CC1 - CM

• Spectrophotometry


  Technique that uses prisms or gratings to isolate a narrow range of wavelength of light
• Nephelometry


  Technique that measures the amount of light scattered by an unknown substance at right angles
• Turbidimetry


  Technique that measures the amount of light blocked (absorbance) by a suspension of particles
• Electrophoresis


  Technique where charged molecules move at different rates when pulled through an electrical field
• Flame Emission Spectrophotometry


  Technique that measures light emitted by excited atoms
• Atomic Absorption Spectrophotometry


  Technique that measures light absorbed by ground-state atoms
• Clinical chemistry is the branch of medical science involved in the analysis of biological materials, usually bodily fluids, to provide diagnostic results on the state of the human body
• Analytes tested in clinical chemistry

  • Lipid profile
  • Cholesterol
  • Bilirubin-Liver Function Test
• Spectrophotometry


  • Uses prisms or gratings to isolate a narrow range of wavelength of light
  • Employs two kinds of light: visible and invisible spectrum
• Wavelength ranges

  • Ultraviolet light < 400 nm (visible spectrum)
  • Infrared light 700 nm > (invisible spectrum)
• Nephelometry


  • Measures the amount of light scattered by an unknown substance at right angles
  • Depends on the wavelength and particle size
• Turbidimetry


  • Measures the amount of light blocked (absorbance) by a suspension of particles
  • Depends on particle size and concentration
• Electrophoresis


  • Charged molecules move at different rates when pulled through an electrical field
  • Cations move to the cathode, anions move to the anode
• Flame Emission Spectrophotometry


  • Measures light emitted by excited atoms
  • Atoms return to ground state: sodium (intense yellow flame), potassium (violet flame), and calcium (brick red flame)
  • Internal standards: lithium or cesium
• Atomic Absorption Spectrophotometry


  • Measures light absorbed by ground-state atoms
  • Routinely used to measure the concentration of trace metals
  • Hollow cathode lamp is the usual light source
• Spectrophotometer


  • Light source
  • Entrance slit
  • Monochromator
  • Exit slit
  • Cuvette
  • Photodetector
  • Meter or read-out device
• Beer-Lambert Law


  • The amount of light absorbed by a solution varies with the concentration of the colored solute
  • Absorbance is directly proportional to concentration
• Calculating concentration using Beer-Lambert Law


  1. A = (ε)(b)(c)

  2. A = absorbance

  3. ε = molar absorptivity (constant for each type of molecule)

  4. b = length of light path (cuvette)

  5. c = concentration of the solution
• Routinely requested clinical chemistry assays

  • Blood glucose
  • Blood lipid profile
• Blood glucose


  • Normally ranges from 70-110 mg/dl (3.9-6.1 mmol/L)
  • Physiologic elevation happens after eating
  • Diabetes is associated with elevated fasting blood glucose
• Glycosylated hemoglobin (HbA1c)


  • Reflects the average blood glucose over a three-month period
  • Measured through affinity chromatography
• Diabetes mellitus


  • Defect in the beta-cells of the pancreas leading to decreased insulin production
  • Manifested by polyuria, polydipsia, and polyphagia
• Methods of blood glucose detection

  • Enzymatic: Glucose oxidase, hexokinase method
  • Non-enzymatic: Nelson-Somogyi, Hagedorn-Jensen, ortho-toluidine
• Lipids


  • Organic substances characterized by insolubility in water and solubility in organic solvents
  • Provide 9 kcal of heat per gram
  • Functions: primary energy source, constituent of cellular membrane
• Classification of lipids


  • Fatty acids
  • Triglycerides
  • Phospholipids
  • Steroids
• Diabetes


  Manifested by polyuria (excessive urination), polydipsia (excessive thirst), and polyphagia (excessive eating)
• Hyperglycemia


  High sugar level
• Hypoglycemia


  Low sugar level
• Methods of Detection


  • Enzymatic: Glucose oxidase, procedure, and hexokinase method (considered as the reference method)
  • Non-enzymatic: Nelson-Somogyi (copper reduction method), Hagedorn-Jensen (iron reduction method), and ortho-toluidine (aromatic amine condensation)
• Lipids


  Organic substances characterized by their general insolubility in water and solubility in organic solvents. Per gram, it may provide 9 kcal of heat.
• Lipids


  • Act as a primary energy source
  • An important constituent of cellular membrane
• Classification of Lipids


  • Fatty Acids
  • Triglycerides (triacylglycerols)
  • Cholesterol
  • Phospholipids
• Fatty Acids


  Simplest form of lipids but not routinely measured. Forms: saturated (solid at room temperature) and unsaturated (liquid at room temperature).
• Triglycerides (triacylglycerols)


  Considered neutral fats consisting of fatty acids and glycerol. Act as the storage form of lipids in the human body.
• When serum samples turned turbid from blood collected after a meal, it is due to the presence of triglycerides.
• Cholesterol


  An example of a steroid alcohol. Necessary for the production of various hormones, vitamin D, and even bile salt which is necessary for fat digestion.
• Measurement of cholesterol is usually requested in conjunction with the diagnosis of cardiovascular diseases.
• Phospholipids


  Most abundant form of lipids, but not routinely measured. Include lecithin (70%), sphingomyelin (20%), and cephalin (10%).
• Lecithin-sphingomyelin (L/S) ratio


  An important parameter to determine fetal-lung maturity and is used to evaluate the safety of a pre-term delivery.
• Lipoproteins


  Lipid transporters which are classified based on their density or through electrophoresis. Generally, as the protein content increases, the lipid content decreases, making its size smaller but denser.