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Related Experiment Videos

Improved HPLC method for carbohydrate-deficient transferrin in serum.

Anders Helander1, Asgeir Husa, Jan-Olof Jeppsson

  • 1Department of Clinical Neuroscience, Karolinska Institutet and Hospital, SE-171 76 Stockholm, Sweden. anders.helander@cns.ki.se

Clinical Chemistry
|October 28, 2003
PubMed
Summary

An improved HPLC method accurately separates and quantifies serum transferrin glycoforms, establishing a reference for carbohydrate-deficient transferrin (CDT) testing. This method aids in diagnosing alcohol abuse and in medico-legal cases.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Clinical Diagnostics

Background:

  • A validated reference method for serum transferrin glycoforms is needed for traceable immunologic assays of carbohydrate-deficient transferrin (CDT).
  • Routine CDT testing relies on immunologic methods requiring traceability to a reference standard.

Purpose of the Study:

  • To describe an improved High-Performance Liquid Chromatography (HPLC) method for the separation and quantification of serum transferrin glycoforms.
  • To establish a reference method for transferrin glycoform analysis, crucial for accurate CDT measurements.

Main Methods:

  • Serum transferrin was iron-saturated and lipoproteins precipitated.
  • Glycoforms were separated using anion-exchange chromatography (SOURCE 15Q column) with salt gradient elution.

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  • Quantification was achieved by measuring absorbance of the iron-transferrin complex at 470 nm.

    • Main Results:

    • The HPLC method reproducibly separated and quantified asialo- to hexasialotransferrin glycoforms, with near-baseline separation for disialo- and trisialotransferrin.
    • Intra- and interassay coefficients of variation for disialotransferrin were below 5%.
    • Asialotransferrin was detected in chronic heavy drinkers, distinguishing them from controls.

    Conclusions:

    • The developed HPLC method meets the criteria for a preliminary reference method for CDT.
    • This method is suitable for analyzing diverse serum transferrin glycoform profiles and confirming CDT results in forensic contexts.