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Erythrocyte galactokinase assay with high performance liquid chromatography
N Mizoguchi1, T Eguchi, N Sakura
1Department of Pediatrics, Hiroshima University School of Medicine, Japan.
Summary
A new assay measures erythrocyte galactokinase activity using HPLC, offering a sensitive and accurate alternative to radioisotopic methods. This blood test is ideal for neonatal screening follow-ups.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Clinical Diagnostics
Background:
- Galactokinase (GALK) deficiency is a rare genetic disorder.
- Accurate measurement of GALK activity is crucial for diagnosing galactosemia subtypes.
- Existing radioisotopic assays require specialized facilities and are less accessible.
Purpose of the Study:
- To develop and validate a novel, non-radioisotopic assay for erythrocyte galactokinase activity.
- To adapt high-performance liquid chromatography (HPLC) for quantifying galactose 1-phosphate (Gal-1-P).
- To assess the suitability of this assay for neonatal screening follow-up.
Main Methods:
- Erythrocyte lysates were incubated with galactose to produce galactose 1-phosphate (Gal-1-P).
- Gal-1-P was quantified using high-performance liquid chromatography (HPLC).
- A post-column fluorometric detection method utilizing arginine for reducing sugars was employed.
Main Results:
- The HPLC assay demonstrated high sensitivity and accuracy, comparable to radioisotopic methods.
- The assay requires only a small blood sample volume.
- The method successfully quantified Gal-1-P, reflecting galactokinase activity.
Conclusions:
- The described HPLC-based assay provides a sensitive, accurate, and accessible method for determining erythrocyte galactokinase activity.
- This assay eliminates the need for radioisotopes, making it safer and more practical.
- It is well-suited as a follow-up diagnostic test in neonatal screening programs for galactosemia.