Urinary Acylglycine Acylglycine LC LC- -MSMS method MSMS method - - PowerPoint PPT Presentation

Urinary Urinary Acylglycine Acylglycine LC LC-

• MSMS method

MSMS method Analytical concerns & clinical use Analytical concerns & clinical use

R.G.Edwards R.G.Edwards

Acylglycine Acylglycine formation formation

• Glycine

Glycine-

• N

N-

• acylase

acylase

• Acyl

Acyl-

• CoA

CoA esters conjugated esters conjugated glycine ( glycine (Crn Crn, , GluN GluN) )

• Polar compounds urine

Polar compounds urine excreted in urine excreted in urine

• Acylglycines

Acylglycines reflect reflect mitochondrial acyl mitochondrial acyl-

• CoA

CoA esters esters

• Acyl

Acyl-

• CoA

CoA dehydrogenases dehydrogenases

• Leu

Leu/ Ile/ Val/ Lys catabolism / Ile/ Val/ Lys catabolism

Propionylglycine Propionylglycine Alloisoleu Suberylglycine Suberylglycine 3 3-

• Me

Me crotonylglycine crotonylglycine

Biosynthesis of Biosynthesis of Acylglycines Acylglycines

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

• Hexanoylglycine

Hexanoylglycine 13

13C

C2

2 (IS)

(IS)

• 9

9 acylglycines acylglycines; propionyl, ; propionyl, tigly tigly, 3 , 3-

• methylcrotonyl,

methylcrotonyl, isovaleryl isovaleryl, , 2 2-

• methylbutyryl,

methylbutyryl, hexanoyl hexanoyl, , octanoyl

• ctanoyl,

, phenylpropionyl phenylpropionyl, , suberyl suberyl

• Current use; NNS MCAD urinary hexanoylglycine

Current use; NNS MCAD urinary hexanoylglycine

Acylglycine Acylglycine excretion excretion

GCDH (decompensated) ~ 202 Glutarylglycine MADD ~ 144 Isobutyryl SCAD, MCAD, MADD ~ 144 Butyryl 75 174.2 C13 Hexanoyl MCAD, {MCT feeds} 74 230.2 Suberyl MCAD 74 206.2 3-phenylpropionyl MCAD, MADD 74 200.3 Octanoyl MCAD, MADD, PCC, BKT, {Valp} 74 172.2 Hexanoyl MADD, SBCAD 74 158.0 2-methylbutyryl IVD, MADD, {EMA-A}, {Valp} 74 158.0 Isovaleryl HMGCL, 3MCC, HLCS, {BKT}, {thiamine def} 74 156.2 3-methylcrotonyl PCC, HLCS, BKT 112 156.1 Tigly (2-mecrotonyl) PCC, MUT, HLCS 74 130.1 Propionyl Enzyme deficiency Daughter ion m/z Parent ion m/z Urine Glycine

3 3-

• Methylcrotonylglycine excretion

Methylcrotonylglycine excretion

MCAD MADD PCC b-KT 3MCC Thiamine def Biotinidase IVD

Leucine Leucine catabolism catabolism

*

? TG

Medium Acyl Medium Acyl-

• CoA

CoA Dehydrogenase deficiency Dehydrogenase deficiency

PG TG 3-MCG IVG 2-MBG HG OG PPG SG

MADD & MCAD MADD & MCAD

PG PG TG TG 3MCG 3MCG IVG 2MBG IVG 2MBG HG HG OG OG PPG PPG SG SG

Ile/Leu catabolism Riboflavin responsive can normalise

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

Isobutyryl CoA CoA dehydrogenase dehydrogenase – – isobutyrylglycine isobutyrylglycine

• EMA encephalopathy

EMA encephalopathy – – isovalerylglycine isovalerylglycine (EMA) (EMA) Elevated C5 acylcarnitine > Elevated C5 acylcarnitine >

• IVD

IVD -

• isovalerylglycine

isovalerylglycine

• SBCAD

SBCAD – – 2 2-

• methylbutyrylglycine (2

methylbutyrylglycine (2-

• EHA)

EHA)

• Equivocal Organic acids &

Equivocal Organic acids & acyls acyls (? (?acylgly acylgly detected) detected)

• ‘

‘Quick screen method Quick screen method’ ’ Pitt et al. Negative ion method; additional Pitt et al. Negative ion method; additional MRM orotic acid, MRM orotic acid, sulphocysteine sulphocysteine etc. etc.