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Differences between enzymatic and diazo methods for measuring direct bilirubin

K Nakayama1

  • 1Department of Laboratory Medicine, Niigata University School of Medicine, Japan.

European Journal of Clinical Chemistry and Clinical Biochemistry : Journal of the Forum of European Clinical Chemistry Societies
|August 1, 1995
PubMed
Summary

Disparities in direct bilirubin measurements between enzymatic and diazo methods stem from conjugated bilirubin. Incomplete oxidation of conjugated bilirubin by bilirubin oxidase causes these measurement differences.

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Area of Science:

  • Clinical Chemistry
  • Biochemistry

Background:

  • Direct bilirubin measurement is crucial for diagnosing liver conditions.
  • Both diazo and enzymatic methods are used, but discrepancies exist.

Purpose of the Study:

  • To investigate the causes of disparities between enzymatic and diazo methods for direct bilirubin measurement.
  • To identify the bilirubin subfractions responsible for these differences.

Main Methods:

  • High-performance liquid chromatography (HPLC) was used to analyze bilirubin subfractions.
  • Spectrophotometry was employed to study bilirubin oxidation by bilirubin oxidase at different pH levels.

Main Results:

  • Disparities were greater in samples containing conjugated bilirubin (Type I) compared to those with mainly unconjugated bilirubin (Type II).

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  • Bile and synthetic conjugated bilirubin showed significant differences between methods.
  • Incomplete oxidation of conjugated bilirubin by bilirubin oxidase at pH 3.7 produced interfering substances, unlike complete oxidation at pH 7.2.

    • Conclusions:

    • Conjugated bilirubin is the primary cause of discrepancies between enzymatic and diazo methods.
    • Incomplete oxidation products of conjugated bilirubin by bilirubin oxidase lead to inaccurate direct bilirubin readings.
    • Clinical interpretation of direct bilirubin values from enzymatic methods requires caution, especially in samples with high conjugated bilirubin levels.