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Introduction

Glu-1-P. >. GALT. UDP Gal. Gal-1-P. UDPGlc. UDPGlc dehydrogenase. UDPGlc. UDP-glucuronate. NAD +. NADH. Introduction. Fluorescent Assay for GALT Activity. GALT activity is determined by how much UDPGlc is consumed during the assay.

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Introduction

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  1. Glu-1-P > GALT UDPGal Gal-1-P UDPGlc UDPGlc dehydrogenase UDPGlc UDP-glucuronate NAD+ NADH Introduction • Fluorescent Assay for GALT Activity GALT activity is determined by how much UDPGlc is consumed during the assay. The consumption of UDPGlc is determined by the difference of UDPGlc amounts before and after the incubation by a fluorescent assay (shown below). Ref: Clin Chim Acta 1966;13(3):369-379

  2. Introduction (cont) • Radioactive Assay for GALT Activity Glu-1-P > GALT UDPGal* Gal*-1-P UDPGlc Gal*: 14C radioactive isotope labeled galactose In the radioactive assay, the enzymatic product UDPGal* is separated from the Gal*-1-P by paper chromatography or ion exchange liquid chromatography, and the paper segments or eluent fractions are counted for radioactivity to determine what percentage of Gal*-1-P is converted to UDPGal* to determine GALT activity Ref: Clin Chim Acta 1967;15:489 –92.

  3. Introduction (cont) • Shortcomings of Fluorescent Assay • Non-specificity due to non-GALT mediated fluorescence • Not sensitive (incapable of measuring low enzyme activity) • Require exogenous enzyme, which increases the complexity of the assay and also the burden of quality control • Shortcomings of Radioactive Assay • Post-incubation sample preparation is laborious • Not very specific • Sensitivity issue, having difficulty in measuring low GALT activity

  4. Question • Radioactive assays are very sensitive in general; why does the radioactive GALT assay have a sensitivity issue? How does dialysis of the red blood cell lysate improve the assay sensitivity so the low GALT activity can be measured? Ref: Clin Chim Acta 1995;235:125–36.

  5. > Glu-1-P GALT UDP-Gal* Gal*-1-P UDPGlu Materials and Methods • Reaction in LC-MS/MS GALT Assay Quantitated by LC-MS/MS Quantitated by LC-MS/MS Gal*: 13C6 labeled galactose.

  6. Materials and Methods (cont) • MS/MS settings

  7. Materials and Methods (cont) • LC-MS/MS Chromatography

  8. Materials and Methods (cont) • Blood Samples Used in Assay Comparison • 15 samples collected in Metabolism Clinic of Children’s Hospital Boston (CHB) in a course of 3 months • Samples include 6 classic galactosemia, 7 variants, and 2 carriers • Blood Samples Used in Assay Application • 33 patients presumed to be classic galactosemia were arranged to visit CHB on the same day • Blood samples were drawn on the same day and all samples assayed for GALT activity within 48 hours after blood collection

  9. Question • Why didn’t the authors use commercially available13C2 labeled Gal-1-P in their LC-MS/MS based GALT assay? • Why would the authors be willing to pay extra money to have someone custom synthesize 13C6 labeled Gal-1-P and use it in the assay?

  10. Results • GALT Activity in Relation to UDPGlc Concentration

  11. Results (cont) • Recovery and Imprecision of the LC-MS/MS Assay

  12. Results (cont) • GALT Activity in Controls and Patients with Galactosemia

  13. Results (cont) • Comparison Between Fluorescent and LC-MS/MS Assay • Fluorescent and LC-MS/MS assay showed good correlation in general, with the former 20% higher than the latter. • When GALT activity is very low or absent, fluorescent assay is not reliable

  14. Question • Why does the effect of UDPGlc concentration on GALT activity not follow typical Michaelis-Menton kinetics?

  15. Conclusion • Features of the LC-MS/MS Assay • Simple post-incubation sample preparation • Excellent sensitivity with LOQ of 0.2% of normal enzyme activity • Wide linearity range to allow quantification of both low enzyme activity and normal enzyme activity • Unmatched specificity: each compound is identified by LC retention time, parent ion m/z and daughter ion m/z • No exogenous enzyme is needed

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