1. © Copyright 2009 by the American Association for Clinical Chemistry Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry Y. Li, A.S. Ptolemy, L. Harmonay, M. Kellogg, and G.T. Berry May 2010 http://www.clinchem.org/cgi/reprint/56/5/772 © Copyright 2010 by the American Association for Clinical Chemistry Journal Club

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Fluorescent Assay for GALT Activity > Gal-1-P UDPGal GALT UDPGlc Glu-1-P UDPGlc UDPGlc dehydrogenase UDP-glucuronate NAD + NADH 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

3. © Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont)  Radioactive Assay for GALT Activity > Gal*-1-P UDPGal* GALT UDPGlc Glu-1-P 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.

4. © Copyright 2009 by the American Association for Clinical Chemistry 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

5. © Copyright 2009 by the American Association for Clinical Chemistry Question Ref: Clin Chim Acta 1995;235:125–36.  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?

6. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods  Reaction in LC-MS/MS GALT Assay > Gal*-1-P UDP-Gal* GALT UDPGlu Glu-1-P Quantitated by LC-MS/MS Gal*: 13 C 6 labeled galactose.

7. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  MS/MS settings Analyte MS/MS transition Cone (Volts) Collision Energy (ev) Parent ion (m/z) Daughter ion (m/z) Glu-1-P a 25979 d 30 [ 13 C 6 ]-Glu-1-P b 2657930 UDPGal/UDPGlc c 565323 e 3425 [ 13 C 6 ]- UDPGal5713233425 a Other hexose-1-phosphates and hexose-6-phosphates, other than Glu-1-P, are also detected by the same MS/MS transition, though may not be in their optimal MS settings; b enzyme substrate [ 13 C 6 ]-Gal-1-P is also detected by the same mass transition; c UDPGal/UDPGlc have identical retention time and the same mass transitions, and therefore cannot be distinguished from each; d negative ion PO 3 - ; e negative ion of uridine monophosphate.

8. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  LC-MS/MS Chromatography

9. © Copyright 2009 by the American Association for Clinical Chemistry 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

10. © Copyright 2009 by the American Association for Clinical Chemistry Question  Why didn’t the authors use commercially available 13 C 2 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 13 C 6 labeled Gal-1-P and use it in the assay?

11. © Copyright 2009 by the American Association for Clinical Chemistry Results  GALT Activity in Relation to UDPGlc Concentration

12. © Copyright 2009 by the American Association for Clinical Chemistry Results (cont)  Recovery and Imprecision of the LC-MS/MS Assay Amount of Hgb(mg) (Relative Amount) AccuracyImprecision (%CV) Measured Enzyme Activity a Recovery (%) Intra- assay Inter- assay 0.56 (100% b )21.8100 b 2.14.5 0.14 (25%)5.397.22.56.7 0.28 (5%)1.0394.8%4.68.2 0.00112 (0.2%)0.0492.1%9.713.2 a unit is  mol/g Hgb/hour; b 100% was arbitrarily assigned.

13. © Copyright 2009 by the American Association for Clinical Chemistry Results (cont) SubjectsSample No. GALT Activity (µmol/g Hgb/hour) MeanRangeSD Normal Control7123.8 15.6- 30.53.8 D/G variant13.6N/A Classical galactosemia*33NDN/A ND: Non-detectable with LOD of 0.07% of normal controls (or 0.01μmol/g Hgb/hr). N/A: not applicable.  GALT Activity in Controls and Patients with Galactosemia

14. © Copyright 2009 by the American Association for Clinical Chemistry 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

15. © Copyright 2009 by the American Association for Clinical Chemistry Question  Why does the effect of UDPGlc concentration on GALT activity not follow typical Michaelis-Menton kinetics?

16. © Copyright 2009 by the American Association for Clinical Chemistry 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