Casetta, B.

Donati, M. A.

Hirano, I.

La Marca, Giancarlo

Malvagia, S.

Pasquini, E.

Pela, Ivana

Zammarchi, Enrico

English

Florence Research

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2006; 41: 586–592Published online 23 February 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.1013Pre- and post-dialysis quantitative dosage of thymidinein urine and plasma of a MNGIE patient by usingHPLC-ESI-MS/MSG. la Marca,1∗ S. Malvagia,1 B. Casetta,2 E. Pasquini,1 I. Pela,1 M. Hirano,3 M. A. Donati1 andE. Zammarchi11 Metabolic and Neuromuscular Unit, A. Meyer Children’s Hospital, Department of Paediatrics, University of Florence, Florence, Italy2 Applied Biosystems, Monza, Italy3 Department of Neurology, Columbia University College of Physicians and Surgeons, New York, USAReceived 13 July 2005; Accepted 12 January 2006Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disordercharacterized by severe gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheralneuropathy and leukoencephalopathy. The disease is due to a thymidine phosphorylase defect. Thisenzyme catalyses the phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. For thisreason, increased levels of thymidine in plasma and urine are found in MNGIE patients. Haemodialysiscan reduce circulating plasma thymidine levels and can be beneficial in some MNGIE patients. Wedeveloped a fast analytical method based on HPLC-ESI-MS/MS capable of identifying pyrimidinenucleotides (thymine, cytosine, uracil) and nucleosides (thymidine, citidine, uridine) in plasma and urineafter direct dilution of the samples without pre-treatment.In the patient studied, we observed a significant reduction of plasmatic and urinary thymidine levelsduring and after dialysis. However, we noted a progressive reduction of the initial thymidine levelafter some dialytic trials. This method will be useful not only for thymidine level follow-up duringdialysis in MNGIE patients but also for the improvement of the diagnosis or diagnostic suspect in otherpyrimidine defects such as dihydropyrimidine dehydrogenase deficiency, dihydropyrimidinase deficiencyand ureidopropionase deficiency. Copyright  2006 John Wiley & Sons, Ltd.KEYWORDS: MNGIE; thymidine; pyrimidine; dialysis; tandem mass spectrometryINTRODUCTIONMitochondrial neurogastrointestinal encephalomyopathy(MNGIE) is an autosomal recessive disorder defined clini-cally by severe gastrointestinal dysmotility, cachexia, ptosis,ophthalmoparesis, peripheral neuropathy and leukoence-phalopathy.1 Skeletal muscle biopsies of patients haverevealed abnormalities of mtDNA and mitochondrial res-piratory chain enzymes.2 The disease is caused by thethymidine phosphorylase defect. This enzyme catalysesphosphorolysis of thymidine to thymine and deoxyri-bose 1-phosphate.3 For this reason, MNGIE patients showincreased levels of thymidine in plasma and urine. The genehas been cloned and mapped by Nishino et al. in 1999.4Some authors hypothesized that mitochondrial deoxyri-bonucleotide triphosphate (dNTP) pool imbalance is likelyresponsible for mutagenesis in mitochondrial DNA.5Hemodialysis can reduce circulating plasma thymidinelevels and can be beneficial in some MNGIE patients.6ŁCorrespondence to: G. la Marca, Department of Paediatrics,A. Meyer Children’s Hospital, Via Luca Giordano 13, 50132Florence, Italy. E-mail: g.lamarca@meyer.itIn recent years several methods for thymidine quan-titation have been developed using HPLC-UV or HPLC-MS/MS.6 – 8 We have developed a fast analytical methodbased on HPLC-ESI-MS/MS, capable of detecting a spec-trum of some pyrimidine metabolites (including thymidine)in plasma and urine. This method can be useful for theaccurate determination of the dosage of thymidine duringdialytic treatments in MNGIE patients.EXPERIMENTALMaterials and methodChemical standard cytosine, cytidine, uracil, uridine,thymine and thymidine were purchased from Sigma Aldrich(St Louis, USA). Labelled standard Me-13C-thymidine, uracil-1,3-15N2, thymine-2H4 and uridine-ribose-1-13C were pur-chased from Cambridge Isotopes Laboratories (Andover,MA, USA); cytosine-2,4-13C2,-15N3, cytidine-15N3 were pur-chased from Aldrich (St Louis, USA).Stock solutions (1–3 mM) were made in H2O C ammo-nium acetate 50 mM.All solvents were HPLC grade (Sigma Aldrich, St Louis,USA).Copyright  2006 John Wiley & Sons, Ltd.Pre- and post-dialysis thymidine in a MNGIE patient 587An Applied Biosystems-Sciex (Toronto, Canada) API4000 triple-quadrupole mass spectrometer equipped witha TurboIonSpray (TIS) source was employed in this study.The quantitation experiments were performed using a seriesPE 200 micropump (Perkin Elmer, USA) coupled to a PEwell-plate autosampler, both fully controlled by the API4000 data system (Analyst 1.4.1 software).The TIS source was operated in positive ion mode at350 °C. Collision-activated dissociation (CAD) MS/MS wasperformed in the LINAC Q2 collision cell, operating withnitrogen at 10 mTorr pressure as the collision gas.The declustering potentials (DP) for all standards (chem-icals and labelled) were in the range of 30–80 V, the collisionenergies (CE) in the range of 12–27 V and the collision exitpotential (CXP) in the range of 5–8 V (laboratory frame). MSand MS/MS spectra were collected in continuous flow modeby connecting the infusion pump directly to the TIS source.A standard solution of 5 ng/µl of each chemical and labelledstandards in H2O C Ammonium Acetate 50 mM was infusedat 5 µl/min. The HPLC method had a gradient starting from100% H2O C ammonium acetate 50 mM (solvent A) up to100% MeOH C solvent A 50 : 50 (solvent B).The column was a Luna C18, 5 µm, 150 mm ð 3 mm(Phenomenex, USA) and the flow rate was 400 µl/min.The injection loop was 40 µl and the injection volume was3 µl.Table 1. Declustering potentials, collision energies, collisionexit potentials and transitions of each compound in positiveESI modeCompoundDP(V)CE(V)CXP(V)Q1(Th)Q3(Th)Cytosine 51 27 8 112.1 95.0Cytosine-2,4-13C2,-15N3 51 27 8 117.1 100.0Cytidine 35 23 8 244.2 112.1Cytidine-15N3 35 23 8 247.2 115.1Uracil 80 24 5 113.1 70.0Uracil-1,3-15N2 80 24 5 115.1 71.0Uridine 30 15 6 245.2 113.1Uridine-ribose-1-13C 30 15 6 246.2 113.1Thymidine 30 12 8 243.2 127.1Me-13C-thymidine 30 12 8 244.2 128.1Thymine 80 24 6 131.1 114.1Thymine-2H4 80 24 6 127.1 110.1The acquired data were processed using the Analyst 1.4.1proprietary software including the ‘Explore’ option for chro-matographic and spectral interpretation. For quantitativedata, MS/MS spectra were collected operating in multiplereaction monitoring (MRM) mode for all compounds. MRM[Thymidine+H]+[13C-Thymidine+H]+[M-116+H]+[M-116+H]+Figure 1. Product ion scan of thymidine and 13C-thymidine.Copyright  2006 John Wiley & Sons, Ltd. J. Mass Spectrom. 2006; 41: 586–592588 G. la Marca et al.transitions, DP, CE and CXP for all pyrimidines are reportedin Table 1.The product ion scan spectra of thymidine and13C-thymidine are reported in Fig. 1.SamplesDietary and drug interferences are the major threat to thediagnosis of purine/pyrimidine defects, as many foods,beverages and drugs can contain, especially, purines. Wecollected the urine and plasma of healthy controls who wererecommended to avoid, 48 h before the collection, coffee,tea, chocolate, soft drinks, bananas, not-home-made sweets,liquorice, red meat and, when possible, antibiotics, aromat-ics (paracetamol, aspirin) and purine/pyrimidine analoguessuch as fluorouracil and allopurinol. The best condition wasTable 2. Intra-day reproducibility of pyrimidines at various concentrationsUnspiked urine (creatinine 3.59 mM)Thymidine Thymine Cytidine Cytosine Uridine UracilExpected value 1 7.5 12.2 0 1 19Average 0.95 7.56 12.43 0.00 0.97 18.7SD 0.12 0.44 0.96 – 0.1 1.07CV% 12.43 5.82 7.72 – 10.31 5.72Accuracy 95.00 100.80 101.89 – 97.00 98.42Spiked urine (creatinine 3.59 mM)Thymidine Thymine Cytidine Cytosine Uridine UracilExpected value 21.36 27.86 22.56 20.36 31.36 39.36Average 20.86 28.44 24.01 19.74 32.01 37.56SD 0.61 1.3 4.3 3.9 0.99 4.02CV% 2.92 4.57 17.91 19.76 3.09 10.70Accuracy 97.68 102.08 106.43 96.95 102.07 95.43Spiked urine (creatinine 2.11 mM)Thymidine Thymine Cytidine Cytosine Uridine UracilExpected value 10.03 15.44 18.08 10.11 10.96 24.47Average 9.45 13.95 19.04 8.87 11.13 25.29SD 0.41 4.6 2.6 1.6 0.32 2.1CV% 4.33 32.97 13.66 18.04 2.88 8.30Accuracy 94.2 90.35 105.31 87.73 101.55 103.35Spiked urine (creatinine 2.11 mM)Thymidine Thymine Cytidine Cytosine Uridine UracilExpected value 20.03 35.44 28.08 20.11 20.96 34.47Average 20.23 33.12 27.44 19.88 22.04 35.82SD 1.08 4.6 1.9 3.1 2.32 3.1CV% 5.31 13.89 6.92 15.59 10.53 8.65Accuracy 100.98 93.45 97.72 98.86 105.15 103.92Spiked urine (creatinine 0.44 mM)Thymidine Thymine Cytidine Cytosine Uridine UracilExpected value 11 16.94 15.28 10.01 10.13 21.11Average 11.29 17.12 16.02 9.91 11.01 20.63SD 0.73 2.7 1.5 1.1 0.57 1.71CV% 6.46 15.77 9.36 11.10 5.18 8.29Accuracy 102.61 101.06 104.84 99.00 108.69 97.73Spiked urine (creatinine 0.44 mM)Thymidine Thymine Cytidine Cytosine Uridine UracilExpected value 26 31.94 30.28 25.01 25.13 46.11Average 25.98 30.12 30.02 25.06 24.44 44.86SD 1.74 4 1.4 3.1 2.57 5.08CV% 6.69 13.28 4.66 12.37 10.52 11.32Accuracy 99.92 94.30 99.14 100.20 97.25 97.29Copyright  2006 John Wiley & Sons, Ltd. J. Mass Spectrom. 2006; 41: 586–592Pre- and post-dialysis thymidine in a MNGIE patient 589to perform analysis on urine collected for 24 h or, whennot possible, on urine randomly collected and normalizedto creatinine. Our reference value for thymidine in urinewas 0.09–1.34 µmol/mmol creatinine (5–95th percentiles,based on controls D 80). Plasma thymidine was normally notdetectable or was present only in traces.Sample preparationsUrine and plasma samples were diluted 1 : 20 with H2O Cammonium acetate 50 mM, added with labelled standards(conc. 100 µM) and directly injected in the LC/MS/MS systemfor analysis. Urine and plasma samples were collected andanalysed on the same day. If a rapid analysis was notpossible, samples were frozen to 20° C immediately toavoid degradation. No addition of preservatives such aschloroform or toluene was made, to avoid electrospraysuppression. For this study different urine samples withdifferent creatinine concentrations were spiked with amixture of chemical standards. Sample dilution rate (20times) and injection volume (3 µl) were selected in ordernot to overload the chromatographic column even after aconsistent number of sample injections (0.3 µl of originalurine was introduced through each single injection). Theinjection volume was selected after several trials withdifferent injection volumes. The dilution step made withH2O C ammonium acetate 50 mM and no organic solventwas intended to pack the injected sample plug at the frontend of the column.In order to assess the robustness of the method, sev-eral urine and plasma samples (with very different densitiesand creatinine concentrations) were processed several times.Samples were processed and read in comparison with aque-ous standard (results are stated without normalization for thecreatinine content). As an example, Table 2 reports the thymi-dine data obtained for three samples with low (0.44 mmol/l),medium (2.11 mmol/l) and high (3.59 mmol/l) creatinineconcentrations. The resulting intra-day reproducibility wasbelow 6.7% for values above 10 µmol/l. In an inter-day repro-ducibility test, the values were better than 3.6% (Table 3).With the proposed parameters, the estimated thymidinelimit of detection (3 ð SD) was less than 0.01 µM (similar val-ues for all other pyrimidines). We found the worst limit ofdetection in the case of uracil of about 0.08 µmol/l.No deterioration in column efficiency was observed afteranalyses of 125 urine and 80 plasma samples.Case reportThe proband was a woman born in 1976 who died at theage of 28. Her parents were consanguineous. No otherfamily members, including an older and a younger brother,had any neurological or gastrointestinal abnormalities.The onset of symptoms was in the first months of lifewith vomiting and diarrohea; when 10 years old, shepresented diarrohea, borborygmia and abdominal pain.Gastrointestinal investigations and intestinal biopsy showedno lesion.At the age of 19 she presented impaired growth: anotherbiopsy showed nonspecific chronic inflammation.At the age of 20 the patient was examined for unsteadygait: Electromyography (EMG) showed neurogenic changesin muscle, and an axonal, mostly sensory, peripheralneuropathy.She developed bowel obstruction and went for operativeintervention with surgical resection several times.At the age of 22, the patient was referred to ourdepartment for abdominal pain, diarrhoea, diverticula anddilatation of the bowel. ECG, echocardiography, audiometryand neuropsychological examinations were normal, as wellas plasma creatine kinase and plasma lactate. The retina wasnormal. Examination revealed mild ptosis, ophthalmoplegia,areflexia, sensory ataxia and diffuse muscle weaknessassociated with pronounced cachexia. She was 152 cm tall(<3rd percentile) and weighed 30 kg (<3rd percentile).Gastrointestinal investigations showed chronic pseudo-obstruction due to dysmotility and dilation. Brain MRIdemonstrated leukoencephalopathy.MNGIE was suspected for specific clinical symptoms(intestinal pseudo-obstructions, leukoencephalopathy andperipheral neuropathy) and was diagnosed by markedlyraised plasma thymidine and severely reduced thymidinephosphorylase activity in leukocytes.6 Sequence analysis ofthe patient’s TP gene revealed homozygosity for the muta-tion g3867a.6 Analysis of muscle mitochondrial enzyme wasnormal, and muscle histochemical analysis did not show anyabnormalities. A tentative therapeutic trial with benzbro-marone was started without any clinical improvement.She underwent three weekly dialysis treatments toreduce thymidine levels. We analysed via our tandem massspectrometric method, plasma and urine samples collectedbefore and after dialysis. We demonstrated a gradualreduction of thymidine levels during dialytic treatment.RESULTS AND DISCUSSIONWe have developed a new and fast analytical methodthat allows the quantification of pyrimidine metabolites inbiological fluids such as urine and plasma.Table 3. Inter-day (6 days) reproducibility on spiked urineSpiked urine (creatinine 0.44 mM)Thymidine Thymine Citidine Cytosine Uridine UracilExpected value 11 16.94 15.28 10.01 10.13 21.11Average 11.07 17.33 15.55 10.81 11.91 21.35SD 0.39 0.7 0.95 1 0.34 1.01CV% 3.52 4.04 6.11 9.25 2.85 4.73Accuracy 100.65 102.30 101.77 107.99 117.57 101.14Copyright  2006 John Wiley & Sons, Ltd. J. Mass Spectrom. 2006; 41: 586–592590 G. la Marca et al.Figure 2. LC-MS/MS pyrimidine standards profile.121 µM1 µMMNGIE patientControlXIC  of + MRMXIC  of + MRM3.6e43.4e43.2e43.0e42.8e42.6e42.4e42.2e42.0e41.8e41.6e41.4e41.2e41.0e48000.06000.04000.02000.00.03.0e42.8e42.6e42.4e42.2e42.0e41.8e41.6e41.4e41.2e41.0e48000.06000.04000.02000.00.00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Max . 3.0e4 cps.Max . 3.6e4 cps.8.5Time, minFigure 3. Urine thymidine level: MNGIE patient versus control.Copyright  2006 John Wiley & Sons, Ltd. J. Mass Spectrom. 2006; 41: 586–592Pre- and post-dialysis thymidine in a MNGIE patient 591Figure 4. Plasma thymidine level in the MNGIE patient.121 µM15.8 µMMNGIE patient pre dialysisMNGIE patient post dialysis3.6e43.4e43.2e43.0e42.8e42.6e42.4e42.2e42.0e41.8e41.6e41.4e41.2e41.0e48000.06000.04000.02000.00.03.0e42.8e42.6e42.4e42.2e42.0e41.8e41.6e41.4e41.2e41.0e48000.06000.04000.02000.00.00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.00.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0Max. 4.2e4 cps.Max. 3.6e4 cps.8.5Time, minTime, min3.2e43.4e43.6e43.8e44.0e44.2e4XIC  of + MRMXIC  of + MRM (2)Figure 5. Comparison between pre- and post-dialysis urine thymidine level in the MNGIE patient.Copyright  2006 John Wiley & Sons, Ltd. J. Mass Spectrom. 2006; 41: 586–592592 G. la Marca et al.Thymidine profile0204060801001201400 4Urine samplingsConc. µMDialysis123µM15.8µMdays1 2 3 5Figure 6. Thymidine level reduction after haemodialysis usedas therapeutic approach.Long term Thymidine profile 0204060801001201 7 10 13 16 19 22Urine samplingsConc. µMmonths4Figure 7. Long-term thymidine profile: the graph shows thatrepetitive dialysis can reduce the maximum basal level ofcirculating thymidine in a MNGIE patient.The proposed method is characterized by a fast and verysimple sample preparation. Calibration is made internallywith aqueous labelled standards. Instrumental analysis timeis less than 17 min. Chromatography combined with thespecificity provided by tandem mass spectrometry detectionallows a fast and specific procedure for the determinationof some pyrimidines. At the moment we are able toidentify pyrimidine nucleotides (thymine, cytosine, uracil)and nucleosides (thymidine, citidine, uridine) (Fig. 2). Thismethod allowed the quantification of urine (Fig. 3) andplasma (Fig. 4) thymidine levels in a MNGIE patient before,during and after dialysis used as therapeutic approach(Figs 5, 6).In the patient studied, we observed a significant reductionof plasmatic and urinary thymidine levels during and afterdialysis. However, we noted a progressive reduction of theinitial thymidine level after some dialytic trials (Fig. 7).This method will be useful not only for thymidinelevel follow-up during dialysis in MNGIE patients but alsofor improvement of the diagnosis or diagnostic suspectin other pyrimidine defects such as dihydropyrimidinedehydrogenase deficiency, dihydropyrimidinase deficiencyand ureidopropionase deficiency.REFERENCES1. Hirano M, Silvestri G, Blake DM, Lombes A, Minetti C, Bonilla E,Hays AP, Lovelace RE, Butler I, Bertorini TE, et al. Mitochondrialneurogastrointestinal encephalomyopathy (MNGIE): clinical,biochemical, and genetic features of an autosomal recessivemitochondrial disorder. Neurology 1994; 44: 721.2. Papadimitriou A, Comi GP, Hadjigeorgiou GM, Bordoni A,Sciacco M, Napoli L, Prelle A, Moggio M, Fagiolari G, Bresolin N,Salani S, Anastasopoulos I, Giassakis G, Divari R, Scarlato G.Partial depletion and multiple deletions of muscle mtDNA infamilial MNGIE syndrome. Neurology 1998; 51: 1086.3. Nishino I, Spinazzola A, Papadimitriou A, Hammans S, Steiner I,Hahn CD, Connolly AM, Verloes A, Guimaraes J, Maillard I,Hamano H, Donati MA, Semrad CE, Russell JA, Andreu AL,Hadjigeorgiou GM, Vu TH, Tadesse S, Nygaard TG, Nonaka I,Hirano I, Bonilla E, Rowland LP, DiMauro S, Hirano M.Mitochondrial neurogastrointestinal encephalomyopathy: anautosomal recessive disorder due to thymidine phosphorylasemutations. Ann. Neurol. 2000; 47: 792.4. Nishino I, Spinazzola A, Hirano M. Thymidine phosphorylasegene mutations in MNGIE, a human mitochondrial disorder.Science 1999; 283: 689.5. Song S, Wheeler LJ, Mathews CK. Deoxyribonucleotide poolimbalance stimulates deletions in HeLa cell mitochondrial DNA.J. Biol. Chem. 2003; 278: 43893.6. Spinazzola A, Marti R, Nishino I, Andreu AL, Naini A, Tadesse S,Pela I, Zammarchi E, Donati MA, Oliver JA, Hirano M. Alteredthymidine metabolism due to defects of thymidinephosphorylase. J. Biol. Chem. 2002; 277: 4128.7. van Kuilenburg AB, van Lenthe H, Loffler M, van Gennip AH.Analysis of pyrimidine synthesis ‘‘de novo’’ intermediates inurine and dried urine filter- paper strips with HPLC-electrospraytandem mass spectrometry. Clin. Chem. 2004; 50: 2117.8. Vidotto C, Fousert D, Akkermann M, Griesmacher A, Muller MM.Purine and pyrimidine metabolites in children’s urine. Clin. Chim.Acta 2003; 335: 27.Copyright  2006 John Wiley & Sons, Ltd. J. Mass Spectrom. 2006; 41: 586–592

Pre and post-dialysis quantitative dosage of thymidine in urine and plasma of a MNGIE patient by using HPLC-ESI-MS/MS

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Pre and post-dialysis quantitative dosage of thymidine in urine and plasma of a MNGIE patient by using HPLC-ESI-MS/MS

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