Clinica Chimica Acta 442 (2015) 73–74

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Letter to the Editor Quantitative amino acid analysis by liquid chromatography–tandem mass spectrometry: Implications for the diagnosis of argininosuccinic aciduria Keywords: Argininosuccinic aciduria Argininosuccinate lyase Urea cycle Quantitative liquid chromatography/tandem mass spectrometry (LC–MS/MS)

Argininosuccinic aciduria (ASA; MIM 207900) is an autosomal recessive disorder caused by the deficiency of the urea cycle enzyme argininosuccinate lyase (ASL; MIM 608310) that catalyzes the breakdown of argininosuccinic acid (ASA) in arginine and fumarate. Clinical presentation is heterogeneous, varying from a neonatal form with severe hyperammonemia, progressing, if untreated, to encephalopathy, coma and death [1], to late-onset forms with or without episodic hyperammonemia [1–5]. The late-onset manifestations include neurocognitive deficiencies (intellectual disability, behavioral abnormalities, seizures), liver disease (hepatitis, cirrhosis) and systemic hypertension, some of which might be unrelated to hyperammonemia. Argininosuccinic aciduria is included in the newborn screening (NBS) uniform panel [6]. The NBS marker is citrulline, while argininosuccinic acid is not routinely monitored by all NBS programs. The disorder is confirmed by increased citrulline and ASA in plasma and urine. Long term treatment consists of a protein restricted diet, oral arginine supplementation and, in some cases, the use of scavengers such as phenylbutyrate or benzoate that improve clinical outcome in the majority of patients [1, 5,7,8]. Patients with ASA, especially the late-onset and milder variant, can be missed by NBS, if ASA is not monitored, because of normal citrulline concentration [9]. In these patients the detection of ASA in plasma can be challenging as well. Traditionally, the argininosuccinic acidrelated compounds (free acid and anhydrides) are identified by Ion Exchange Chromatography (IEC), although the identification lacks analyte specificity and requires additional analysis [10,11]. Tandem mass spectrometry-based methods for amino acid analysis have increased specificity and sensitivity. Here, we evaluate the sensitivity and specificity for ASA using a quantitative liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for amino acid analysis. This method allows detection of both ASA and ASA anhydrides without any need for further sample preparation, even at very low concentrations which would be undetectable by traditional IEC. Additionally, we reviewed 5 years (2009–13) of IEC results obtained by our clinical laboratory to evaluate the need for increased sensitivity and specificity for this analyte. Briefly, 45 amino acids and related compounds were labeled using the aTraq™ reagents from AB Sciex [12–14]; the chromatographic separation was obtained using ABSciex amino acid analysis column (C18, 4.6 × 150 mm, 5 μm); the LC–MS/MS system was an API 4000 tandem

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mass-spectrometer coupled with a Shimadzu HPLC, operated in scheduled selective reaction monitoring mode (SRM). Amino acid quantitation was obtained using specific internal standards, pre-labeled as part of the aTraq kit, together with a 6-point external calibration curve for each amino acid [13]. The assay analytical performance was similar to previously published data [13,14]. The new method shows a good correlation with the IEC method (Biochrom 20 plus or Biochrom 30 Amino Acid Analyzers [11]) (slopes between 0.93 and 1.04, y-intercepts between − 7.0 and +7.0, R2 N 0.96). Reference intervals for plasma and urine were established using samples from normal controls according to protocol approved by the Institutional Review Board of the University of Utah. Statistical analysis was performed using EP® evaluator software [Release 10 (2013), Data Innovations LLC] and/or GraphPad Prism® software [ver 5.04 (2010), GraphPad Software Inc]. Specific transitions were used to detect labeled free ASA and its internal standard (respectively: 439.2 N 121.1 and 431.2 N 113.1), and unlabeled ASA anhydrides (273.1 N 70.1) [15]; the free ASA external calibration curve (r = 0.99914) was used for both compounds. The low limit of quantification (LLOQ) for free ASA with the LC–MS/MS method was 2 μmol/l (coefficient of variation = 13.4%; recovery = 108.9%). To evaluate the clinical sensitivity of the LC–MS/MS method and compare it to the IEC method, we analyzed 35 plasma samples from 22 patients with ASA [M12, F10], including samples before and after diagnosis and treatment. In Fig. 1A, we compare total ASA concentration, free acid plus anhydride compounds, which represented 20% of the argininosuccinic acid-related compounds on average (N = 35; Spearman r = 0.9797, 95% confidence interval = 0.9595–0.9899; P b 0.0001). Although the two methods correlate very well at higher ASA levels, they do not correlate well at low concentrations (b60 μmol/l; N = 14; Spearman r = 0.7489, 95% confidence interval = 0.3469– 0.9184; P = 0.0021). A significant bias is present, with LC–MS/MS values being more than 30% higher (Deming regression slope = 1.611; intercept = −59.11; bias = 33.2%). This bias most likely reflects the difference in sensitivity between the two methods. ASA is also characterized by elevated citrulline; therefore we extended the comparison to this amino acid. Fig. 1B shows the comparison between the values of citrulline in the same cohort of samples obtained with the 2 methods (N = 35; Spearman r = 0.9766, 95% confidence interval = 0.9528– 0.9885; P b 0.0001). The 2 methods correlate very well both at high and at low citrulline concentrations (b60 μmol/l; N = 10; Spearman r = 0.9758; P b 0.0001). Remarkably, using the LC–MS/MS method we were able to identify ASA in all patients, even when ASA had not been detected by IEC. ASA is a specific biomarker, only elevated in plasma from ASA patients, and undetectable in plasma from ASA carriers and normal subjects. We tested analyte specificity by confirming the absence of ASA free acid and anhydrides in plasma in confirmed heterozygotes (N = 3), in 100 normal controls, and in 74 clinical samples reported as normal by IEC. ASA is present in the urine of normal subjects, although at much lower concentrations than in ASA patients. One hundred twenty nine samples of normal urines were tested to confirm previously reported age-dependent normal ranges [13]. Taken

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Letter to the Editor

Fig. 1. Plasma A) ASA (free acid and anhydride compounds) and B) citrulline concentrations by IEC (x axis) vs LC–MS/MS analysis (y axis) in 35 plasma samples from 22 confirmed ASA patients. In the lower right corner, only the low concentration (x b 60 μmol/l) data points are included (N = 14 for ASA; N = 10 for citrulline). Solid line indicates linear regression. Two-tailed P values for the Spearman correlation analysis are indicated.

together, these data suggest that the quantitative amino acid analysis by LC–MS/MS has a better diagnostic yield for argininosuccinic aciduria, allowing direct quantification of ASA levels at very low concentration. Between 2009 and 2013, we analyzed 342 plasma samples and 23 urine samples for amino acid analysis from 44 patients with argininosuccinic aciduria [mean age 8.19 ± 10.60 y; 25 M and 19 F]. The vast majority of the samples were sent as follow-up testing to monitor treatment. Few samples were diagnostic (9 samples). Plasma ASA levels varied greatly both among patients and among different samples from the same patient, reflecting treatment, metabolic control, and disease variability. Urinary excretion of ASA was markedly increased in all ASA samples (N 5000 μmol/g creatinine). In few plasma specimens from known patients, plasma ASA was undetectable by IEC. In 26 (out of 334; 7.8%) plasma samples, citrulline was normal, with low ASA levels (b60 μmol/l) in most of these samples. This has important diagnostic implications, since in the absence of clinical suspicion for ASA (normal citrulline) and/or other helpful tests, the lab may miss an ASA patient using the routine IEC analysis. Our data suggest that the ASA can be missed in patients' plasma samples with routine IEC amino acid analysis, with potential negative implications for the diagnosis. Our retrospective analysis confirmed the need for an improved sensitivity and specificity for ASA. Accurate and specific quantification of this analyte can be obtained by LC–MS/MS. In conclusion, the identification of small amounts of ASA in patients, but not in carriers, of this disease, without any further sample manipulation, greatly improves the odds of identifying mild or atypical cases. References [1] Erez A, Nagamani SCS, Lee B. Argininosuccinate lyase deficiency—argininosuccinic aciduria and beyond. Am J Med Genet C Semin Med Genet 2011;157:45–53. [2] Mori T, Nagai K, Mori M, et al. Progressive liver fibrosis in late-onset argininosuccinate lyase deficiency. Pediatr Dev Pathol 2002;5:597–601. [3] Tuchman M, Lee B, Lichter-Konecki U, et al. Urea cycle disorders consortium of the rare diseases clinical research network; cross-sectional multicenter study of patients with urea cycle disorders in the united states. Mol Genet Metab 2008;94:397–402. [4] Brunetti-Pierri N, Erez A, Shchelochkov O, Craigen W, Lee B. Systemic hypertension in two patients with ASL deficiency: a result of nitric oxide deficiency? Mol Genet Metab 2009;98:195–7. [5] Ficicioglu C, Mandell R, Shih VE. Argininosuccinate lyase deficiency: long-term outcome of 13 patients detected by newborn screening. Mol Genet Metab 2009;98:273–7. [6] Watson MS, Mann MY, Lloyd-Puryear MA, Rinaldo P, Howell RR, editors. Newborn screening: toward a uniform screening panel and system, 8(Suppl.). Genet Med; 2006. p. 1S–11S.

[7] Nagamani SC, Shchelochkov OA, Mullins MA, et al. A randomized controlled trial to evaluate the effects of high-dose versus low-dose of arginine therapy on hepatic function tests in argininosuccinic aciduria. Mol Genet Metab 2012;107(3):315–21. [8] Mercimek-Mahmutoglu S, Moeslinger D, Häberle J, et al. Long-term outcome of patients with argininosuccinate lyase deficiency diagnosed by newborn screening in Austria. Mol Genet Metab 2010;100(1):24–8. [9] Feuchtbaum L, Carter J, Dowray S, Currier RJ, Lorey F. Birth prevalence of disorders detectable through newborn screening by race/ethnicity. Genet Med 2012;14:937–45. [10] Ratner S, Kunkemueller M. Separation and properties of argininosuccinate and its two anhydrides and their detection in biological materials. Biochemistry 1966;5:1821–32. [11] Shih VE. Amino acid analysis in physician's guide to the laboratory diagnosis of metabolic diseases. London: Chapman&Hall; 1996. [12] Kaspar H, Dettmer K, Chan Q, et al. Urinary amino acid analysis: a comparison of iTRAQ-LC–MS/MS, GC–MS, and amino acid analyzer. 2009 J Chromatogr B Analyt Technol Biomed Life Sci 2009;877(20-21):1838–46. [13] Held PK, White L, Pasquali M. Quantitative urine amino acid analysis using liquid chromatography tandem mass spectrometry and aTRAQ reagents. J Chromatogr B Analyt Technol Biomed Life Sci 2011;879(26):2695–703. [14] Filee R, Schoos R, Boemer F. Evaluation of physiological amino acids profiling by tandem mass spectrometry. JIMD rep; 2013[Nov 5 epub ahead of print]. [15] Piraud M, Vianey-Saban C, Bourdin C, et al. A new reversed-phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism. Rapid Commun Mass Spectrom 2005;19(12):1587–602.

I. De Biase⁎ T. Yuzyuk N. Longo M. Pasquali Departments of Pathology, University of Utah ARUP Laboratories ARUP Institute for Clinical and Experimental Pathology; Salt Lake City, Utah, USA ⁎Corresponding author at: University of Utah School of Medicine Assistant Medical Director, Biochemical Genetics and Supplemental Newborn Screening, ARUP Laboratories 500 Chipeta Way, Salt Lake City, Utah 84108, United States. Tel.: +1 801 583 2787. E-mail address: [email protected] (I. De Biase). A. Liuc ARUP Institute for Clinical and Experimental Pathology; Salt Lake City, Utah, USA 23 December 2014