Clinical Chemistry - 12 Electrolytes

CLINICAL CHEMISTRY WATER ELECTROLYTES OUTLINE • • • Electrolytes Water o Osmolality o Clinically Significant Electrolytes o Concentration of Cations and Anions in Extracellular and Intracellular Water o Sodium o Potassium o Chloride o Bicarbonate o Magnesium o Calcium o Phosphate o Lactate Anion Gap • • • • • • • • • ELECTROLYTES • • • Electrolytes o Ions capable of carrying an electric charge (+/- charge) 2 Classifications: o According to the charge it carries ▪ Anion: ions that carry negative (-) charge and move toward the anode • Anode - positively-charged field • E.g.: Chloride (Cl-), Bicarbonate (HCO3-), Phosphate (PO4-) ▪ Cations: ions that carry positive (+) charge and move toward the cathode • Cathode - negatively-charged field • E.g.: sodium (Na+), potassium (K+), magnesium (Mg2+), calcium (Ca2+) o According to its location/Distribution ▪ Intracellular: found inside/within the cell • E.g.: K+, PO4 ▪ Extracellular: found outside the cell, plasma or interstitial fluid • Extracellular intravascular - inside the blood vessel/ within the plasma • Extracellular extravascular - outside the blood vessel/within the interstitial fluid • E.g.: Na+, ClFunctions of Electrolytes & Electrolytes Involved o Volume and osmotic regulation (Na+, Cl-, K+) ▪ Movement of water in and out of the cell o Myocardial rhythm & contractility (K+, Mg2+, Ca2+) ▪ Heart activity o Neuromuscular Excitability (K+, Mg2+, Ca2+) ▪ Nerve transmission and synapse regulation o Cofactors in enzyme activation (Mg2+, Ca2+, Zn2+) ▪ Act as activators in enhance enzyme activity o Regulation of adenosine triphosphatase (ATPase) ion pumps (Mg2+) o Acid-base balance (HCO3-, K+, Cl-) ▪ For pH regulation o Production & use of ATP from glucose (Mg2+, PO4-) ▪ For energy • • 40-75% of body weight Essential for overall body functions Contains and carries electrolytes Major component of plasma Can directly affect concentration of electrolytes in body Function: o Transport nutrients to the cell (through the plasma) o Determines cell volume (cytosol is water) o Removes waste product (through urine & feces) o Acts as body’s coolant (heat released through sweat) Location: o Intracellular Fluid (ICF): 2/3 or 67% of body’s water o Extracellular Fluid (ECF): 1/3 or 33% of body’s water ▪ Plasma (intravascular) ▪ Interstitial fluid (extravascular) Distribution of Body Water in Adult Compartment Percent (%) of Percent (%) of Body Weight Total Body Water Extracellular Plasma 5 8 Interstitial 15 25 Intracellular 40 67 Total Body Water 60 100 Electrolyte Concentration is maintained by: o Passive Transport: passive movement of ions across a membrane ▪ No energy/ATP required ▪ Movement of ion controlled by concentration gradient ▪ Movement is from an area higher concentration to an area of lower concentration to maintain homeostasis/balance of ions o Active Transport: mechanism that requires energy to move ions across cellular membranes ▪ ATP is required ▪ ATPase-dependent ion pumps ▪ Ex. Movement of sodium and potassium across the membrane through the sodium-potassium ATPase ion pump Level of electrolytes is affected by the following o Dietary intake of electrolytes ▪ Diet is a major factor electrolyte level ▪ Most electrolytes are derived from diet o Hormone function and activity ▪ Hormones play vital role in the excretion and reabsorption of various electrolytes o Function of intestines and kidneys ▪ Regulation and control of electrolytes ▪ Electrolytes are absorbed in intestine through diet • GIT disorders/malabsorption syndrome has defective absorption affecting electrolyte absorption ▪ Abnormal kidneys affect normal retention/ reabsorption and excretion of electrolytes OSMOLALITY • • Definition: physical property of a solution based on concentration of solutes per kilogram of solvent (mmol/kg) Blood osmolality is regulated by: o Thirst sensation o Arginine vasopressin hormone (AVP) • • o Other Factors ▪ ANP • Aka Atrial Natriuretic Peptide/Factor (ANF) • Produced by the heart • Hormone that increases excretion of sodium in the kidneys • Has antagonistic function with aldosterone • Promotes sodium excretion • Aldosterone - promote sodium reabsorption ▪ AVP: (ADH) • Promotes water retention/reabsorption in the kidney ▪ GFR: • Aka Glomerular Filtration Rate • With volume expansion, GFR increases • With volume depletion, GFR decreases to sustain available volume • Directly proportional to the plasma volume ▪ Electrolytes: • Increased plasma sodium will increase urinary sodium excretion • Aldosterone - promote sodium conservation • ANP - promote sodium removal • Determination of Osmolality and Osmolal gap o Specimen: serum or urine o Osmolality: ▪ Indicates number of molecules per Kg of solvent ▪ Any substance dissolve in a solvent will: • ↓ the freezing point by 1.858C • ↑ the boiling point by 0.52C • ↓ vapor pressure (Dew point) by 0.3 mmHg • ↑ the osmotic pressure by 17,000 mmHg ▪ Main contributors of plasma osmolality are • glucose • urea • sodium • chloride Clinical Significance o Thirst Sensation: ▪ Response to consume more fluids ▪ When there is a need to consume more fluid, thirst sensation in the hypothalamus is stimulated to increase fluid intake ▪ Prevents water deficit or dehydration ▪ When plasma osmolality decrease, there is a need to consume more fluid to prevent dehydration ▪ Water is important in all biochemical reactions and metabolic activities o Arginine vasopressin hormone (AVP) ▪ Aka: Antidiuretic hormone (ADH) ▪ Function: increase water reabsorption in kidneys ▪ Suppressed in excess water load: ↓ POSM (dilute) • Lower plasma osmolality (POSM) • Causes frequent urination to lower water load ▪ Activated in water deficit: ↑ POSM (concentrated) • Higher solute than solvent • Conserves, retains, and reabsorbs water in kidneys to increase/maintain plasma volume in the circulation ▪ Caffeine & alcohol (ethyl) can suppress AVP and increase diuresis (urination) Regulation of Blood Volume o Renin-angiotensin-aldosterone system (RAAS) ▪ ↓ blood volume/pressure → Renin (kidneys) → Angiotensin I → Angiotensin II → Vasoconstriction and ↑ aldosterone (↑ Na+ ) ▪ Activated when blood volume and blood pressure are decreased (hypovolemic & hypotension) ▪ Hypovolemia - corrected by Na retention/ conservation (high Na = high blood volume) ▪ Hypotension - corrected by vasoconstriction CLINICALLY SIGNIFICANT ELECTROLYTES 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 ▪ ▪ ▪ ▪ 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 SODIUM Kidneys release Renin • Renin - enzyme will act with angiotensinogen as substrate When renin acts, angiotensinogen will be converted to angiotensin I (inactive form) When angiotensin I is circulated in the lungs, ACE will be released and catalyze conversion of angiotensin I to angiotensin II (active from) Angiotensin II will promote: • Vasoconstriction of blood vessels (↑ BP) • Stimulation of adrenal gland to release aldosterone (promote Na retention) DESCRIPTION AND REGULATION • • • The most abundant cation in the ECF Major Extracellular cation Regulated by Na+,K+ -ATPase ion pump o Na+,K+ -ATPase ion pump moves 3 Na+ ions out of the cell in exchange for 2 K+ ions o Active transport mechanism o Sodium and potassium movement maintain balance (electroneutrality) of cations in and out of cell o • • • • • Plasma concentration depends in renal regulation o Intake of water in response to thirst ▪ ↑ Na = ↑ POSM = activation of thirst center ▪ Intake of water = ↓ Na ▪ 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 ▪ Water loss = ↓ plasma vol. = ↑ Na ▪ AVP activation = water reabsorption/conservation (to normalize/lower plasma sodium) o The blood volume status, which affects Na excretion through: ▪ Aldosterone: promotes increased sodium reabsorption in kidneys, important in homeostasis ▪ Angiotensin II: stimulate adrenal gland to increase release aldosterone to conserve sodium ▪ ANP: antagonist of aldosterone, promote urinary excretion of sodium, important in homeostasis The plasma sodium concentration depends greatly on Reference values: 135-145 mmol/L Threshold critical value: 160 mmol/L and 120 mmol/L o >160 mmol/L = hypernatremia o <120 mmol/L = hyponatremia CSF Sodium: 135 - 150 mmol/L o Sodium can be found in CSF though passing on BBB • CLINICAL APPLICATIONS • Hyponatremia o Low plasma sodium levels o Cause of Hyponatremia (↓ Na+) ▪ ↑ Sodium Loss • Hypoadrenalism (↓ aldosterone) • Potassium deficiency o In kidneys, aldosterone promotes Na reabsorption & K excretion o When K is low, K should be conserved and reabsorbed while excreting sodium (inverse relationship bet. K & Na) • Diuretic use (Thiazide) • Ketonuria (Na lost with ketones) • Salt-losing nephropathy • Prolonged vomiting or diarrhea ▪ ↑ Water Retention • Renal failure (dilution of Na) • Nephrotic syndrome (↓ COP-PV, ↑ AVP) o ↑ AVP = ↑ water retention • Congestive Heart Failure (CHF) • Hepatic cirrhosis ▪ Water Imbalance • Syndrome of Inappropriate Antidiuretic Hormone (SIADH) (↑ AVP, ↑ H2O retention) • Pseudohyponatremia Classification of Hyponatremia by Osmolality ▪ With Low Osmolality • Increased sodium loss • Increased water retention (dilutional effect) ▪ With Normal Osmolality • Normal osmolality due to compensation of other substances • Increased non-sodium cations • Lithium excess • Increased gamma globulins • Severe hyperkalemia • Severe hypermagnesemia • Severe hypercalcemia • Pseudohyponatremia • Hyperlipidemia • Hyperproteinemia • Pseudohyperkalemia ▪ With High Osmolality • Sodium did not influence osmolality but other substances are affecting POSM • Hyperglycemia • Mannitol infusion Hypernatremia o Increased plasma sodium levels o Cause of Hypernatremia (↑ Na+) ▪ Excess Water Loss • Diabetes insipidus (↓ AVP) • 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 DETERMINATION OF SODIUM • • Specimen o Serum, plasma (lithium heparin, ammonium heparin, and lithium oxalate o False ↑ with marked/significantly increased hemolysis ▪ Sodium is present inside the cell Methods o Flame Emission Spectrophotometry (FES): ▪ Yellow after excitation o AAS: not commonly used ▪ 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 o Colorimetric/chemical method: Albanese-Lein ▪ Cupric sulfate and NaOH ▪ Uses magnesium uranyl acetate ▪ End product: yellow complex ▪ POTASSIUM DESCRIPTION AND REGULATION • • • • • • • • Aka Kalium Major Intracellular Cation o Most abundant inside the cell Functions: Regulation of neuromuscular excitability and contraction of heart, ICF volume, H concentration o ↑ K+: ↑ cell excitability → muscle weakness o ↓ K+: ↓ cell excitability → arrythmia or paralysis o Controls majority of heart activity 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 Na+, K+ -ATPase pump o ↓ function ↓ cellular entry → ↑ potassium (hyperkalemia) → hypoxia, digoxin overdose, hypomagnesemia, propanolol (β-blocker) o ↑ function ↑ cellular entry → ↓ plasma potassium (hypokalemia) → insulin, epinephrine o For every 3 Na ions, 2 K ions move in out of cell ↑ with exercise, hyperosmolality (DM), cellular breakdown o Patients are discouraged to do arm exercise (flex, fist clenching) o Prolonged tourniquet application is not allowed o Hemolysis will increase plasma K Reference values: 3.5-5.2 mmol/L Threshold critical values: o 6.5 mmol/L: hyperkalemia o 2.5 mmol/L: hypokalemia CLINICAL APPLICATION • • 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 Methods o FES: violet after excitation o AAS (not commonly performed) o ISE: valinomycin membrane ▪ Reference method for K determination ▪ Valinomycin - antibiotic with high affinity with K • Increases specificity for K o Colorimetric/chemical Method: Lockhead & Purcell (turbidimetric using sodium tetraphenylboron for ppt) CHLORIDE DESCRIPTION AND REGULATION • • • • • • Major Extracellular Anion Predominant in plasma Similar with sodium Involve in maintaining osmolality blood volume and electric neutrality (chloride shift) o Chloride and bicarbonate are involved in maintaining the negative charge balance in and out of the cell o When bicarbonate exits, chloride enters to maintain electric balance Rate limiting component in Na+ reabsorption o When sodium is reabsorbed, chloride is passively reabsorbed o When sodium is excreted, chloride is passively excreted o Sodium & chloride in plasma, exist as salt o sodium & chloride can be excreted through skin via perspiration/sweat Reference values: 98-107 mmol/L CLINICAL APPLICATIONS • • Hyperchloremia (↑ Cl-) o Cause of Hyperchloremia ▪ Excess Loss of HCO3• GI Losses • RTA • Metabolic acidosis Hypochloremia (↓ Cl-) o Cause of Hypochloremia ▪ Excess Loss of Cl • Prolonged vomiting • Diabetic ketoacidosis • Aldosterone deficiency • Salt-losing pyelonephritis • • • DETERMINATION OF BICARBONATE • 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 ▪ 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 • PHYSIOLOGY AND REGULATION • • • • • • BICARBONATE (HCO3-) REGULATION AND CLINICAL APPLICATIONS • • • Measures decreased absorbance of oxidized NAD MAGNESIUM Colorimetric: ▪ Reaction: reddish color 2nd Most Abundant anion in the ECF o 2nd to chloride 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 Along with chloride, maintains negative ionic charge neutrality of the plasma and the cell (electroneutrality) o Bicarbonate exits, chloride enters the cells Specimen: o Serum, plasma (heparin) o False ↓ if 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: ▪ ▪ • • Reabsorbed in the kidneys o 85% is reabsorbed in proximal convoluted tubule o 15% is reabsorbed in distal convoluted tubule ↑ HCO3 o Metabolic Alkalosis ▪ Severe vomiting ▪ Hypokalemia ▪ Hypoventilation ▪ Excessive alkali intake ↓ HCO3 o Metabolic Acidosis ▪ Hyperventilation • 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: 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 in serum o Complexed with PO4- and citrated: 5% Parathyroid Hormone (PTH) o Produced in the parathyroid gland o Responsible for increased renal reabsorption of Mg o Increases intestinal absorption of Mg ▪ Mg can be derived from diet Aldosterone and thyroxine (T4) o Promotes ▪ Increased renal excretion of Mg & K ▪ Sodium absorption CLINICAL APPLICATIONS • 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) REGULATION AND DISTRIBUTION • • • • • • • • DETERMINATION OF MAGNESIUM • • • Specimen: o Serum, plasma (lithium heparin) 24 hour iurine o Hemolysis cause false elevation Methods o Calgamite Method ▪ Mg2+ + calmagite → reddish-violet (532 nm) o 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 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 CALCIUM • • 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 (activation of coagulation factors to promote coagulations) Absorbed in upper small intestine in the presence of vit D o Without vit D, Ca will just be part of the excreta o Vit. D in active form is utilized 99% in bone and teeth and 1% in blood and ECF Calcium in the blood is distributed as: o Ionized Ca+2 ▪ Unbound or freeform ▪ Physiological active form of calcium ▪ 45% of Total Calcium in serum/plasma o Protein bound Ca+2 ▪ Present in albumin (transporter) ▪ 40% of Total Calcium in blood o Complex Ca+2 ▪ Bound to anions (HCO3, PO4, Lactate) ▪ 15% of Total Calcium in serum 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 ▪ Calcium in the blood enters the bone to promote calcification of the bone ▪ Blood calcium levels decreases due to Ca deposition to 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 • 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 activates vit. D to promote calcium absorption o However, excessive UV exposure can be harmful which can cause skin disorders o Melanin serves as a protection against UV rays o Melanin-poor race (Caucasians) are more prone to the harmful effects of UV rays 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 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 PHOSPHATE 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) Intracellular PO4 = organic phosphate Extracellular PO4 (plasma, serum, ECF) = inorganic PO4 Component of phospholipids, nucleic acids, creatine phosphate, and ATP Levels of phosphate is inversely related to calcium In small intestine, phosphate in diet is absorbed maximally at the jejunum Two forms: o Organic: principal anion within the cell o Inorganic: ion part of blood buffers ▪ Measured in laboratory tests Growth hormone (GH) ↓ renal excretion of phosphate • • If Ca is absorbed, PO4 is excreted If PO4 is absorbed, Ca is excreted • • • • • • • DETERMINATION OF CALCIUM • • Specimen o Serum, plasma (lithium heparin), 24 hour urine o Hemolysis cause false elevation Methods o AAS ▪ Reference method for Ca & Mg determination o ISE (liquid membrane electrode) o Ortho-cresolphthalein complexone (CPC) o Arsenzo III dye o EDTA titration method ▪ Bacarra ▪ Dower ▪ Zobel ▪ EDTA - for Ca chelation 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 CLINICAL APPLICATIONS • • Hypophosphatemia o Cause of Hypophosphatemia ▪ Hyperparathyroidism (↑ renal excretion) ▪ Vit. D Deficiency/ antacid use (↓ intestinal abs) Hyperphosphatemia o Cause of Hyperphosphatemia ▪ Hypoparathyroidism ▪ Lymphoblastic leukemia, intensive exercise, neoplastic disorders ▪ hypervitaminosis DETERMINATION OF PHOSPHATE • • Specimen o Serum, plasma (lithium heparin), 24-hour urine o Hemolysis cause false increase Methods o Ammonium phosphomolybdate complex (340 nm) o Fiske-Subbarow Method (600 nm) o Reaction: ▪ Phosphorus + Ammonium molybdate → Ammonium phosphomolybdate (purple) ▪ o o Ammonium phosphomolybdate + aminonaphtholsulfonic acid → Molybdenum blue (reduced ammonium phosphomolybdate) Reducing Agent: Pictol (Amino Napthol Sulfonic Acid) Other Reducing Agents: ▪ Elon (methyl amino phenol) ▪ Ascorbic acid ▪ Senidine (N-Phenyl-P Phenylenediamine Hydrochloride) ANION GAP • • LACTATE DESCRIPTION AND REGULATION • • Indicator of severity of oxygen deprivation (hypoxia) o ↓ tissue O2 = ↑ plasma lactate o Hypoxemia - low blood O2 o Hypoxia - low tissue O2 • • CLINICAL APPLICATIONS • • Hypoxic Conditions (Type A) o Lactate Acidosis ▪ Shock ▪ MI ▪ Severe congestive heart failure ▪ Pulmonary edema, severe blood loos Metabolic Origin (Type B) o Diabetes mellitus o Severe infection o Leukemia o Liver or renal disease o Toxins (ethanol, methanol, or salicylate poisoning DETERMINATION OF LACTATE • Enzymatic methods: lactate oxidase & peroxidase Mathematical approximation of difference bet. concentration of unmeasured cations and unmeasured anions Formulas : 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 Generally healthy indivuals with increased anion gap, the increased anion gap is used to check for accuracy and performance of machine during measurement (QC)