Cc 2 Lab

• Cations


  Positively charged ions
• Anions


  Negatively charged ions
• Sodium


  • Most abundant cation in the ECF
  • Renal threshold: 110-130 mmol/L
  • Also called "natrium"
  • Major extracellular cation in the body
  • Plasma concentration depends greatly on the intake and excretion of water
  • Reference value: 135-145 mmol/L
  • CSF level of sodium: 136-150 mmol/L
• Sodium


  • Transmission and conduction of nerve impulses
  • Regulation of body fluid levels
  • Na++ shifts into cells and K+ shifts out of the cells (sodium pump)
  • Assists with regulation of acid-base balance
• Sodium: Flame Emission Photometry


  1. Measure the light emitted by a single atom burned in a flame

  2. Involves excitation of electrons

  3. The light intensity of the characteristic wavelength produced by each of the atoms is directly proportional to the number of atoms that are emitting energy

  4. Na++ produces a YELLOW color in a flame
• Sodium: Atomic Absorption Spectrophotometry


  1. Measures the light absorbed by atoms dissociated by heat

  2. Uses a hollow cathode lamp

  3. The flame serves to dissociate the element from its chemical bonds and place it in a ground state
• Sodium: Ion Selective Electrode


  1. An electrochemical transducer capable of responding to one given ion

  2. Very sensitive and selective for the ion it measures

  3. Indirect ISE (w/ sample dilution)

  4. Direct ISE (w/o sample dilution)

  5. ISE Membrane for Na++: glass aluminum silicate
• Sodium: Colorimetry (Albanese-Lein)


  1. Sodium + Zinc uranyl acetate -> Sodium uranyl precipitate

  2. Sodium uranyl precipitate + H2O -> YELLOW

  3. Decrease in the absorbance = proportional to the Na content
• Sodium: "PROFAME Diagnostics" Procedure


  1. Label the tubes for the unknown with the patient's name including the test to be performed

  2. Mix and read absorbance after 1 minute incubation
• Patient Preparation: there are no special preparations
• Specimen Type: serum, plasma (lithium heparin, ammonium heparin, and lithium oxalate), and urine (preferable a 24-hr collection)
• Serum or plasma should be freshly drawn and urine (random/timed) should be properly collected
• Serum or plasma should be physically separated from contact with cells within two hours from the time of collection
• Separated serum or plasma should not remain at RT longer than 8 hours
• Urine assays are performed within 2 hours of collection. For timed specimens, the collection container should be kept in the refrigerator or on ice during the timed period. No preservative is required.
• Sodium: Reference Value


  • Conversion Factor: 1.0
  • Serum/Plasma: 136-145 mmol/L
  • CSF: 40-220 mmol/day
  • Urine (24-hr): 136-150 mmol/L
  • Threshold Critical Value: Hypernatremia - 160 mmol/L, Hyponatremia - 120 mmol/L

  
• Sodium: Sources of Interferences

  • Contaminated glasswares
  • Systematic error in measurement
  • In-vitro hemolysis (most common)
  • Unpurified water contains traces of sodium ions which will erroneously elevate sodium levels
• Sodium: Clinical Significance | Hypernatremia

  • Excess water loss: Diabetes insipidus, Renal tubular disorder, Prolonged diarrhea, Profuse sweating, Severe burns, Vomiting, Fever, Hyperventilation
  • Increased intake or retention: Hyperaldosteronism (Conn's Disease), Sodium bicarbonate infusion, Increased oral or IV intake of NaCl, Ingestion of seawater

  
• Sodium: Clinical Significance | Hyponatremia

  • Increase sodium loss: Diuretic use, Saline infusion
  • Increased water retention: Renal failure, Nephrotic syndrome, Aldosterone deficiency, Cancer, Syndrome of inappropriate ADH secretion (SIADH), Hepatic cirrhosis, Primary polydipsia, CNS abnormalities, Myxedema

  
• Potassium


  • Major intracellular cation
  • Essential for normal membrane excitability for nerve impulse
  • Also known as "kalium"
  • Only 2% of the body's total potassium that circulates in the plasma
  • RBC Potassium concentration is around 105 mmol/L
• Potassium: Methods of Measurement


  1. Flame Emission Photometry: Potassium produces a PURPLE color in a flame

  2. Ion Selective Electrode: ISE Membrane for Potassium: VALINOMYCIN

  3. Atomic Absorption Spectrophotometry
• Potassium: Colorimetry (Lockhead and Purcell)


  1. Potassium + Sodium cobaltinitrite -> Sodium Potassium Cobaltinitrite

  2. Sodium Potassium Cobaltinitrite + phenol -> BLUE
• Potassium: "PROFAME Diagnostics" Procedure


  1. Label the tubes for the unknown with the patient's name including the test to be performed

  2. Mix and read absorbance after 1 minute incubation
• Specimen Type: Non-hemolyzed Serum, Heparinized plasma; and Urine
• Blood specimens should also be separated from the red cells shortly after the collection to prevent any leakage of potassium. Sample is stable for at least 5 days if stored at 2-8 degree celsius
• Potassium: Sources of Interferences

  • Sample hemolysis
  • Thrombocytosis
  • Prolonged tourniquet application
  • Fist clenching
  • Blood stored on ice
  • IV fluid
  • High blast counts
  • Recentrifugation of SST

  
• Potassium: Clinical Significance | Hyperkalemia

  Decreased Renal Excretion: Acute and chronic renal failure, Severe dehydration, Addison's disease (Hypoaldosteronism)
• Potassium: Clinical Significance | Hypokalemia

  • Gastrointestinal Loss: Gastric suction and laxative abuse, Intestinal tumor and malabsorption, Cancer and radiotherapy, Vomiting and diarrhea
  • Renal loss: Diuretics, Hyperaldosteronism, Cushing syndrome, Leukemia, Bartter's syndrome, Gitelman's syndrome, Liddle's syndrome, Malignant hypertension

  
• Calcium


  • Most abundant cation in the body
  • 99% is in the bones, 1% is in the blood and circulated in ECF
  • Involved in coagulation, enzyme activities and maintenance of blood pressure
  • Absorbed in the duodenum and the absorption is in acidic pH
• Calcium: Functions


  • Blood coagulation
  • Enzyme activity
  • Excitability of skeletal and cardiac muscle
  • Maintenance of blood pressure
• Calcium: Hormones


  • 1,25 Dihydroxycholecalciferol (Vitamin D/Calcitriol): Mobilization of Ca++ from the bones/osteoclastic resorption in the bone, Stimulates absorption of Ca++ in the gut and reabsorption in the kidneys
  • PTH: Increases the Ca++ level by mobilizing bone Ca++, Activated the process of bone resorption, Suppresses urinary loss of Ca++, Stimulated the conversion of inactive vitamin D to active vitamin D3 in the kidney
  • Calcitonin: Inhibits PTH and Vitamin D3 which results to decreased Ca++ level, Inhibits bone resorption, Promotes urinary excretion of Ca++
• Calcium: Methods of Measurement

  • Ion Selective Electrode (ISE)
  • Flame Emission Photometry (FEP)
  • Atomic Absorption Spectrophotometry
  • Precipitation of Calcium: Clark and Collip Precipitation, Ferro Ham Chloranilic Acid Precipitation
  • Formation of colored complex: Alizarin, O-cresolphthalein, Calcein (Diehl-Ellingboe method), Ammonium purpurate, Nuclear fast red
  • Removal of calcium complex titration
• Ion Selective Electrode (ISE)

  Indicator electrode: Liquid membrane with calcium binding agent (or calcium ionophore)
• Flame Emission Photometry (FEP)

  Calcium produces an ORANGE color in a flame
• Atomic Absorption Spectrophotometry (AAS)


  Reference Method
• Ca Assay: Clark and Collip Precipitation


  The Ca++ is precipitated as oxalate directly from the specimen, and after washing, the precipitate is dissolved in acid and the liberated oxalic acid is titrated with KMnO4 solution until a FAINT PINK COLOR which persists for about a minute is observed.
• Ca Assay: Ferro Ham Chloranilic Acid Precipitation


  Ca++ in the sample is precipitated as Ca++ chloranilate by saturated solution of sodium chloranilate. The excess Chloranilic acid is removed by washing the precipitate with isopropyl alcohol and the precipitate is then treated with EDTA, which chelated calcium and releases Chloranilic acid which yields a PURPLE COLOR.
• Ca Assay: O-cresolphthalein Complexone Method

  1. Calcium reacts with o-cresolphthalein complexone to form a purple color complex in alkaline medium

  2. The intensity of color measured photometrically between 540 and 600 nm

  3. Maximum absorbance at 575 nm

  4. Directly proportional to the amount of calcium

  5. 8-hydroxyquinoline is incorporated into the reagent to preferentially bind magnesium and prevent interferences from magnesium cations

  6. For the reaction mixture it will contain 8-hydroxyquinoline which will bind magnesium, ethanol which decreases the absorbance of the bla
• Ca Assay: Ferro Ham Chloranilic Acid Precipitation


  1. Ca++ in the sample is precipitated as Ca++ chloranilate by saturated solution of sodium chloranilate

  2. The excess Chloranilic acid is removed by washing the precipitate with isopropyl alcohol

  3. The precipitate is then treated with EDTA, which chelated calcium and releases Chloranilic acid which yields a PURPLE COLOR
• Ca Assay: O-cresolphthalein Complexone Method

  1. Calcium reacts with o-cresolphthalein complexone to form a purple color complex in alkaline medium

  2. The intensity of color measured photometrically between 540 and 600 nm

  3. Maximum absorbance at 575 nm

  4. Directly proportional to the amount of calcium

  5. 8-hydroxyquinoline is incorporated into the reagent to preferentially bind magnesium and prevent interferences from magnesium cations

  6. The reaction mixture will contain 8-hydroxyquinoline which will bind magnesium, ethanol which decreases the absorbance of the blank and urea that will decrease the turbidity of a lipemic sample and will eventually increase the color intensity