blood loss o Metabolic Origin (Type B) ▪ Diabetes mellitus ▪ Severe infection ▪ Leukemia ▪ Liver or renal disease ▪ Toxins (ethanol, methanol, or salicylate poisoning) Only increase in lactate is clinically significant o Decrease in lactate is considered as physiologic • Formulas for Anion Gap: o AG = Na - (Cl + HCO3) ▪ Reference values: 7 - 16 mmol/L o AG = (Na + K) - (Cl + HCO3) ▪ Reference values: 10 - 20 mmol/L ↑ Anion gap o >20 mEq/L o ↑ unmeasured anions o Conditions: ▪ Uremia ▪ Ketotic states (DM, alcoholism, starvation) ▪ Lactic acidosis ▪ Toxin ingestion (salicylates, methanol, ethylene glycol) ▪ Dehydration (↑ plasma protein) ▪ Metabolic alkalosis ▪ Renal disease ↓ Anion gap o <10 mEq/L o ↓ unmeasured anions (hypoalbuminemia) o ↑ in unmeasured cations ▪ Hypermagnesemia ▪ Hypercalcemia ▪ Multiple myeloma ▪ Polyclonal gammopathy DETERMINATION OF LACTATE • Enzymatic methods: o Lactate oxidase o Peroxidase - coupling enzyme REFERENCE VALUES OF ELECTROLYTES Electrolyte Reference Values Threshold Critical Sodium (Na+) 135-145 mmol/L 120 & 160 mmol/L Potassium (K+) 3.5-5.2 mmol/L 2.5 & 6.5 mmol/L Chloride (Cl-) 98-107 mmol/L Magnesium (Mg2-) 0.63 - 1.0 mmol/L or 1.26 - 2.10 mEq/L Calcium (Ca2+) 8.6-10 mg/dL ANION GAP • • • • • Mathematical approximation of difference bet. concentration of unmeasured cations and unmeasured anions Indicates an increase in one or more unmeasured ions in serum ↑ anion gap = ↑ unmeasured anions ↓ anion gap = ↓ unmeasured anions, ↑ unmeasured cations Generally healthy indivuals with increased anion gap, the increased anion gap is used to check for accuracy and performance of analyzers during measurement (QC) o Denotes poor quality of analyzers 8