- Email: [email protected]
MS: A new tool for the diagnosis of peroxisomal biogenesis disorders
Clinica Chimica Acta 398 (2008) 86–89
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Urine acylcarnitine analysis by ESI–MS/MS: A new tool for the diagnosis of peroxisomal biogenesis disorders Guglielmo Duranti a,e,1, Sara Boenzi a,1, Cristiano Rizzo a, Lucilla Ravà b, Vincenzo Di Ciommo b, Rosalba Carrozzo c, Maria Chiara Meschini c, David W. Johnson d, Carlo Dionisi-Vici a,⁎ a
Division of Metabolism, Bambino Gesù Research Institute, Piazza S. Onofrio 4, 00165 Rome, Italy Unit of Epidemiology and Statistics, Bambino Gesù Research Institute, Piazza S. Onofrio 4, 00165 Rome, Italy Unit of Molecular Medicine, Bambino Gesù Research Institute, Piazza S. Onofrio 4, 00165 Rome, Italy d Department of Genetic Medicine, Women's and Children's Hospital, North Adelaide, Australia e Department of Human Movement and Sport Sciences, Rome University of Movement Sciences IUSM, Piazza Lauro de Bosis 15, 00194 Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 4 April 2008 Received in revised form 6 August 2008 Accepted 21 August 2008 Available online 28 August 2008 Keywords: Peroxisomal biogenesis disorders Zellweger syndrome Infantile Refsum disease Tandem mass spectrometry ESI–MS/MS Acylcarnitines
a b s t r a c t Background: Patients with peroxisomal biogenesis disorders (PBDs) have an abnormal profile of circulating acylcarnitines (i.e. elevated C16:0-DC-, C18:0-DC-, C24:0-, C26:0-carnitine). We developed an ESI–MS/MS method for quantification of urine acylcarnitines and tested its reliability for the diagnosis of PBDs. Methods: Urine from 7 patients with PBDs (5 Zellweger syndrome, 2 infantile Refsum disease), from 2 patients with D-bifunctional protein (D-BP) deficiency, and from 130 healthy controls were analysed by ESI– MS/MS, using a multiple reactions monitoring (MRM) method, and quantified with labelled internal standards. Acylcarnitine levels between groups were analyzed by the STATA™ statistics data analysis and compared by the non parametric Mann–Whitney test. Results: In PBDs, the urinary excretion of long-chain dicarboxylylcarnitines (C14:0-DC-, C16:0-DC-, and C18:0DC-carnitine), and of very long-chain monocarboxylylcarnitines (C22:0-, C24:0-, C26:0-carnitine) were significantly elevated compared to controls (p b 0.0001). Interestingly, among PBDs the most severe abnormalities of acylcarnitine profile were observed in patients with Zellweger syndrome. One patient with D-BP showed similar abnormalities to PBDs, while in the other only C16:0-DC-carnitine was markedly elevated. Conclusions: This study shows that MRM ESI–MS/MS acylcarnitine analysis unequivocally discriminates patients with PBDs and D-BP deficiency from controls, representing a reliable and sensitive method for the diagnosis that requires a short-time analysis with high sample through-put. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Peroxisomes are cellular organelles that catalyse a wide range of essential functions, playing a key role in the metabolism of a variety of lipids that also include β-oxidation of very long-chain fatty acids (VLCFA) and of long-chain dicarboxylic fatty acids [1–3]. Inherited defects of peroxisome metabolism are clinically and genetically heterogeneous and can be roughly classified into two main groups, peroxisome biogenesis disorders (PBDs) and single peroxisomal enzyme deficiencies [4,5]. Abbreviations: VLCFA, very long-chain fatty acids; PBDs, peroxisomal biogenesis disorders; GC/MS, gas chromatography/mass spectrometry; CoA, coenzyme-A; ESI–MS/ MS, electrospray ionization tandem mass spectrometry; MRM, multiple reaction monitoring; D-BP, D-bifunctional protein; C14:0-DC-carnitine, tetracanedioylcarnitine; C16:0-DC-carnitine, hexadecanedioylcarnitine; C18:0-DC-carnitine, octadecanedioylcarnitine; C22:0-carnitine, behenoylcarnitine; C24:0-carnitine, lignoceroylcarnitine; C26:0-carnitine, cerotoylcarnitine; CVs, Coefficients of variation; LoD, limit of detection; LoQ, limit of quantification. ⁎ Corresponding author. Division of Metabolism, Bambino Gesù Children's Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy. Tel.: +39 6 68592225; fax: +39 6 68592736. E-mail address: [email protected] (C. Dionisi-Vici). 1 These authors contributed equally to this work. 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.08.018
Based on the clinical presentation, PBD patients can be categorized in the following major groups according to the spectrum of disease severity: classical Zellweger syndrome, neonatal adrenoleukodystrophy, rhizomelic chondrodysplasia punctata, and infantile Refsum disease. The most common diagnostic tool for peroxisomal disorders is the quantification of plasma VLCFA by gas chromatography/mass spectrometry (GC/MS), a time-consuming method with low instrument through-put due to sample preparation, derivatisation and analysis. Further complex biochemical investigations in body fluids and cultured fibroblasts as well as molecular analyses are needed to confirm the diagnosis [6]. Trans-esterification of coenzyme-A (CoA) esters with carnitine is an effective mechanism to buffer cellular organelles from the accumulation of acyl-CoA esters. We have previously shown that patients with peroxisomal biogenesis disorders (PBDs) have a characteristic profile of plasma and whole blood acylcarnitines with elevation of long-chain dicarboxylylcarnitines [i.e. hexadecanedioylcarnitine (C16:0-DC-carnitine), and octadecanedioylcarnitine (C18:0-DC-carnitine)] and of very long-chain monocarboxylylcarnitines [i.e. lignoceroylcarnitine (C24:0carnitine), and cerotoylcarnitine (C26:0-carnitine)], as detected by electrospray ionization tandem mass spectrometry (ESI–MS/MS) [7].
G. Duranti et al. / Clinica Chimica Acta 398 (2008) 86–89
Based on this observation, we developed a new multiple reaction monitoring (MRM) ESI–MS/MS method for quantification of acylcarnitine in urine and tested its reliability for the diagnosis of PBDs. 2. Materials and methods 2.1. Subjects Morning urine spot samples were collected from seven patients with proven PBDs. Five patients (pt. 1–5), all died within the first year of life presented with a classical Zellweger phenotype (i.e. craniofacial abnormalities, severe neurological dysfunction, profound hypotonia, seizures, abnormal brain MRI, eye abnormalities, liver dysfunction, adrenal gland insufficiency, and renal cysts). Two had a less severe clinical picture, consistent with infantile Refsum disease. Patient 6, who survived until the age of 5 years, presented with mild facial abnormalities, developmental delay, seizures, pigmentary retinopathy, and white matter abnormalities at brain MRI. Patient 7, presents a milder phenotype characterized by neurosensorial deafness, adrenal insufficiency, periventricular white matter hyperintensity and growth retardation. She is alive at the age of 8 years and presents only a minimal cognitive impairment. Urine was also collected from 2 patients (pt. 8–9) with isolated D-bifunctional protein [D-BP] deficiency. Both showed severe neurological impairment, seizures, pigmentary retinopathy, and neurosensorial deafness. All patients underwent a full biochemical assessment, including plasma VLCFA, phytanic and pristanic acids, bile acid profile, plasmalogens in erythrocytes, and urine organic acid GC/MS analysis. Those belonging to PBDs group showed an abnormal profile of all circulating metabolites. Patients with D-BP deficiency had normal phytanic acid. All patients showed dicarboxylic- and epoxy-dicarboxylic-aciduria. Biochemical diagnosis was confirmed with extensive studies in cultured fibroblasts (R.J.A. Wanders, Amsterdam, The Netherlands). As controls, spot morning urine samples were obtained from 130 healthy age-matched subjects. Patients and controls did not receive carnitine supplementation. The study was approved by the Institutional Review Board and informed consent was obtained from the controls' and patients' families. 2.2. Sample preparation Working methanol solutions containing 0.04 μmol 2[H]9-hexadecanedioylcarnitine [8], 0.04 μmol 2[H]4-cerotoylcarnitine (synthesized by H.J. ten Brink, VU Medical Center, Amsterdam, The Netherlands) and 10 μmol of 2[H]3-creatinine (CDN. Isotopes, Pointe Claire, Canada) were prepared daily from internal standards stock solutions containing 5 μmol labelled acylcarnitines and 1.25 mmol labelled creatinine. Urine was applied on a neonatal screening filter paper WS 903 (Whatman, Dassel, Germany) and dried. A filter paper disc urine spot of 5 mm diameter, corresponding to 3.5 µL urine, was first extracted by agitation for 30 min at room temperature with 420 µL working methanol solution into a 1.5 mL Eppendorf tube (Eppendorf, Hamburg, Germany) and then evaporated under nitrogen stream. 100 µL of butanolic-HCl (Regis Technologies, Morton Grove, IL, USA) were added to each sample, heated at 65 °C for 60 min, and evaporated under nitrogen stream. Samples were reconstituted in 200 µL water/acetonitrile/formic acid (50:50:0.025 v/v/v) solvent. 2.3. Tandem mass spectrometry 30 µL of the butilated sample were automatically injected by an autosampling system (PE 200 series autosampler, Perkin Elmer, Norwalk, CT, USA) into a liquid chromatography pump (PE LC 200 series, Perkin Elmer, Norwalk, CT, USA) with solvent flow rate of 50 µL/min of water/acetonitrile/formic acid (50:50:0.025 v/v/v) to a triple quadruple tandem mass spectrometer (Sciex API 365; PE Sciex Instrument, Concord, ON, Canada). The run-time was 3.5 min for each sample. MRM ESI–MS/MS method was performed in positive ionisation. The collision energy was 44 eV. Specific MRM transitions were selectively analysed for long-chain dicarboxylylcarnitines [514/85 for tetracanedioylcarnitine (C14:0-DC-carnitine), 542/85 for C16:0-DC-carnitine, and 570/ 85 for C18:0-DC-carnitine], for very long-chain monocarboxylylcarnitines [540/85 for behenoylcarnitine (C22:0-carnitine), 568/85 for C24:0-carnitine and 596/85 for C26:0carnitine] and for creatinine [114/44 for creatinine and 117/47 for 2[H]3-creatinine]. Calculation of the concentration was done by comparing acylcarnitine ion intensities with corresponding labelled internal standards with Neonatal Script software (PE Sciex Instrument, Concord, ON, Canada ). The long-chain dicarboxylylcarnitines were compared to 2[H]9-hexadecanedioylcarnitine, and the very long-chain monocarboxylylcarnitines were compared to 2[H]4-cerotoylcarnitine. Values were expressed as μmol/mmol creatinine. Results of creatinine obtained with ESI–MS/MS were also compared with those obtained by a Hitachi analysis (Jaffe method). 2.4. Precision, accuracy, linearity and limit of detection MRM ESI–MS/MS method was validated for linearity, recovery, run-to-run, withinrun accuracy and limit of detection analysis. The method was set as established for
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tandem mass spectrometry in clinical chemistry by following the C50-A and the EP15A2 of CLSI [9,10] guidelines. Control urine was screened prior to spiking to ensure that it was free of endogenous interference of C16:0-DC-carnitine. For linearity and recovery assays, 0.01, 0.04, 0.08, 0.16 and 0.32 μmol of C16:0-DCcarnitine were added to control urine and tested. Median values of four replicates in 5 days were used by Ordinary Least Squares for regression analysis. For method validation three concentrations [0.04 μM C16:0-DC-carnitine (low); 0.08 μM C16:0-DCcarnitine (medium); 0.16 μM C16:0-DC-carnitine (high)] were added to control urine with 20 mM creatinine level. Assays were performed in 5 days in four replicates as defined in case of unknown coefficient of variation. For limit of detection (LoD) and quantification (LoQ) we follows the NCCLS protocol [11] by testing 60 times within the same day a control sample (without added C16:0DC-carnitine) and 60 times a 0.004 μmol/L C16:0-DC-carnitine sample. LoB was calculated as the 95th percentile of a series of results obtained in the control sample without added C16:0-DC-carnitine. Type I and II errors were set to α = β = 5% [12]. To determine LoD we used the following formula LoD = LoB + 1.645 × SD, where SD is the estimated standard deviation of the sample distribution at 0.004 μmol/L concentration C16:0-DC-carnitine. The test results of LoD were used to estimate bias and imprecision. The standard error for LoD [SE(LoD)] when α and β error were set to 0.05 were calculated with the Linnet and Kondratovich formula [12]. The difference of the main of the replicate and the accepted reference values is an estimate of bias. Total error at that level was calculated as Total Error = Bias + 2 × SD. LoQ was calculated as ten times standard deviation of the LoB. The prepared standard samples were stable for more than 24 h at room temperature, with a 5.5% mean percentage decrease in concentration. 2.5. Statistical analysis Data were analyzed by STATA™ statistics data analysis. Median of acylcarnitine values were compared through Mann–Whitney U-test. The level of significance was set at p ≤ 0.05.
3. Results The linearity study showed that the curve was linear from 0.01 to 0.32 μmol for C16:0-DC-carnitine (slope: 0.9748 ± 0.0636; intercept: 0.0019 ± 0.0048; R2 = 0.9965 ± 0.0037). The mean recoveries across different C16:0-DC-carnitine concentrations were 95.9% (0.04 μmol/L), 94.2% (0.08 μmol/L) and 93.9% (0.16 μmol/L), respectively. Table 1 shows the results of run-to-run and within-run accuracies; CVs at different concentrations of C16:0-DC-carnitine were all below 10%. The limit of detection (LoD) for C16:0-DC-carnitine was 0.0026 μmol/L with a standard error of 0.0003, the calculated Bias was −0.0003 and total error 0.0009. The limit of quantification (LoQ) was 0.0103 μmol/L. Urinary creatinine values correlated with those obtained using an automatic Hitachi analyser (Jaffe method) (R = 0.9999). Quantitative values of urinary long-chain dicarboxylylcarnitines and very long-chain monocarboxylylcarnitines obtained from patients and controls are shown in Table 2 and Fig. 1. In all urine samples from PBD patients a highly significant increase of long-chain dicarboxylylcarnitines and of very long-chain monocarboxylylcarnitines was observed (p b 0.0001). Median values (μmol/mmol creatinine) for each compound were as follows: C14:0-DC-carnitine 0.92, C16:0-DC-carnitine 0.62, C18:0-DC-carnitine 0.28, C22:0-carnitine 0.05, C24:0-carnitine 0.05, C26:0-carnitine 0.02. As shown in Table 2, patients with Zellweger syndrome had a more pronounced excretion of long-chain dicarboxylylcarnitines compared to those with infantile Refsum disease. Urinary long-chain dicarboxylylcarnitines were also significantly elevated in D-BP deficient patients (p b 0.05). However, the very longchain monocarboxlylcarnitines were elevated only in one of the two patients studied (Table 2). Table 1 Run-to-run and within-run accuracy with different C16:0-DC-carnitine concentrations: low (0.04 μmol/L), medium (0.08 μmol/L), high (0.16 μmol/L) C16:0-DC-carnitine Mean total SD run-to-run total % CV SD within-run total % CV
0.04 μmol/L
0.08 μmol/L
0.16 μmol/L
0.038 0.003 8.31 0.003 7.74
0.075 0.005 6.78 0.005 6.20
0.150 0.012 7.67 0.011 7.48
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G. Duranti et al. / Clinica Chimica Acta 398 (2008) 86–89
Table 2 Urinary acylcarnitine levels (μmol/mmol creatinine) obtained by MRM ESI–MS/MS analysis from patients with peroxisomal biogenesis disorders, with D-bifunctional protein deficiency, and from 130 healthy controls C14:0-DC- C16:0-DC- C18:0-DC- C22:0C24:0carnitine carnitine carnitine carnitine carnitine Peroxisomal biogenesis disorders Zellweger syndrome Pt. 1 1.35 0.62 Pt. 2 0.91 1.08 Pt. 3 2.09 0.97 Pt. 4 0.97 0.93 Pt. 5 0.92 0.34 Infantile Refsum disease Pt. 6 0.63 0.32 Pt. 7 0.59 0.36
C26:0carnitine
0.28 0.29 0.31 0.40 0.16
0.06 0.05 0.07 0.08 0.04
0.04 0.08 0.09 0.07 0.05
0.05 0.04 0.01 0.13 0.02
0.12 0.12
0.04 0.04
0.03 0.03
0.02 0.02
D-bifunctional protein deficiency Pt. 8 0.34 0.18 Pt. 9 0.85 0.83
0.04 0.27
0.02 0.04
0.01 0.03
nd 0.03
Controls (n = 130) Median 0.07 Mean±SD 0.11 ± 0.10 Highest value 0.31
nd 0.01 ± 0.02 0.03
nd nd 0.01
nd 0.01±0.01 0.03
nd nd nd
nd 0.01 ± 0.01 0.03
nd: not detectable.
Fig. 2 shows the MRM ESI–MS/MS acylcarnitine profile from a Zellweger patient compared with a control. 4. Discussion ESI–MS/MS is a powerful analytical platform used in many fields of laboratory diagnostics and therapeutic drug monitoring. The analysis of acylcarnitines using ESI–MS/MS has shown to be a powerful tool for
Fig. 2. Urinary acylcarnitine profiles obtained by MRM ESI–MS/MS analysis from a patient with a peroxisomal biogenesis disorder (A), and from a control (B). Specific MRM transitions (Q1/Q3) are as follows: 514/85, C14:0-DC-carnitine; 542/85, C16:0-DCcarnitine; 570/85, C18:0-DC-carnitine; 540/85, C22:0-carnitine; 568/85, C24:0-carnitine; 596/85, C26:0-carnitine. Q1/Q3 masses of internal standards (IS), 551/85 2[H]9hexadecanedioylcarnitine, 600/85 2[H]4-cerotoylcarnitine.
Fig. 1. Box plot showing quantitative values of urinary long-chain dicarboxylylcarnitines (A) and very long-chain monocarboxylylcarnitines (B) in 130 controls (empty bars), in 7 patients with peroxisomal biogenesis disorders (squared bars), and in two patients with D-bifunctional protein deficiency ( ).
•
the diagnosis of many inherited metabolic diseases. Urinary acylcarnitine analysis by ESI–MS/MS can be used to diagnose most of the organic acidurias and fatty acid oxidation disorders [13,14] but so far has never been applied to detect PBDs. Based on our previous results showing an abnormal profile of circulating acylcarnitines in PBDs [7], we have tested whether similar abnormalities were also detectable in urine, a biological fluid more easily obtainable compared to blood. The use of MRM with labelled internal standards allowed high accuracy for quantitative acylcarnitine determination, enabling optimal compensation for losses during sample preparation and signal-intensity fluctuations due to matrix effects. Through this method, we were able to detect an unequivocally abnormal pattern of urine acylcarnitines in PBDs, and we enlarged the spectrum of abnormal carnitine species compared to the blood study [7]. All presented elevated urinary excretion of C14:0-DC-, C16:0-DC-, C18:0-DC-carnitine and of C22:0-, C24:0-, C26:0-carnitine esters. Interestingly, patients with the most severe phenotype (i.e. Zellweger syndrome) presented more pronounced abnormalities compared to those with a less severe clinical picture (i.e. infantile Refsum disease). In the two patients with D-BP deficiency, we observed different patterns. One presented with a profile similar to those observed in PBDs. In the other patient, only C16:0-DC-carnitine was markedly elevated, whereas the other carnitine esters were within control range
G. Duranti et al. / Clinica Chimica Acta 398 (2008) 86–89
or only mildly increased. These variations might be explained with a different residual activity of D-BP among the two patients, or by a different contribution of the second peroxisomal bifunctional protein enzyme (i.e. L-bifunctional protein) [2,15]. In conclusion, our study shows that MRM ESI–MS/MS acylcarnitine analysis unequivocally discriminates patients with PBDs and D-BP deficiency from controls, representing a reliable and sensitive method for the diagnosis that requires a short-time analysis with high sample through-put. References [1] Wanders RJ, Vreken P, Ferdinandusse S, et al. Peroxisomal fatty acid alpha- and beta-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem Soc Trans 2001;29:250–67. [2] Ferdinandusse S, Denis S, Van Roermund CW, Wanders RJ, Dacremont G. Identification of the peroxisomal beta-oxidation enzymes involved in the degradation of long-chain dicarboxylic acids. J Lipid Res 2004;45:1104–11. [3] Reddy JK, Hashimoto T. Peroxisomal ß-oxidation and peroxisome proliferatoractivated receptor: an adaptive metabolic system. Annu Rev Nutr 2001;21:193–230. [4] Gould SJ, Raymond GV, Valle D. The peroxisome biogenesis disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. 8th ed. New York, NY: McGraw-Hill; 2001. p. 3181–217. [5] Wanders RJA, Barth PG, Heymans HAS. Single peroxisomal disorders enzyme deficiencies. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. 8th ed. New York, NY: McGraw-Hill; 2001. p. 3219–56.
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[6] Steinberg SJ, Dodt G, Raymond GV, Braverman NE, Moser AB, Moser HW. Peroxisome biogenesis disorders. Biochim Biophys Acta 2006;1763:1733–48. [7] Rizzo C, Boenzi S, Wanders RJ, Duran M, Caruso U, Dionisi-Vici C. Characteristic acylcarnitine profiles in inherited defects of peroxisome biogenesis: a novel tool for screening diagnosis using tandem mass spectrometry. Pediatr Res 2003;53:1013–8. [8] Johnson DW. Synthesis of dicarboxylic acylcarnitines. Chem Phys Lipids 2004;129: 161–71. [9] Chase DH, Barr JR, Duncan MW, et al. Mass spectrometry in the clinical laboratory: general principles and guidance; approved guideline. CLSI document C50-A, vol. 27. Clinical and Laboratory Standard Institute; 2007. p. 1–94. [10] Carey RN, Anderson FP, George H, et al. User demonstration of performance for precision and accurancy; approved guidelines–second edition. CLSI document EP15-A2, vol. 25. Clinical and Laboratory Standard Institute; 2005. p. 1–49. [11] Tholen DW, Linnet K, Kondratovich M, et al. Protocols for determination of limits of detection and limits of quantification; approved guideline. NCCLS document EP17A, vol. 24. National Committee of Clinical and Laboratory Standards; 2004. p. 1–39. [12] Linnet K, Kondratovich M. Partly nonparametric approach for determining the limit of detection. Clin Chem 2004;50:732–40. [13] Kobayashi H, Hasegawa Y, Endo M, Purevsuren J, Yamaguchi S. ESI–MS/MS study of acylcarnitine profiles in urine from patients with organic acidemias and fatty acid oxidation disorders. J Chromatogr B Analyt Technol Biomed Life Sci 2007;855:80–7. [14] Mueller P, Schulze A, Schindler I, Ethofer T, Buehrdel P, Ceglarek U. Validation of an ESI–MS/MS screening method for acylcarnitine profiling in urine specimens of neonates, children, adolescents and adults. Clin Chim Acta 2003;327:47–57. [15] Jiang LL, Kurosawa T, Sato M, Suzuki Y, Hashimoto T. Physiological role of D-3hydroxyacyl-CoA dehydratase/ D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein. J Biochem 1997;121:506–13.
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Urine acylcarnitine analysis by ESI–MS/MS: A new tool for the diagnosis of peroxisomal biogenesis disorders Guglielmo Duranti a,e,1, Sara Boenzi a,1, Cristiano Rizzo a, Lucilla Ravà b, Vincenzo Di Ciommo b, Rosalba Carrozzo c, Maria Chiara Meschini c, David W. Johnson d, Carlo Dionisi-Vici a,⁎ a
Division of Metabolism, Bambino Gesù Research Institute, Piazza S. Onofrio 4, 00165 Rome, Italy Unit of Epidemiology and Statistics, Bambino Gesù Research Institute, Piazza S. Onofrio 4, 00165 Rome, Italy Unit of Molecular Medicine, Bambino Gesù Research Institute, Piazza S. Onofrio 4, 00165 Rome, Italy d Department of Genetic Medicine, Women's and Children's Hospital, North Adelaide, Australia e Department of Human Movement and Sport Sciences, Rome University of Movement Sciences IUSM, Piazza Lauro de Bosis 15, 00194 Rome, Italy b c
a r t i c l e
i n f o
Article history: Received 4 April 2008 Received in revised form 6 August 2008 Accepted 21 August 2008 Available online 28 August 2008 Keywords: Peroxisomal biogenesis disorders Zellweger syndrome Infantile Refsum disease Tandem mass spectrometry ESI–MS/MS Acylcarnitines
a b s t r a c t Background: Patients with peroxisomal biogenesis disorders (PBDs) have an abnormal profile of circulating acylcarnitines (i.e. elevated C16:0-DC-, C18:0-DC-, C24:0-, C26:0-carnitine). We developed an ESI–MS/MS method for quantification of urine acylcarnitines and tested its reliability for the diagnosis of PBDs. Methods: Urine from 7 patients with PBDs (5 Zellweger syndrome, 2 infantile Refsum disease), from 2 patients with D-bifunctional protein (D-BP) deficiency, and from 130 healthy controls were analysed by ESI– MS/MS, using a multiple reactions monitoring (MRM) method, and quantified with labelled internal standards. Acylcarnitine levels between groups were analyzed by the STATA™ statistics data analysis and compared by the non parametric Mann–Whitney test. Results: In PBDs, the urinary excretion of long-chain dicarboxylylcarnitines (C14:0-DC-, C16:0-DC-, and C18:0DC-carnitine), and of very long-chain monocarboxylylcarnitines (C22:0-, C24:0-, C26:0-carnitine) were significantly elevated compared to controls (p b 0.0001). Interestingly, among PBDs the most severe abnormalities of acylcarnitine profile were observed in patients with Zellweger syndrome. One patient with D-BP showed similar abnormalities to PBDs, while in the other only C16:0-DC-carnitine was markedly elevated. Conclusions: This study shows that MRM ESI–MS/MS acylcarnitine analysis unequivocally discriminates patients with PBDs and D-BP deficiency from controls, representing a reliable and sensitive method for the diagnosis that requires a short-time analysis with high sample through-put. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Peroxisomes are cellular organelles that catalyse a wide range of essential functions, playing a key role in the metabolism of a variety of lipids that also include β-oxidation of very long-chain fatty acids (VLCFA) and of long-chain dicarboxylic fatty acids [1–3]. Inherited defects of peroxisome metabolism are clinically and genetically heterogeneous and can be roughly classified into two main groups, peroxisome biogenesis disorders (PBDs) and single peroxisomal enzyme deficiencies [4,5]. Abbreviations: VLCFA, very long-chain fatty acids; PBDs, peroxisomal biogenesis disorders; GC/MS, gas chromatography/mass spectrometry; CoA, coenzyme-A; ESI–MS/ MS, electrospray ionization tandem mass spectrometry; MRM, multiple reaction monitoring; D-BP, D-bifunctional protein; C14:0-DC-carnitine, tetracanedioylcarnitine; C16:0-DC-carnitine, hexadecanedioylcarnitine; C18:0-DC-carnitine, octadecanedioylcarnitine; C22:0-carnitine, behenoylcarnitine; C24:0-carnitine, lignoceroylcarnitine; C26:0-carnitine, cerotoylcarnitine; CVs, Coefficients of variation; LoD, limit of detection; LoQ, limit of quantification. ⁎ Corresponding author. Division of Metabolism, Bambino Gesù Children's Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy. Tel.: +39 6 68592225; fax: +39 6 68592736. E-mail address: [email protected] (C. Dionisi-Vici). 1 These authors contributed equally to this work. 0009-8981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2008.08.018
Based on the clinical presentation, PBD patients can be categorized in the following major groups according to the spectrum of disease severity: classical Zellweger syndrome, neonatal adrenoleukodystrophy, rhizomelic chondrodysplasia punctata, and infantile Refsum disease. The most common diagnostic tool for peroxisomal disorders is the quantification of plasma VLCFA by gas chromatography/mass spectrometry (GC/MS), a time-consuming method with low instrument through-put due to sample preparation, derivatisation and analysis. Further complex biochemical investigations in body fluids and cultured fibroblasts as well as molecular analyses are needed to confirm the diagnosis [6]. Trans-esterification of coenzyme-A (CoA) esters with carnitine is an effective mechanism to buffer cellular organelles from the accumulation of acyl-CoA esters. We have previously shown that patients with peroxisomal biogenesis disorders (PBDs) have a characteristic profile of plasma and whole blood acylcarnitines with elevation of long-chain dicarboxylylcarnitines [i.e. hexadecanedioylcarnitine (C16:0-DC-carnitine), and octadecanedioylcarnitine (C18:0-DC-carnitine)] and of very long-chain monocarboxylylcarnitines [i.e. lignoceroylcarnitine (C24:0carnitine), and cerotoylcarnitine (C26:0-carnitine)], as detected by electrospray ionization tandem mass spectrometry (ESI–MS/MS) [7].
G. Duranti et al. / Clinica Chimica Acta 398 (2008) 86–89
Based on this observation, we developed a new multiple reaction monitoring (MRM) ESI–MS/MS method for quantification of acylcarnitine in urine and tested its reliability for the diagnosis of PBDs. 2. Materials and methods 2.1. Subjects Morning urine spot samples were collected from seven patients with proven PBDs. Five patients (pt. 1–5), all died within the first year of life presented with a classical Zellweger phenotype (i.e. craniofacial abnormalities, severe neurological dysfunction, profound hypotonia, seizures, abnormal brain MRI, eye abnormalities, liver dysfunction, adrenal gland insufficiency, and renal cysts). Two had a less severe clinical picture, consistent with infantile Refsum disease. Patient 6, who survived until the age of 5 years, presented with mild facial abnormalities, developmental delay, seizures, pigmentary retinopathy, and white matter abnormalities at brain MRI. Patient 7, presents a milder phenotype characterized by neurosensorial deafness, adrenal insufficiency, periventricular white matter hyperintensity and growth retardation. She is alive at the age of 8 years and presents only a minimal cognitive impairment. Urine was also collected from 2 patients (pt. 8–9) with isolated D-bifunctional protein [D-BP] deficiency. Both showed severe neurological impairment, seizures, pigmentary retinopathy, and neurosensorial deafness. All patients underwent a full biochemical assessment, including plasma VLCFA, phytanic and pristanic acids, bile acid profile, plasmalogens in erythrocytes, and urine organic acid GC/MS analysis. Those belonging to PBDs group showed an abnormal profile of all circulating metabolites. Patients with D-BP deficiency had normal phytanic acid. All patients showed dicarboxylic- and epoxy-dicarboxylic-aciduria. Biochemical diagnosis was confirmed with extensive studies in cultured fibroblasts (R.J.A. Wanders, Amsterdam, The Netherlands). As controls, spot morning urine samples were obtained from 130 healthy age-matched subjects. Patients and controls did not receive carnitine supplementation. The study was approved by the Institutional Review Board and informed consent was obtained from the controls' and patients' families. 2.2. Sample preparation Working methanol solutions containing 0.04 μmol 2[H]9-hexadecanedioylcarnitine [8], 0.04 μmol 2[H]4-cerotoylcarnitine (synthesized by H.J. ten Brink, VU Medical Center, Amsterdam, The Netherlands) and 10 μmol of 2[H]3-creatinine (CDN. Isotopes, Pointe Claire, Canada) were prepared daily from internal standards stock solutions containing 5 μmol labelled acylcarnitines and 1.25 mmol labelled creatinine. Urine was applied on a neonatal screening filter paper WS 903 (Whatman, Dassel, Germany) and dried. A filter paper disc urine spot of 5 mm diameter, corresponding to 3.5 µL urine, was first extracted by agitation for 30 min at room temperature with 420 µL working methanol solution into a 1.5 mL Eppendorf tube (Eppendorf, Hamburg, Germany) and then evaporated under nitrogen stream. 100 µL of butanolic-HCl (Regis Technologies, Morton Grove, IL, USA) were added to each sample, heated at 65 °C for 60 min, and evaporated under nitrogen stream. Samples were reconstituted in 200 µL water/acetonitrile/formic acid (50:50:0.025 v/v/v) solvent. 2.3. Tandem mass spectrometry 30 µL of the butilated sample were automatically injected by an autosampling system (PE 200 series autosampler, Perkin Elmer, Norwalk, CT, USA) into a liquid chromatography pump (PE LC 200 series, Perkin Elmer, Norwalk, CT, USA) with solvent flow rate of 50 µL/min of water/acetonitrile/formic acid (50:50:0.025 v/v/v) to a triple quadruple tandem mass spectrometer (Sciex API 365; PE Sciex Instrument, Concord, ON, Canada). The run-time was 3.5 min for each sample. MRM ESI–MS/MS method was performed in positive ionisation. The collision energy was 44 eV. Specific MRM transitions were selectively analysed for long-chain dicarboxylylcarnitines [514/85 for tetracanedioylcarnitine (C14:0-DC-carnitine), 542/85 for C16:0-DC-carnitine, and 570/ 85 for C18:0-DC-carnitine], for very long-chain monocarboxylylcarnitines [540/85 for behenoylcarnitine (C22:0-carnitine), 568/85 for C24:0-carnitine and 596/85 for C26:0carnitine] and for creatinine [114/44 for creatinine and 117/47 for 2[H]3-creatinine]. Calculation of the concentration was done by comparing acylcarnitine ion intensities with corresponding labelled internal standards with Neonatal Script software (PE Sciex Instrument, Concord, ON, Canada ). The long-chain dicarboxylylcarnitines were compared to 2[H]9-hexadecanedioylcarnitine, and the very long-chain monocarboxylylcarnitines were compared to 2[H]4-cerotoylcarnitine. Values were expressed as μmol/mmol creatinine. Results of creatinine obtained with ESI–MS/MS were also compared with those obtained by a Hitachi analysis (Jaffe method). 2.4. Precision, accuracy, linearity and limit of detection MRM ESI–MS/MS method was validated for linearity, recovery, run-to-run, withinrun accuracy and limit of detection analysis. The method was set as established for
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tandem mass spectrometry in clinical chemistry by following the C50-A and the EP15A2 of CLSI [9,10] guidelines. Control urine was screened prior to spiking to ensure that it was free of endogenous interference of C16:0-DC-carnitine. For linearity and recovery assays, 0.01, 0.04, 0.08, 0.16 and 0.32 μmol of C16:0-DCcarnitine were added to control urine and tested. Median values of four replicates in 5 days were used by Ordinary Least Squares for regression analysis. For method validation three concentrations [0.04 μM C16:0-DC-carnitine (low); 0.08 μM C16:0-DCcarnitine (medium); 0.16 μM C16:0-DC-carnitine (high)] were added to control urine with 20 mM creatinine level. Assays were performed in 5 days in four replicates as defined in case of unknown coefficient of variation. For limit of detection (LoD) and quantification (LoQ) we follows the NCCLS protocol [11] by testing 60 times within the same day a control sample (without added C16:0DC-carnitine) and 60 times a 0.004 μmol/L C16:0-DC-carnitine sample. LoB was calculated as the 95th percentile of a series of results obtained in the control sample without added C16:0-DC-carnitine. Type I and II errors were set to α = β = 5% [12]. To determine LoD we used the following formula LoD = LoB + 1.645 × SD, where SD is the estimated standard deviation of the sample distribution at 0.004 μmol/L concentration C16:0-DC-carnitine. The test results of LoD were used to estimate bias and imprecision. The standard error for LoD [SE(LoD)] when α and β error were set to 0.05 were calculated with the Linnet and Kondratovich formula [12]. The difference of the main of the replicate and the accepted reference values is an estimate of bias. Total error at that level was calculated as Total Error = Bias + 2 × SD. LoQ was calculated as ten times standard deviation of the LoB. The prepared standard samples were stable for more than 24 h at room temperature, with a 5.5% mean percentage decrease in concentration. 2.5. Statistical analysis Data were analyzed by STATA™ statistics data analysis. Median of acylcarnitine values were compared through Mann–Whitney U-test. The level of significance was set at p ≤ 0.05.
3. Results The linearity study showed that the curve was linear from 0.01 to 0.32 μmol for C16:0-DC-carnitine (slope: 0.9748 ± 0.0636; intercept: 0.0019 ± 0.0048; R2 = 0.9965 ± 0.0037). The mean recoveries across different C16:0-DC-carnitine concentrations were 95.9% (0.04 μmol/L), 94.2% (0.08 μmol/L) and 93.9% (0.16 μmol/L), respectively. Table 1 shows the results of run-to-run and within-run accuracies; CVs at different concentrations of C16:0-DC-carnitine were all below 10%. The limit of detection (LoD) for C16:0-DC-carnitine was 0.0026 μmol/L with a standard error of 0.0003, the calculated Bias was −0.0003 and total error 0.0009. The limit of quantification (LoQ) was 0.0103 μmol/L. Urinary creatinine values correlated with those obtained using an automatic Hitachi analyser (Jaffe method) (R = 0.9999). Quantitative values of urinary long-chain dicarboxylylcarnitines and very long-chain monocarboxylylcarnitines obtained from patients and controls are shown in Table 2 and Fig. 1. In all urine samples from PBD patients a highly significant increase of long-chain dicarboxylylcarnitines and of very long-chain monocarboxylylcarnitines was observed (p b 0.0001). Median values (μmol/mmol creatinine) for each compound were as follows: C14:0-DC-carnitine 0.92, C16:0-DC-carnitine 0.62, C18:0-DC-carnitine 0.28, C22:0-carnitine 0.05, C24:0-carnitine 0.05, C26:0-carnitine 0.02. As shown in Table 2, patients with Zellweger syndrome had a more pronounced excretion of long-chain dicarboxylylcarnitines compared to those with infantile Refsum disease. Urinary long-chain dicarboxylylcarnitines were also significantly elevated in D-BP deficient patients (p b 0.05). However, the very longchain monocarboxlylcarnitines were elevated only in one of the two patients studied (Table 2). Table 1 Run-to-run and within-run accuracy with different C16:0-DC-carnitine concentrations: low (0.04 μmol/L), medium (0.08 μmol/L), high (0.16 μmol/L) C16:0-DC-carnitine Mean total SD run-to-run total % CV SD within-run total % CV
0.04 μmol/L
0.08 μmol/L
0.16 μmol/L
0.038 0.003 8.31 0.003 7.74
0.075 0.005 6.78 0.005 6.20
0.150 0.012 7.67 0.011 7.48
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Table 2 Urinary acylcarnitine levels (μmol/mmol creatinine) obtained by MRM ESI–MS/MS analysis from patients with peroxisomal biogenesis disorders, with D-bifunctional protein deficiency, and from 130 healthy controls C14:0-DC- C16:0-DC- C18:0-DC- C22:0C24:0carnitine carnitine carnitine carnitine carnitine Peroxisomal biogenesis disorders Zellweger syndrome Pt. 1 1.35 0.62 Pt. 2 0.91 1.08 Pt. 3 2.09 0.97 Pt. 4 0.97 0.93 Pt. 5 0.92 0.34 Infantile Refsum disease Pt. 6 0.63 0.32 Pt. 7 0.59 0.36
C26:0carnitine
0.28 0.29 0.31 0.40 0.16
0.06 0.05 0.07 0.08 0.04
0.04 0.08 0.09 0.07 0.05
0.05 0.04 0.01 0.13 0.02
0.12 0.12
0.04 0.04
0.03 0.03
0.02 0.02
D-bifunctional protein deficiency Pt. 8 0.34 0.18 Pt. 9 0.85 0.83
0.04 0.27
0.02 0.04
0.01 0.03
nd 0.03
Controls (n = 130) Median 0.07 Mean±SD 0.11 ± 0.10 Highest value 0.31
nd 0.01 ± 0.02 0.03
nd nd 0.01
nd 0.01±0.01 0.03
nd nd nd
nd 0.01 ± 0.01 0.03
nd: not detectable.
Fig. 2 shows the MRM ESI–MS/MS acylcarnitine profile from a Zellweger patient compared with a control. 4. Discussion ESI–MS/MS is a powerful analytical platform used in many fields of laboratory diagnostics and therapeutic drug monitoring. The analysis of acylcarnitines using ESI–MS/MS has shown to be a powerful tool for
Fig. 2. Urinary acylcarnitine profiles obtained by MRM ESI–MS/MS analysis from a patient with a peroxisomal biogenesis disorder (A), and from a control (B). Specific MRM transitions (Q1/Q3) are as follows: 514/85, C14:0-DC-carnitine; 542/85, C16:0-DCcarnitine; 570/85, C18:0-DC-carnitine; 540/85, C22:0-carnitine; 568/85, C24:0-carnitine; 596/85, C26:0-carnitine. Q1/Q3 masses of internal standards (IS), 551/85 2[H]9hexadecanedioylcarnitine, 600/85 2[H]4-cerotoylcarnitine.
Fig. 1. Box plot showing quantitative values of urinary long-chain dicarboxylylcarnitines (A) and very long-chain monocarboxylylcarnitines (B) in 130 controls (empty bars), in 7 patients with peroxisomal biogenesis disorders (squared bars), and in two patients with D-bifunctional protein deficiency ( ).
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the diagnosis of many inherited metabolic diseases. Urinary acylcarnitine analysis by ESI–MS/MS can be used to diagnose most of the organic acidurias and fatty acid oxidation disorders [13,14] but so far has never been applied to detect PBDs. Based on our previous results showing an abnormal profile of circulating acylcarnitines in PBDs [7], we have tested whether similar abnormalities were also detectable in urine, a biological fluid more easily obtainable compared to blood. The use of MRM with labelled internal standards allowed high accuracy for quantitative acylcarnitine determination, enabling optimal compensation for losses during sample preparation and signal-intensity fluctuations due to matrix effects. Through this method, we were able to detect an unequivocally abnormal pattern of urine acylcarnitines in PBDs, and we enlarged the spectrum of abnormal carnitine species compared to the blood study [7]. All presented elevated urinary excretion of C14:0-DC-, C16:0-DC-, C18:0-DC-carnitine and of C22:0-, C24:0-, C26:0-carnitine esters. Interestingly, patients with the most severe phenotype (i.e. Zellweger syndrome) presented more pronounced abnormalities compared to those with a less severe clinical picture (i.e. infantile Refsum disease). In the two patients with D-BP deficiency, we observed different patterns. One presented with a profile similar to those observed in PBDs. In the other patient, only C16:0-DC-carnitine was markedly elevated, whereas the other carnitine esters were within control range
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or only mildly increased. These variations might be explained with a different residual activity of D-BP among the two patients, or by a different contribution of the second peroxisomal bifunctional protein enzyme (i.e. L-bifunctional protein) [2,15]. In conclusion, our study shows that MRM ESI–MS/MS acylcarnitine analysis unequivocally discriminates patients with PBDs and D-BP deficiency from controls, representing a reliable and sensitive method for the diagnosis that requires a short-time analysis with high sample through-put. References [1] Wanders RJ, Vreken P, Ferdinandusse S, et al. Peroxisomal fatty acid alpha- and beta-oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem Soc Trans 2001;29:250–67. [2] Ferdinandusse S, Denis S, Van Roermund CW, Wanders RJ, Dacremont G. Identification of the peroxisomal beta-oxidation enzymes involved in the degradation of long-chain dicarboxylic acids. J Lipid Res 2004;45:1104–11. [3] Reddy JK, Hashimoto T. Peroxisomal ß-oxidation and peroxisome proliferatoractivated receptor: an adaptive metabolic system. Annu Rev Nutr 2001;21:193–230. [4] Gould SJ, Raymond GV, Valle D. The peroxisome biogenesis disorders. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. 8th ed. New York, NY: McGraw-Hill; 2001. p. 3181–217. [5] Wanders RJA, Barth PG, Heymans HAS. Single peroxisomal disorders enzyme deficiencies. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. 8th ed. New York, NY: McGraw-Hill; 2001. p. 3219–56.
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