Urinary Acylglycine Acylglycine LC LC- -MSMS method MSMS method Urinary Analytical concerns & clinical use Analytical concerns & clinical use R.G.Edwards R.G.Edwards
Acylglycine formation formation Acylglycine • Glycine Glycine- -N N- -acylase acylase • • Acyl Acyl- -CoA CoA esters conjugated esters conjugated • glycine (Crn Crn, , GluN GluN) ) glycine ( • Polar compounds urine Polar compounds urine • excreted in urine excreted in urine • Acylglycines Acylglycines reflect reflect • mitochondrial acyl- -CoA CoA mitochondrial acyl esters esters • Acyl Acyl- -CoA CoA dehydrogenases dehydrogenases • • Leu Leu/ Ile/ Val/ Lys catabolism / Ile/ Val/ Lys catabolism •
Biosynthesis of Acylglycines Acylglycines Biosynthesis of Alloisoleu 3 3- -Me Me crotonylglycine crotonylglycine Propionylglycine Propionylglycine Suberylglycine Suberylglycine
Method Characteristics Method Characteristics • Micromass Micromass Quattro Micro, Waters 2795 HPLC Quattro Micro, Waters 2795 HPLC • • Negative ion Negative ion • • Urine Urine ‘ ‘Dilute & shoot Dilute & shoot’ ’, 20 , 20 µ µ L L • • 2.5 min, isocratic 80% 2.5 min, isocratic 80% AcN AcN • 13 C • Hexanoylglycine Hexanoylglycine 13 C 2 (IS) • 2 (IS) • 9 9 acylglycines acylglycines; propionyl, ; propionyl, tigly tigly, 3 , 3- -methylcrotonyl, methylcrotonyl, isovaleryl isovaleryl, , • 2- -methylbutyryl, methylbutyryl, hexanoyl hexanoyl, , octanoyl octanoyl, , phenylpropionyl phenylpropionyl, , 2 suberyl suberyl • Current use; NNS MCAD urinary hexanoylglycine Current use; NNS MCAD urinary hexanoylglycine •
Acylglycine excretion excretion Acylglycine Parent ion Daughter ion Urine Glycine Enzyme deficiency m/z m/z Propionyl PCC, MUT, HLCS 130.1 74 Tigly (2-mecrotonyl) 156.1 112 PCC, HLCS, BKT 3-methylcrotonyl 156.2 74 HMGCL, 3MCC, HLCS, {BKT}, {thiamine def} Isovaleryl IVD, MADD, {EMA-A}, {Valp} 158.0 74 2-methylbutyryl 158.0 74 MADD, SBCAD Hexanoyl 172.2 74 MCAD, MADD, PCC, BKT, {Valp} Octanoyl 200.3 74 MCAD, MADD 3-phenylpropionyl 206.2 74 MCAD Suberyl 230.2 74 MCAD, {MCT feeds} C13 Hexanoyl 174.2 75 Butyryl 144 ~ SCAD, MCAD, MADD Isobutyryl 144 ~ MADD Glutarylglycine GCDH (decompensated) 202 ~
3- -Methylcrotonylglycine excretion Methylcrotonylglycine excretion 3 3MCC ? TG * b-KT Leucine Leucine catabolism catabolism Thiamine Biotinidase MCAD MADD PCC def IVD
Medium Acyl- -CoA CoA Dehydrogenase deficiency Dehydrogenase deficiency Medium Acyl SG PPG IVG HG OG 2-MBG PG TG 3-MCG
MADD & MCAD MADD & MCAD Riboflavin responsive can normalise HG IVG OG 2MBG PPG SG 3MCG PG TG PPG HG SG Ile/Leu catabolism IVG OG 2MBG TG 3MCG PG
Analytical issues Analytical issues • Separation of IVG/ 2MBG (TG/3MBG) Separation of IVG/ 2MBG (TG/3MBG) • • Deterioration of suberylglycine Deterioration of suberylglycine • • Alternative source of pure standards ( Alternative source of pure standards (synth synth SBC) SBC) • • Appropriate control material Appropriate control material - - ? Spiked, patient ? Spiked, patient • • Mixed standards for all or some Mixed standards for all or some glycines glycines • • Addition of Addition of butyryl butyryl/ / isobutyryl isobutyryl glycine glycine •
Clinical & Diagnostic Use Clinical & Diagnostic Use Elevated C4 acylcarnitine Elevated C4 acylcarnitine • • SCAD SCAD – – { {butyrylglycine butyrylglycine} (EMA +/ } (EMA +/- - MeSuccinate MeSuccinate) ) • Isobutyryl CoA CoA dehydrogenase dehydrogenase – – isobutyrylglycine isobutyrylglycine • Isobutyryl • EMA encephalopathy – – isovalerylglycine isovalerylglycine (EMA) (EMA) • EMA encephalopathy Elevated C5 acylcarnitine > Elevated C5 acylcarnitine > • IVD - - isovalerylglycine isovalerylglycine • IVD • SBCAD – – 2 2- -methylbutyrylglycine (2 methylbutyrylglycine (2- -EHA) EHA) • SBCAD • Equivocal Organic acids & acyls acyls (? (?acylgly acylgly detected) detected) • Equivocal Organic acids & • ‘Quick screen method Quick screen method’ ’ Pitt et al. Negative ion method; additional Pitt et al. Negative ion method; additional • ‘ MRM orotic acid, sulphocysteine sulphocysteine etc. etc. MRM orotic acid,
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