Principles

Labs perform many tests to assess protein status and nitrogen balance. Understanding the principles is key to accurate testing

• Total Protein
• Albumin
• Prealbumin (Transthyretin)
• Globulins
• Urea (Blood Urea Nitrogen - BUN)
• Creatinine
• Uric Acid
• Ammonia
• Cerebrospinal Fluid (CSF) Protein
• Urine Protein
• Protein Electrophoresis (Serum and Urine)
• Immunofixation Electrophoresis (IFE)
• Tumor Markers (PSA, AFP, CEA, CA-125, etc.)

Total Protein

• Principle: The Biuret Reaction is the basis for most total protein assays. In an alkaline solution, peptide bonds react with copper(II) ions (Cu2+) to form a colored complex. The intensity of the colored complex is directly proportional to the number of peptide bonds, and therefore the protein concentration, in the sample
• Reactions
  • Peptide Bonds + Cu2+ (in Alkaline Solution) → Colored Complex
• Components of the Biuret Reagent
  • Copper(II) Sulfate (CuSO4): Provides the Cu2+ ions
  • Sodium Potassium Tartrate: Chelates the Cu2+ ions to prevent precipitation
  • Sodium Hydroxide (NaOH): Provides the alkaline environment
  • Potassium Iodide (KI): Prevents reduction of Cu2+
• Detection: The intensity of the colored complex is measured spectrophotometrically at a wavelength between 540-560 nm
• Advantages: Simple, widely available, and relatively inexpensive
• Disadvantages: Susceptible to interferences from lipemia, hemolysis, and icterus

Albumin

• Principle: Dye-binding methods are commonly used. Albumin binds to a dye (e.g., bromcresol green or bromcresol purple) at a specific pH, causing a change in the dye’s absorbance. The change in absorbance is directly proportional to the albumin concentration
• Dyes Used
  • Bromcresol Green (BCG): At a pH of 4.2, BCG binds to albumin, causing a color change
  • Bromcresol Purple (BCP): At a pH of 5.2, BCP binds to albumin, causing a color change
• Reactions
  • Albumin + Dye → Albumin-Dye Complex (Colored)
• Detection: The absorbance of the Albumin-Dye Complex is measured spectrophotometrically
• Advantages: Simple, rapid, and automated
• Disadvantages: Susceptible to interferences from certain medications and elevated bilirubin levels

Prealbumin (Transthyretin)

• Principle: Immunochemical methods (e.g., nephelometry, turbidimetry) are used to measure prealbumin. Antibodies specific to prealbumin are used to capture and quantify prealbumin in a blood sample
• Methods
  • Nephelometry
  • Turbidimetry
• Reactions
  • Prealbumin + Prealbumin-Specific Antibody → Antibody-Prealbumin Complex
• Detection: The amount of antibody-prealbumin complex formed is measured, and is proportional to the prealbumin concentration
• Advantages: Specific and automated
• Disadvantages: Can be affected by interfering substances and requires specialized equipment

Globulins

• Principle: Globulins are typically not measured directly, but are calculated by subtracting albumin from total protein
  • Globulins = Total Protein - Albumin

Urea (Blood Urea Nitrogen - BUN)

• Principle: Several methods are available for measuring BUN. Two common methods are the enzymatic method and the direct chemical method
• Enzymatic Method (Urease Method)
  • Urease hydrolyzes urea to ammonia and carbon dioxide. The ammonia is then reacted with a chromogen to produce a colored product
  • Reactions
    • Urea + H2O –(Urease)–> 2 NH3 + CO2
    • NH3 + Reagent → Colored Product
• Direct Chemical Method (Diacetyl Monoxime Method)
  • Urea reacts with diacetyl monoxime in the presence of acid and a catalyst to form a colored product
  • Reactions
    • Urea + Diacetyl Monoxime → Colored Product
• Detection: The intensity of the colored product is measured spectrophotometrically
• Advantages: Both methods are widely available and relatively inexpensive
• Disadvantages: Susceptible to interferences from ammonia and other nitrogen-containing compounds

Creatinine

• Principle: The Jaffe Reaction is commonly used. Creatinine reacts with picric acid in an alkaline solution to form a colored Janovski complex. The rate of color formation is measured spectrophotometrically
• Reactions
  • Creatinine + Picric Acid (in Alkaline Solution) → Janovski Complex (Colored)
• Methods
  • Kinetic Jaffe Method: Measures the rate of color formation, reducing interferences
  • Compensated Jaffe Method: Uses a blank to correct for interferences
  • Enzymatic Methods: Use enzymes to convert creatinine to measurable products
• Detection: The intensity of the colored complex is measured spectrophotometrically
• Advantages: Simple and widely available
• Disadvantages: Susceptible to interferences from bilirubin, glucose, and certain medications

Uric Acid

• Principle: Several methods are available for measuring uric acid. Two common methods are the enzymatic method and the chemical method
• Enzymatic Method (Uricase Method)
  • Uricase oxidizes uric acid to allantoin, and the decrease in absorbance at 290 nm is measured
  • Reactions
    • Uric Acid + O2 + H2O –(Uricase)–> Allantoin + H2O2 + CO2
  • Coupled enzyme reactions can be used to quantify the H2O2 produced
• Chemical Method (Phosphotungstic Acid Method)
  • Uric acid reduces phosphotungstic acid to tungsten blue in an alkaline solution
  • Reactions
    • Uric Acid + Phosphotungstic Acid → Tungsten Blue
• Detection: The decrease in absorbance at 290 nm (enzymatic method) or the intensity of the tungsten blue (chemical method) is measured spectrophotometrically
• Advantages: The enzymatic method is more specific and less susceptible to interferences
• Disadvantages: The chemical method is less expensive but more susceptible to interferences

Ammonia

• Principle: Ammonia reacts with a chromogen in the presence of alkali to form a colored product
• Methods
  • Enzymatic Method: Ammonia reacts with α-ketoglutarate and NADPH in the presence of glutamate dehydrogenase (GLDH) to form glutamate and NADP+. The decrease in absorbance at 340 nm is measured
  • Ion-Selective Electrode (ISE) Method: Ammonia is measured using an ion-selective electrode
• Reactions
  • NH4+ + α-Ketoglutarate + NADPH –(GLDH)–> Glutamate + NADP+ + H2O
• Detection: The decrease in absorbance at 340 nm (enzymatic method) is measured spectrophotometrically or the potential difference is measured (ISE)
• Advantages: The enzymatic method is more sensitive and specific
• Disadvantages: Ammonia is volatile and can be easily contaminated

Cerebrospinal Fluid (CSF) Protein

• Principle: Similar to total protein methods, the Biuret Reaction or dye-binding methods (e.g., Coomassie Brilliant Blue) are used to measure total protein in CSF
• Special Considerations
  • CSF protein levels are much lower than serum protein levels, requiring more sensitive methods
  • Samples should be free of blood contamination

Urine Protein

• Principle: Several methods are available for measuring urine protein. Two common methods are the turbidimetric method and the dye-binding method
• Turbidimetric Method
  • Protein is precipitated by adding a reagent (e.g., sulfosalicylic acid, trichloroacetic acid), and the turbidity of the solution is measured
  • Reactions
    • Protein + Precipitating Reagent → Turbidity
• Dye-Binding Method
  • Similar to serum albumin methods, protein binds to a dye (e.g., Coomassie Brilliant Blue) causing a change in absorbance
  • Reactions
    • Protein + Dye → Protein-Dye Complex (Colored)
• Detection: The turbidity or the intensity of the colored complex is measured spectrophotometrically
• Advantages: The dye-binding method is more sensitive and less susceptible to interferences
• Disadvantages: Turbidimetric methods are less sensitive

Protein Electrophoresis (Serum and Urine)

• Principle: Proteins are separated based on their electrical charge and size
• Procedure
  • Sample is applied to a support medium (e.g., agarose gel or cellulose acetate)
  • An electric field is applied, causing proteins to migrate at different rates
  • Proteins are stained with a protein-specific dye
  • The separated protein bands are visualized and quantified
• Clinical Significance
  • Helps identify abnormal protein patterns, such as:
    • Monoclonal Gammopathies (e.g., multiple myeloma)
    • Nephrotic Syndrome
    • Liver Disease
    • Inflammatory Conditions
• Protein electrophoresis fractions * Albumin * Alpha-1 Globulins * Alpha-2 Globulins * Beta Globulins * Gamma Globulins

Immunofixation Electrophoresis (IFE)

• Principle: Similar to protein electrophoresis, but after electrophoresis, the gel is overlaid with specific antibodies that bind to specific proteins. The antibody-protein complexes are then visualized
• Clinical Significance
  • Used to identify monoclonal proteins in serum and urine
  • Helps diagnose multiple myeloma and other plasma cell disorders

Tumor Markers (PSA, AFP, CEA, CA-125, etc.)

• Principle: Immunoassays (e.g., ELISA, chemiluminescence) are used to measure tumor markers. Antibodies specific to the tumor marker are used to capture and quantify the tumor marker in a blood sample
• Methods
  • ELISA
  • Chemiluminescence Immunoassay (CLIA)
• Reactions
  • Tumor Marker + Tumor Marker-Specific Antibody → Antibody-Tumor Marker Complex
• Detection: The amount of antibody-tumor marker complex formed is measured, and is proportional to the tumor marker concentration
• Clinical Significance
  • PSA: Prostate-Specific Antigen; prostate cancer
  • AFP: Alpha-Fetoprotein; liver cancer, germ cell tumors
  • CEA: Carcinoembryonic Antigen; colorectal cancer, lung cancer
  • CA-125: Cancer Antigen 125; ovarian cancer

Cardiac Markers (Troponin, CK-MB, Myoglobin)

• Principle: Immunoassays (e.g., ELISA, chemiluminescence) are used to measure cardiac markers. Antibodies specific to the cardiac marker are used to capture and quantify the cardiac marker in a blood sample
• Methods
  • ELISA
  • Chemiluminescence Immunoassay (CLIA)
• Reactions
  • Cardiac Marker + Cardiac Marker-Specific Antibody → Antibody-Cardiac Marker Complex
• Detection: The amount of antibody-cardiac marker complex formed is measured, and is proportional to the cardiac marker concentration
• Clinical Significance
  • Troponin: Myocardial Infarction
  • CK-MB: Myocardial Infarction
  • Myoglobin: Myocardial Infarction

Key Terms

• Biuret Reaction: A chemical reaction used to measure total protein
• Dye-Binding Method: A method to measure albumin using dyes
• Turbidimetry: A method to measure the turbidity of a solution
• Nephelometry: A method to measure the amount of light scattered at an angle
• Urease Method: An enzymatic method to measure blood urea nitrogen (BUN)
• Jaffe Reaction: A chemical reaction used to measure creatinine
• Uricase Method: An enzymatic method to measure uric acid
• Ion-Selective Electrode (ISE): A method to measure the concentration of ions
• Protein Electrophoresis: A method to separate proteins based on their electrical charge and size
• Immunofixation Electrophoresis (IFE): A method to identify monoclonal proteins
• ELISA (Enzyme-Linked Immunosorbent Assay): An immunoassay that uses enzyme-labeled antibodies
• Chemiluminescence Immunoassay (CLIA): An immunoassay that uses chemiluminescent labels
• Tumor Marker: A substance produced by cancer cells or other cells in response to cancer
• Cardiac Marker: A substance released into the blood when the heart is damaged
• Spectrophotometry: A method to measure the absorbance of light by a solution
• Albumin: A major protein in the blood
• Globulin: A group of proteins in the blood
• Urea: A nitrogen-containing compound that is the major end product of protein metabolism
• Uric Acid: A nitrogen-containing compound that is the major end product of purine metabolism
• Creatinine: A waste product of muscle metabolism
• Ammonia: A toxic nitrogen-containing compound
• BUN: Blood Urea Nitrogen