MSMS measurement of new biomarkers of liver and kidney functions and brain damage

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1 MSMS measurement of new biomarkers of liver and kidney functions and brain damage Asian Pacific Conference of Chromatography & Mass Spectrometry th -16 th January 2010 R Neil Dalton & Charles Turner WellChild Laboratory King s College, London/ Evelina Children s Hospital

2 ADMA asymmetric dimethylarginine SDMA symmetric dimethylarginine NAA N-acetylaspartate

3 Dimethylarginines Synthesised during posttranslational methylation of arginine residues Primarily nuclear proteins Transcriptional control? Released by proteolysis

4 ADMA endogenous inhibitor of nitric oxide synthase SDMA physiologically inactive metabolised by dimethylarginine diaminohydrolase (DDAH) end-product of metabolism marker of endothelial dysfunction increased in renal disease cleared by the kidney The cardiovascular risk marker

5 First measured clinically by Carnegie et al (1977) urine ADMA and SDMA cation exchange chromatography with ninhydrin detection range of pathological conditions investigated liver disease increased

6 No clinical utility without analytical simplicity Classical measurement not simple!

7 Classical measurement 200μl 5ml plasma solid phase extraction drying OPD derivatisation drying 15min ion pair HPLC fluorescence detection

8

9 Is rapid isotope dilution MSMS of dimethylarginines, particularly ADMA, possible? Isobaric Fragmentation specificity? Previous attempts unsuccessful Implied: even with MSMS, likely that derivatisation and significant chromatography essential

10 Fragmentation specificity +MS2 (203.00) CE (30): 26 MCA scans from Sample 1 (TuneSampleName) of dimethylarginine_initproduct_pos.wiff (Turbo Spray) Max. 6.4e5 cps. +MS2 (203.08) CE (30): 26 MCA scans from Sample 1 (TuneSampleName) of sdma_initproduct_pos.wiff (Turbo Spray) Max. 6.7e5 cps. 6.4e5 6.0e e5 6.5e e5 5.5e e5 5.0e5 5.0e5 4.5e5 4.5e5 4.0e5 4.0e5 3.5e5 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 3.0e5 2.5e e5 m/z 46.2 m/z e5 1.0e e5 5.0e m/z, amu e m/z, amu ADMA product ion (m/z 203.1) scan SDMA product ion (m/z 203.1) scan

11 sample preparation 50μl plasma 50μl stable isotope internal standards 50pmol each of 2 H 6 -ADMA and 2 H 6 -SDMA 200μl acetonitrile Mix, centrifuge 14,000 rpm for 2min, transfer supernatant to 96 deep well plate for MSMS analysis

12 5μl supernatant automatically injected onto a teicoplanin guard column with solvent flowing at 250μl/min Solvent - acetonitrile:water 1:1 with 25% formic acid MSMS - SCIEX API4000 Positive ion MRMs m/z 203.1/46.2 & 203.1/172.2 Stable isotopes m/z 209.1/52.2 & 209.1/175.2

13 ADMA TIC: from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 1.4e4 cps e4 1.0e4 500 TIC XIC of +MRM (2 pairs): Exp 1, 203.1/46.2 amu from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray), S... Max cps XIC of +MRM (2 pairs): Exp 2, 203.1/172.1 amu from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray),... Max cps ADMA SDMA Analyte Conc. / IS Conc SDMA Analyte Conc. / IS Conc.

14 Signal:noise ratios at 0.25µmol/l: ADMA 20 SDMA 100 Cross-talk between ADMA, SDMA, and their standards <0.1% Mean recovery (n=20): ADMA 0.5µmol/l, 92% SDMA 1µmol/l, 100%

15 Intra-assay variation (n=10) (mean, coefficient of variation %): ADMA 0.37µmol/l, 2.1% SDMA 0.44µmol/l, 3.5% Inter-assay variation (n=41) (mean, coefficient of variation %): ADMA 0.39µmol/l, 7.4%; 1.15µmol/l, 5.8%; 3.96µmol/l, 5.1% SDMA 0.51µmol/l, 6.4%; 1.39µmol/l, 4.6%; 4.35µmol/l, 6.6%

16 Samples from large trial looking for biomarkers of preeclampsia Serial samples at 12, 16, 20, and 24 weeks outcomes determined No change in plasma ADMA and concentrations and values c.20% of those reported in Savvidou et al

17 Plasma ADMA as a biomarker Predictor of pre-eclampsia? Marker of endothelial function? The cardiovascular risk marker? Increased in renal failure Prognostic of rate of decline of renal function Plasma SDMA as a biomarker?

18 TIC: from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 1.4e4 cps. TIC: from Sample 52 (L King 10/3/95) of ADMASV5 GFR 2004-Sept-29.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 2.5e4 cps. 1.4e4 1.0e4 TIC e4 2.0e4 1.5e4 TIC e XIC of +MRM (2 pairs): Exp 1, 203.1/46.2 amu from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray), S... Max cps ADMA XIC of +MRM (2 pairs): Exp 2, 203.1/172.1 amu from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray),... Max cps XIC of +MRM (2 pairs): Exp 1, 203.1/46.2 amu from Sample 52 (L King 10/3/95) of ADMASV5 GFR 2004-Sept-29.wiff (Turbo Spray), S... Max cps ADMA XIC of +MRM (2 pairs): Exp 2, 203.1/172.1 amu from Sample 52 (L King 10/3/95) of ADMASV5 GFR 2004-Sept-29.wiff (Turbo Spray),... Max. 1.8e4 cps e SDMA e4 1.0e4 500 SDMA Control subject Patient with renal failure

19 Plasma ADMA as a biomarker Predictor of pre-eclampsia? Marker of endothelial function? The cardiovascular risk marker? Increased in renal failure Prognostic of rate of decline of renal function Plasma SDMA as a biomarker renal function?

20 First measured clinically by Carnegie et al (1977) range of pathological conditions investigated liver disease increased Teerlink et al Plasma ADMA as a biomarker of liver disease?

21 TIC: from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 1.4e4 cps. TIC: from Sample 28 (hepucl18) of ADMASV5 hepaticucl 2004-Oct-14.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 3.5e4 cps. 1.4e4 1.0e4 TIC e4 3.0e4 2.0e4 TIC e XIC of +MRM (2 pairs): Exp 1, 203.1/46.2 amu from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray), S... Max cps ADMA XIC of +MRM (2 pairs): Exp 2, 203.1/172.1 amu from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray),... Max cps XIC of +MRM (2 pairs): Exp 1, 203.1/46.2 amu from Sample 28 (hepucl18) of ADMASV5 hepaticucl 2004-Oct-14.wiff (Turbo Spray)... Max cps ADMA XIC of +MRM (2 pairs): Exp 2, 203.1/172.1 amu from Sample 28 (hepucl18) of ADMASV5 hepaticucl 2004-Oct-14.wiff (Turbo Spray)... Max. 2.0e4 cps e SDMA e4 1.0e4 SDMA Control subject Patient with chronic liver failure

22 Mean Median Range Number (µmol/l) (µmol/l) (µmol/l) Control a 1 -anti-trypsin deficiency Autoimmune hepatitis Alagille s syndrome* Acute liver failure Choledocal cyst Biliary atresia* Giant cell hepatitis Glycogen storage disease Haemangioma* Hepatitis B infection Hepatitis C infection Neonatal hepatitis* Post transplant, no rejection Progressive familial IH cholestasis Wilson s disease *P<5

23 AH Adult patients with chronic liver cirrhosis Mookerjee et al, 2007

24 Liver transplantation Adult patients pre and 1h post transplantation ADMA (um) 2.0 p< Mookerjee et al, 2007 Healthy Volunteer Pre OLT Post OLT

25 Liver transplantation Adult patient pre, during and post transplantation ADMA (um) Pre-anhepatic Anhepatic (T=0) Reperfusion: Portal Venous Reperfusion: Arterial Post-OLT (2hr) Post OLT (4 hr) 1 0 Mookerjee et al, Time (hr)

26 Plasma ADMA as a biomarker Carnegie and Teerlink s observations confirmed Plasma ADMA increases in liver disease Liver major site of DDAH activity Plasma ADMA a measure of effective hepatic blood flow Secondarily dependent on rate of proteolysis

27 Are there consequences of high plasma ADMA? Inhibition of NOS? Endothelial function? Cardiovascular risk? Progression of renal disease?

28 Plasma SDMA as a glomerular filtration rate marker? TIC: from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 1.4e4 cps. TIC: from Sample 52 (L King 10/3/95) of ADMASV5 GFR 2004-Sept-29.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 2.5e4 cps. 1.4e4 1.0e4 TIC e4 2.0e4 1.5e4 TIC e XIC of +MRM (2 pairs): Exp 1, 203.1/46.2 amu from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray), S... Max cps ADMA XIC of +MRM (2 pairs): Exp 2, 203.1/172.1 amu from Sample 7 (new LQC) of ADMASV5 UCLhepatic 2004-Oct-21.wiff (Turbo Spray),... Max cps XIC of +MRM (2 pairs): Exp 1, 203.1/46.2 amu from Sample 52 (L King 10/3/95) of ADMASV5 GFR 2004-Sept-29.wiff (Turbo Spray), S... Max cps ADMA XIC of +MRM (2 pairs): Exp 2, 203.1/172.1 amu from Sample 52 (L King 10/3/95) of ADMASV5 GFR 2004-Sept-29.wiff (Turbo Spray),... Max. 1.8e4 cps e SDMA e4 1.0e4 500 SDMA Control subject Patient with renal failure

29 Routine Inutest GFR in 257 children Plasma SDMA (µmol/l) y = 219x R 2 = Inulin GFR (ml/min/1.73m2) MSMS plasma creatinine (µmol/l) y = x R 2 = Inulin GFR (ml/min/1.73m2) Plasma SDMA v Inutest GFR Plasma creatinine v Inutest GFR

30 Routine Inutest GFR, data in children with GFR >90 ml/min/1.73m y = -014x R 2 = 063 MSMS plasma creatinine (µmol/l) y = x R 2 = Age (years) Age (years) Plasma SDMA v age Plasma creatinine v age

31 Routine Inutest GFR in 257 children y = x 50 y x R 2 R= Inulin GFR (ml/ min/ 1.73m2) egfr (ml/min/1.73m 2 ) y = x R 2 = Inulin GFR (ml/min/1.73m 2 ) SDMA egfr (46000/SDMA (nmol/l)) Creatinine egfr (31*ht(cm)/creatinine (μmol/l)

32 78 children with chronic liver disease 1.4 Correlation with cystatin C, R 2 = patients with CKD (age 55-65) 101 age & sex matched controls plasma SDMA ( mol/l) ( y = 0.535x R 2 = Correlation with cystatin C, R 2 = cystatin C (mg/l)

33 Serum SDMA (µmol/l) y = x R 2 = GFR (ml/min/1.73m2) Sensitivity (true positives) Specificity (false positives) No discrimination SDMA (µmol/l) MSMS creatinine (µmol/l) Creatinine mg/dl serum creatinine (µmol/l) y = 2961x R 2 = Sensitivity (true positives) No discrimination MS Creat egfr SDMA GFR (ml/min/1.73m2) Specificity (false positives) Plasma creatinine and SDMA comparison with formal GFR in 309 adult patients

34 Plasma SDMA as a biomarker Meta-analysis of SDMA as a biomarker of GFR Keilstein et al, 2007 Plasma SDMA a measure of glomerular filtration rate Independent of body size Secondarily dependent on rate of proteolysis

35 NAA N-acetylaspartate is the major organic component of brain tissue Increased concentrations measured on urine organic acid analysis in Canavan s disease (aspartoacylase deficiency) Increased excretion in non-accidental injury/subdural haemorrhage Plasma NAA as a biomarker of brain injury?

36 NAA NAA acquisition included with XIC of +MRM (2 pairs): Exp 1, 203.1/46.2 amu from Sample 309 (NAA QC med) of NAS GFR 2008-September-09.wiff (Turbo Spray) Max. 3.1e4 cps. 6.6e4 5.0e XIC of +MRM (2 pairs): Exp 2, 203.1/172.1 amu from Sample 309 (NAA QC med) of NAS GFR 2008-September-09.wiff (Turbo Spray) Max. 3.0e4 cps. 6.7e4 5.0e4 ADMA SDMA XIC of +MRM (3 pairs): Exp 3, 176.3/88.3 amu from Sample 309 (NAA QC med) of NAS GFR 2008-September-09.wiff (Turbo Spray), e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 Max cps XIC of +MRM (3 pairs): Exp 3, 176.3/88.3 amu from Sample 309 (NAA QC med) of NAS GFR 2008-September-09.wiff (Turbo Spray),... Max cps. 2.0e5 1.0e5 NAA e5 8.0e4 6.0e4 4.0e4 2.0e NAA Blue m/z 176.3/88.3 Red m/z 179.3/91.3 Green m/z 179.3/89.3

37 /NAA /NAA sample preparation 50μl plasma 50μl stable isotope internal standards 50pmol each of 2 H 6 -ADMA, 2 H 6 -SDMA, 2 H 3 -NAA, and 13 C 2, 15 N-NAA 200μl acetonitrile Mix, centrifuge 14,000 rpm for 2min, transfer supernatant to 96 deep well plate for MSMS analysis

38 /NAA 5μl supernatant automatically injected onto a Chirobiotic T, 100*2.1mm column with guard column with solvent flowing at 250μl/min Solvent - acetonitrile:water 1:1 with 25% formic acid MSMS - SCIEX API5000 Positive ion MRMs m/z 203.1/46.2, 203.1/172.2, and 176.3/88.3 Stable isotopes m/z 209.1/52.2, 209.1/175.2, 179.3/91.3, and 179.3/89.3

39 NAA XIC of +MRM (3 pairs): Exp 3, 176.3/88.3 amu from Sample 308 (NAA QC low) of NAS GFR 2008-September-09.wiff (Turbo Spray),... Max cps. XIC of +MRM (3 pairs): Exp 3, 176.3/88.3 amu from Sample 308 (NAA QC low) of NAS GFR 2008-September-09.wiff (Turbo Spray),... Max cps. 2.3e4 2.2e4 4.0e4 2.0e4 1.8e4 3.5e4 1.6e4 3.0e4 1.4e4 2.5e4 1.2e4 1.0e4 2.0e e e XIC of +MRM (3 pairs): Exp 3, 176.3/88.3 amu from Sample 310 (NAA QC high) of NAS GFR 2008-September-09.wiff (Turbo Spray),... Max. 1.8e4 cps. XIC of +MRM (3 pairs): Exp 3, 176.3/88.3 amu from Sample 310 (NAA QC high) of NAS GFR 2008-September-09.wiff (Turbo Spray),... Max. 1.8e4 cps. 2.5e4 2.4e4 2.2e4 4.5e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e e4 3.5e4 3.0e4 2.5e4 1.0e4 2.0e e e m/z 176.3/88.3 & 179.3/91.3 m/z 176.3/88.3 & 179.3/89.3

40 /NAA 22 neonates with suspected hypoxic ischaemic encephalopathy (HIE) Plasma sample within first 6h post partum Sequential sampling in patient with obvious clinical HIE Sequential creatinines (μmol/l) Analyte (nmol/l) ADMA SDMA NAA Mean Median Min Max Day Day Day , 265, 211, 160

41 NAA Plasma NAA as a biomarker Preliminary data but requires further validation Plasma NAA a measure of brain injury?

42 /NAA Rapid, high sensitivity, MSMS analysis of 3 new biomarkers Plasma ADMA a measure of effective hepatic blood flow Plasma SDMA a measure of glomerular filtration rate Independent of body size Plasma NAA a measure of brain injury?

43 Acknowledgements Charles Turner Beth Mallard, Nadia Wahbi Stavros Loukogeorgakis, Patrick Vallance Paul Cheeseman, Giorgina Vergani Nathan Davis, Stephen Hodges, Raj Mookerjee, Rajiv Jalan Caroline Booth, Emma Rigby, Sue Rigden David Williams, Martin Landray Carlo Donadio The WellChild Trust Guy s & St Thomas Charity Guy s & St Thomas NHS Foundation Trust