Lecture 2/21

• hypoproteinemia - protein loss

  • Excretion caused by renal disease
  • Leakage into GI tract - caused by inflammation of GI tract
  • Blood loss in wounds
• causes of hypoproteinemia include protein loss, inadequate intake, decreased synthesis and accelerated catabolism
• hypoproteinemia - inadequate intake

  • protein deficient diet
  • damage to GI tract
• hypoproteinemia - decreased synthesis

  • liver disease
  • inherited immunodeficiency disorders
• hypoproteinemia - accelerated catabolism

  • burns
  • trauma
• hyperproteinemia, although not as common, can be caused by dehydration, infection, and disorders of excessive production
• hyperproteinemia - dehydration

  • causes a relative hyperproteinemia
• hyperproteinemia - infection

  • gamma globulin increase
• hyperproteinemia - disorders of excess production

  • mostly results from paraproteins
  • e.g. multiple myeloma, Waldenstrom's macroglobulinemia, chronic inflammation, collagen vascular disorders, and some neoplasms
• multiple myeloma - produce IgG or IgA light chains
• Waldenstrom's macroglobulinemia - produces IgM
• total protein

  • is run on serum
• • interferences of total protein
  • hemolysis
  • lipemia (in some protein methods)
• hemolysis will cause interference in total protein as contents of RBCs contain proteins
• normal total protein levels

  • ambulatory adults: 6.5-8.3 g/dL
  • recumbent adults: 6.0-7.8 g/dL
• protein levels are lower at birth, but by age 3, will have adult levels of protein
• Kjeldahl - historic method of protein analysis to which our current methodology comes from; originated in 1870s
• Kjeldahl

  • trichloroacetic acid is an organic acid that removes proteins that have nitrogens in them by precipitating them out, does not remove nitrogens that are not proteins
  • analytes that are NOT proteins are not precipitated out
• ammonium borate - NH4H2BO3
• proteins we have analyzed using Kjeldahl method contains 15.1-16.8% nitrogen
• basic steps to Kjeldahl

  • add trichloroacetic acid to sample
  • centrifuge
  • remove supernatant
  • convert sediment to ammonium borate
  • titrate ammonium borate with HCl
  • divide amount of HCl by .16 to acquire result
• Kjeldahl advantages

  • very precise and accurate compared to other methods
• Kjeldahl disadvantages

  • time consuming, labor intensive, removing supernatant and precipitate requires too much skill
• • Refractometry - based on refractive index of dissolved solids
  • Since majority of dissolved solids are protein in plasma, this method is viable
  • Refractometer must be calibrated with fluid that has the same proportion of other dissolved solids
• Refractometry for protein analysis

  • Pros: Fast and easy 
  • Cons: Temperature dependent
  • Interferences: lipemia, icterus, hemolysis 
• • Biuret method for protein analysis
  • Cupric ions join to peptide bonds
  • Violet colored chelate is formed if two or more bonds are present and the medium is alkaline
  • Reagent contains antioxidant and a chemical to prevent copper precipitation in alkaline solutions
• Biuret method for protein analysis

  • Pink to reddish violet color is read at 540 nm
• Biuret method for protein analysis

  • Interferences:
  • Hemolysis, lipemia, bilirubin give false increase
  • High nitrogen content gives false decrease
• the biuret method is the most widely used method of protein analysis in the clinical lab
• • Dye binding for protein analysis
  • Used in electrophoresis - what we are using: amido black
  • Dye used to stain bands in electrophoresis
• • Ultraviolet absorption for protein analysis
  • Used mostly in research labs 
  • Used at 210 nm, which is the wavelength at which peptide bonds will absorb only if confirmation is right