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Chemistry

Principles

Labs play a critical role in diagnosing and monitoring conditions related to heme metabolism. Tests include bilirubin, iron studies, porphyrins, and more

  • Key Tests
    • Bilirubin (Total, Direct, Indirect)
    • Urine Bilirubin
    • Fecal Bilirubin
    • Iron Studies (Serum Iron, TIBC, Transferrin Saturation, Ferritin)
    • Porphyrins (Urine, Blood, Fecal)
    • Lead

Bilirubin

  • Principle: The Diazo Reaction is the basis for most bilirubin assays. Bilirubin reacts with a diazonium salt (e.g., diazotized sulfanilic acid) in an acidic solution to form a colored azobilirubin pigment, which is measured spectrophotometrically
  • Types of Bilirubin Measured
    • Total Bilirubin: Measures both conjugated (direct) and unconjugated (indirect) bilirubin
    • Direct Bilirubin: Measures conjugated bilirubin directly (reacts with diazo reagent in aqueous solution)
    • Indirect Bilirubin: Calculated by subtracting direct bilirubin from total bilirubin (represents unconjugated bilirubin)
  • Reactions
    1. Bilirubin + Diazotized Sulfanilic Acid → Azobilirubin (Colored Product)
  • Enhancers/Accelerators: To measure total bilirubin, a reagent such as methanol or caffeine is added to solubilize the unconjugated bilirubin, allowing it to react with the diazo reagent
  • Detection: The intensity of the colored azobilirubin product is directly proportional to the bilirubin concentration in the sample and is measured spectrophotometrically
  • Advantages: Widely available, relatively inexpensive, and well-established method
  • Disadvantages: Susceptible to interferences from lipemia, hemolysis, and certain medications

Urine Bilirubin

  • Principle: The Diazo Reaction is used. Bilirubin in urine reacts with a diazonium salt to form a colored product
  • Method: Dipstick or Ictotest tablets
  • Reactions
    1. Bilirubin + Diazonium Salt → Colored Product
  • Detection: The color change on the dipstick or tablet is compared to a color chart to estimate the bilirubin concentration in the urine
  • Advantages: Rapid and simple screening test
  • Disadvantages: Less sensitive than serum bilirubin assays, only detects conjugated bilirubin, susceptible to false negatives from ascorbic acid (vitamin C)

Fecal Bilirubin

  • Principle: Quantitative measurement of fecal bilirubin is not routinely performed. However, the presence or absence of bilirubin breakdown products (urobilinogen and stercobilin) can be assessed
  • Method: Chemical tests to detect urobilinogen and stercobilin
  • Reactions
    1. Urobilinogen + Ehrlich’s Reagent → Pink-Red Color
  • Detection: The presence of a colored product indicates the presence of urobilinogen and stercobilin
  • Advantages: Can provide information about bile flow and intestinal function
  • Disadvantages: Not quantitative, affected by diet and medications

Iron Studies

  • Principle: A panel of tests used to evaluate iron metabolism and storage
  • Tests Included
    • Serum Iron: Measures the amount of iron bound to transferrin in the blood
    • Total Iron-Binding Capacity (TIBC): Measures the total amount of iron that can be bound by transferrin
    • Transferrin Saturation: Calculated as (Serum Iron / TIBC) x 100
    • Ferritin: Measures the amount of iron stored in tissues

Serum Iron

  • Principle: Iron is released from transferrin by an acidic buffer and reduced to Fe2+. The Fe2+ reacts with a chromogen to form a colored complex, which is measured spectrophotometrically
  • Reactions
    1. Transferrin-Fe3+ + Acidic Buffer → Transferrin + Fe3+
    2. Fe3+ + Reducing Agent → Fe2+
    3. Fe2+ + Chromogen → Colored Complex
  • Detection: The intensity of the colored complex is directly proportional to the iron concentration in the sample
  • Advantages: Widely available and relatively inexpensive
  • Disadvantages: Affected by diurnal variation and recent iron intake

Total Iron-Binding Capacity (TIBC)

  • Principle: Excess iron is added to saturate all binding sites on transferrin. The unbound iron is removed, and the total iron is then measured using a similar method to serum iron
  • Reactions
    1. Transferrin + Excess Fe3+ → Transferrin-Fe3+ (Saturated)
    2. Removal of Unbound Fe3+
    3. Transferrin-Fe3+ + Acidic Buffer → Transferrin + Fe3+
    4. Fe3+ + Reducing Agent → Fe2+
    5. Fe2+ + Chromogen → Colored Complex
  • Detection: The intensity of the colored complex is directly proportional to the TIBC
  • Advantages: Provides an estimate of transferrin concentration
  • Disadvantages: More complex than serum iron measurement

Transferrin Saturation

  • Principle: Calculated from serum iron and TIBC
    • Transferrin Saturation = (Serum Iron / TIBC) x 100
  • Interpretation
    • Low Transferrin Saturation: Suggests iron deficiency
    • High Transferrin Saturation: Suggests iron overload

Ferritin

  • Principle: Immunochemical methods (e.g., ELISA, chemiluminescence) are used to measure ferritin. Antibodies specific to ferritin are used to capture and quantify the ferritin in a blood sample
  • Methods
    • Enzyme-Linked Immunosorbent Assay (ELISA)
    • Chemiluminescence Immunoassay (CLIA)
  • Reactions
    1. Ferritin + Ferritin-Specific Antibody → Antibody-Ferritin Complex
  • Detection: The amount of antibody-ferritin complex formed is measured, and is proportional to the ferritin concentration in the sample
  • Advantages: Specific and sensitive
  • Disadvantages: Can be affected by inflammation

Porphyrins

  • Principle: Porphyrins are extracted from urine, blood, or feces and separated by chromatography. They are then quantified by spectrophotometry or fluorometry
  • Methods
    • Spectrophotometry
    • Fluorometry
    • High-Performance Liquid Chromatography (HPLC)
  • Procedure
    1. Extraction: Porphyrins are extracted from the sample using organic solvents
    2. Separation: Porphyrins are separated by HPLC based on their chemical properties
    3. Detection: Porphyrins are detected and quantified by measuring their absorbance or fluorescence
  • Advantages: Can identify and quantify specific porphyrins
  • Disadvantages: Complex and requires specialized equipment
  • Urine Porphyrins: Reflects porphyrin excretion
  • Blood Porphyrins: Detects porphyrins present in blood
  • Fecal Porphyrins: Measures porphyrin excretion in feces

Lead

  • Principle: Atomic absorption spectrometry (AAS) or inductively coupled plasma mass spectrometry (ICP-MS) are used to measure lead in whole blood
  • Methods
    • Atomic Absorption Spectrometry (AAS)
    • Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
  • Procedure
    1. Sample Preparation: Blood is treated to release lead
    2. Analysis: The sample is introduced into the instrument, and lead is measured based on its absorption or emission of light
  • Advantages: Highly sensitive and accurate
  • Disadvantages: Requires specialized equipment and trained personnel

Key Terms

  • Diazo Reaction: A chemical reaction used to measure bilirubin
  • Spectrophotometry: A method to measure the absorbance of light by a solution
  • Chromogen: A substance that produces a colored product
  • ELISA (Enzyme-Linked Immunosorbent Assay): An immunoassay that uses enzyme-labeled antibodies
  • CLIA (Chemiluminescence Immunoassay): An immunoassay that uses chemiluminescent labels
  • HPLC (High-Performance Liquid Chromatography): A method to separate and quantify compounds
  • Atomic Absorption Spectrometry (AAS): A method to measure the concentration of elements
  • Inductively Coupled Plasma Mass Spectrometry (ICP-MS): A highly sensitive method to measure the concentration of elements
  • Porphyrins: Intermediates in heme synthesis
  • Ferritin: A protein that stores iron
  • Transferrin: A protein that transports iron in the blood
  • TIBC (Total Iron-Binding Capacity): A measure of the blood’s capacity to bind iron
  • Heme: the iron-containing porphyrin ring of hemoglobin and other respiratory pigments