Donati, Ma

La Marca, Giancarlo

Malvagia, S

Morrone, Amelia

Pasquini, E

Zammarchi, Enrico

English

Florence Research

Uncorrected ProofRAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom. 2003; 17: 1–6Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1248To the Editor-in-ChiefSir,Rapid diagnosis of medium chainAcyl Co-A dehydrogenase (MCAD)deficiency in a newborn by liquidchromatography/tandem mass spec-trometryMedium chain Acyl-CoA dehydro-genase (MCAD) deficiency is the mostcommon b-oxidation defect.1,2 Its pre-sentation varies from acute neonatalto late-onset and even to adulthoodonset,3,4 and in some cases it is asymp-tomatic for an entire lifetime.5 Thesymptoms are characterised by hypo-glycaemia, vomiting, seizures, lethargy,coma, till death. The metabolic disorderis frequently caused by fasting or viralfebrile illness. Moreover, MCAD defi-ciency can be responsible for suddeninfant death syndrome (SIDS),6 for per-manent neurological damage due tohypoglycaemic encephalopathy,7 andfor Reye-like syndrome.8,9Molecular analysis of the MCADgene in clinically affected patients hasrevealed that 80% of patients arehomozygous for a common mutation985A!G, and 18% are heterozygousfor this mutation.7,10 The delayed, ornot performed, diagnosis of MCADdeficiency at presentation causes thedeaths of about 20–25% of patients.11,12Because of its frequency (1:10000–1:17000 newborns)1,13–15 and high mor-tality, neonatal screening by electro-spray ionisation tandem massspectrometry (ESI-MS/MS) is highlyrecommended.13,16–18 However, invery rapid and acute neonatal onset,frequently within 3 days of life,3,19neonatal screening could be too latewith reference to the worsening of thegravity of illness. Therefore, clinicalsymptoms must lead to laboratory testsand to diagnosis as soon as possible.MCAD deficiency diagnosis can beperformed by analysing the acylcarni-tine profile from a dried blood spot orplasma spot.24–33 Acylcarnitine analy-sis was originally performed by high-performance liquid chromatography(HPLC), gas chromatography/massspectrometry (GC/MS),21 or GC oforganic acids after hydrolysis of carni-tine esters. These processes are time-consuming (typical run times are>30 min) and labour-intensive, thuslimiting high-throughput performance.We describe here a patient with acutesevere presentation in his third day oflife. In this case a very rapid diagnosisby LC/MS/MS allowed a completerecovery before the decompensationbecame very important. After dia-gnosis, LC/MS/MS analyses are alsofundamental for follow-up purpo-ses during long-term managementtherapy.The patient, a male, was born at termto non-consanguineous parents fromTuscany, with a weight of 3360 g. Abrother and a sister are healthy. Noclinical abnormalities were found inthe first day of life. After 48 h of life, hebecame hypotonic and hyporeactive.Abnormal laboratory findings inclu-ded metabolic aciduria (blood gasparameters were pH 7.28; pCO2 28.3;HCO3 13.1; EB 11.8), high values oftransaminases and lactate, hyperam-monaemia (291 mM; normal <32 mM),and hypoglycaemia (10 mg/dL; nor-mal 70–120 mg/dL). Because of thepresence of these symptoms, a samplefrom blood spot paper was immedi-ately prepared and immediately ana-lysed by LC/MS/MS. The diagnosis ofMCAD deficiency, due to accumula-tion of medium chain acylcarnitines(see later), was made about 1 h afterdrawing blood.Acylcarnitines and labeled stan-dards of amino acids were purchasedfrom Cambridge Isotope Laboratories(Andover, MA, USA); a stock solutionwas made in methanol. The standardconcentrations are in the range 500–2500 mmol/L for amino acids and in therange 7.6–152 mmol/L for acylcarni-tines. In order to obtain working solu-tions, daily dilutions (1:100) were madeusing methanol. All chemicals andsolvents were of the highest purityavailable from commercial sources,and were used without any furtherpurification.A dried blood spot was punched intoa 1.5-mL tube and 200mL of methanolcontaining labeled standards wereadded. The sample was shaken on avortex system for 20 min, and was thendried under a nitrogen flow at 508C.The extracted acylcarnitines and aminoacids were derivatised to butyl estersusing n-butanol plus HCl (3 M) at 658Cfor25 min.Afterderivatisation thesam-ple was dried under a nitrogen flow at558C and then recovered by 200 mL ofwater/acetonitrile (1:1) containing0.1% formic acid. 40mL of the dilutedsample were injected in flow injectionanalysis (FIA) mode for the MS/MSexperiments.An Applied Biosystems-Sciex(Toronto, Canada) API 2000 triple-quadrupole mass spectrometer equip-ped with a TurboIonSpray source wasemployed for this study. The Tur-boIonSpray source was operated inpositive ion mode with a needle poten-tial of þ5900 V and with a ‘‘turbo’’ gasflow of 10 L/min of air heated at 1508C(nominal heating-gun temperature).Mass calibration and resolutionadjustments on the resolving quadru-poles were performed automatically byusing a 104 mol/L solution of PPGintroduced via the built-in infusionpump. The peak width was set on bothresolving quadrupoles at 0.7 Th (mea-sured at half height) for all MS and MS/MS experiments.Collision-activated dissociation(CAD) MS/MS was performed in theLINAC Q2 collision cell, operatingwith 10 mTorr pressure of nitrogen ascollision gas. The declustering poten-tial (DP) and collision energy (CE) wereautomatically optimized for acylcarni-tines and amino acids using the Ana-lyst 1.1 software. The resulting DP wasþ18 V, and optimal CE was found to be20 eV (laboratory frame) in the case ofamino acids. A DP ramp (10–55 V) anda CE ramp (35–50 eV) were needed inthe case of acylcarnitines.MS and MS/MS spectra were col-lected in continuous flow mode byconnecting the infusion pump directlyto the TurboIonSpray source. A stan-dard solution of 10 ng/mL of eachamino acid and acylcarnitine inwater/acetonitrile (1:1) containing0.1% formic acid was infused at 10 mL/min. The quantitation experimentsCopyright # 2003 John Wiley & Sons, Ltd.RCMLetter to the Editor1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465Uncorrected Proofwere performed using a series 1100Agilent Technologies (Waldbronn,Germany) CapPump coupled to anAgilent Micro ALS autosampler,both fully controlled by the API 2000data system. Mobile phase flow ratewas 30 mL/min using water/acetoni-trile (1:1) containing 0.1% formic acid.The eluent from the column wasdirected to the TurboIonSpray probe.The acquired data were processedusing the Analyst 1.1 proprietary soft-ware including the ‘Explore’ option(for chromatographic and spectralinterpretation) and the ChemoViewsoftware (for quantitative informationgeneration).Genomic DNA was extracted fromthe patient’s and from his parents’peripheral blood lymphocytes usingstandard methods. The genomic frag-ments covering all 12 exons and theexon/intron boundaries of the MCAD-gene were amplified by a set of primerslocated in flanking intronic sequences.PCR amplification was performedunder the following conditions: initialdenaturation at 948C for 4 min wasfollowed by 30 cycles with denatura-tion at 948C for 30 s, annealing tem-perature of 638C for 30 s, and extensionat 728C for 2 min. All the amplificationreactions were performed in a totalvolume of 25 mL containing 2.5 U Poly-Taq DNA polymerase (Polymed, Sam-buca-Firenze, Italy), 25 mM of dNTPs,200 ng forward primer, 200 ng of thereverse primer and 1XPCR reactionbuffer.PCR fragments were separated ona 2% agarose gel containing ethidiumbromide and the bands were visualisedby an UV transilluminator. DNAproducts were purified using aNucleospin extract kit (Macherey-Nagel, Du¨ren, Germany), followingthe manufacturer’s protocol. The dou-ble-stranded purified products wereused for direct sequencing with thesame PCR amplification primers. Thesequencing reactions were performedwith Big Dye terminator cycle sequen-cing ready reaction kit reagents(Applied Biosystems, Foster City, CA,USA). The reactions were run on an ABIPRISM 310 sequencer and were ana-lysed using Sequencing analysis soft-ware, version 3.3.Figure 1 shows a typical MS/MSprofile of a healthy newborn. Figure 2shows the comparison between a nor-mal acylcarnitine profile versus oneaffected by a MCAD deficiency. Theanomalous ions corresponding to veryhigh concentrations of hexanoyl car-nitine (C6), octanoyl carnitine (C8),decanoyl carnitine and decenoyl carni-tine (C10:1) are clearly observed at m/z316, 344, 372 and 370, respectively, onlyin the MCADD-affected profile. Theamino acid profile was normal. Afterdiagnosis the patient was suppliedintravenously with glucose and withinsulin as emergency therapy. Theseadministrations gave high cellularenergy availability, halting lipolithicpathway activation. In addition, a sup-plementation f l-carnitine was neededto remove accumulation of toxic inter-mediates. Figure 3 shows the MCADdeficiency acylcarnitine profile duringemergency therapy (8, 18, and 48 hpost-diagnosis). The use of an LC/MS/MS system during the first 48 hpost-diagnosis permitted the evalua-tion of the removal of the toxic acylcar-nitines (Table 1). After the first 48 h, aFigure 1. ESI-Q1MS/MS profiles of acylcarnitines and amino acids of a healthy newborn.Q1Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1–62 Letter to the Editor1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465Uncorrected ProofFigure 2Q1. Normal acylcarnitine profile compared with one from a patient with MCAD deficiency.Figure 3Q1. Acylcarnitine profile of a patient with MCAD deficiency during emergency therapy.Q1Q1Letter to the Editor 3Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1–61234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465Uncorrected Proofnew management regime was started,consisting of an administration of amilk specific for b-oxidation defects,containing no fats but only carbohy-drates and proteins, and in addition analiquot of human milk containing fats,that provided 15–16% of the total dailyintake of kilocalories. Figure 4 showsthe acylcarnitine MS/MS profiles dur-ing the 48 h after the start of thismanagement regime. The values ofthe increase in medium chain acylcar-nitines allowed the evaluation that, inthis patient, the MCAD residue activitywas probably very low. Therefore, themanagement regime was correctedwith respect to the human milk admin-istration, changing to 8% the contribu-tion of fats to the total daily intake ofkilocalories.Figure 5 shows the MCADD-affectedacylcarnitine profile following the defi-nitive long-term management regime,in comparison with a normal profile.Molecular analysis indicated thatthe patient was homozygous for theknown K329E mutation. This mutationis due to the transition 985A!G,which maps on exon 11 of the MCADgene. His parents were heterozygousfor this mutation. The K329E aminoacid change is the most commonmutation in the Caucasoid group ofhumans. Some authors have estimatedits frequency to be about 90% of allMCAD mutations.22,23In conclusion, the newborn screen-ing method is characterised by a simplesample preparation and the instrumen-tal analysis time is less than 3.3 min. Inthis case we performed the diagnosisabout 1 h after receiving the blood spotpaper, allowing the start of an emer-gency therapy immediately. The lim-itation of Italian newborn screening isthat the drawing blood procedure mustbe performed after 48 h post-birth,which does not allow diagnosis ofsome acute neonatal onset pathologies.In these cases the driving force is clini-cal symptoms which indicate a poten-tial metabolic disease. After thismanifestation of clinical symptoms afundamental role is played by an LC/Table 1. Decrease in levels of medium chain acylcarnitines during emergencytherapyC6 C8 C10 C10:1Diagnosis 3.35 26.7 3.03 0.8978 hours PD 2.6 19 1.79 0.6518 hours PD 1.43 3.28 0.269 0.22548 hours PD 0.457 1.22 0.104 0.12Normal values* 0.06–0.24 0.02–0.23 0.03–0.2 0.05–0.24C6, Hexanoyl carnitine; C8, octanoyl carnitine; C10, decanoyl carnitine; C10:1, decenoylcarnitine; PD, post-diagnosis.* 5th–95th percentile.Figure 4Q1. Restoration of medium chain acylcarnitines during the 48 h after the start of the management regime.Q14 Letter to the EditorCopyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1–61234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465Uncorrected ProofMS/MS system that permits a very fastidentification of defects in comparisonwith other analytical techniques.Giancarlo la Marca*, Sabrina Malvagia,Maria Alice Donati, Amelia Morrone,Elisabetta Pasquiniand Enrico ZammarchiMetabolic and Neuromuscolar Unit,1st Pediatric Clinic,A. Meyer Children’s Hospital,University of Florence,Florence 50132, Italy*Correspondence to:G. laMarca,Metabolicand Neuromuscolar Unit,1st Pediatric Clinic, A. Meyer Children’sHospital, University of Florence,Florence 50132, Italy.E-mail: giancalm@yahoo.comREFERENCES1. 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Mass Spectrom.1994; 8: 129.Received 20 June 2003Revised 1 October 2003Accepted 1 October 20036 Letter to the EditorCopyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 1–61234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465Uncorrected ProofAuthor Query Form (RCM/1248)Special Instructions: Author please write responses to queries directly on Galley proofs and thenfax back. Alternatively please list responses in an e-mail.Q1: Author: Please check thequality of all Figures.Q2: Author: Please providecitation of ref. 20 in text.1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

Rapid diagnosis of medium chain Acyl Co-A dehydrogenase (MCAD) deficiency in a newborn by liquid chromatography/tandem mass spectrometry.

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Rapid diagnosis of medium chain Acyl Co-A dehydrogenase (MCAD) deficiency in a newborn by liquid chromatography/tandem mass spectrometry.

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