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A 9-month-old boy with seizures and discrepant urine tryptophan concentrations.

CASE

A 9-month-old boy with a history of seizures underwent a neurologic and biochemical-genetic evaluation. The brain MRI results were compatible with a diagnosis of Leigh disease, also known as subacute necrotizing encephalomyelopathy, a rare neurometabolic disorder that affects the central nervous system. The patient had been prescribed several antiepileptic medications, including levetiracetam, lamotrigine, phenobarbital, vigabatrin, and topiramate. Metabolic screening for free amino acids was performed on the child's urine, with concentrations quantified with an automated amino acid analyzer (Hitachi L-8800). This commercially available system couples ion-exchange liquid chromatography with postcolumn ninhydrin derivatization before UV detection. This analysis revealed a very large peak with a retention time consistent with the elution of tryptophan (Fig. 1). The calculated urinary excretion was 125 204 [micro]mol/g creatinine. In addition, the urinary concentrations of [gamma]-aminobutyric acid (GABA), [5] [beta]-alanine, [beta]-aminoisobutyric acid, and glutamine were also increased substantially. The concentrations of the remaining amino acids were within their respective reference intervals. For confirmation, we submitted a urine aliquot to an external reference laboratory for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis revealed a tryptophan excretion of 71 [micro]mol/g creatinine (reference interval, 15-302 [micro]mol/g creatinine).

DISCUSSION

The gross discrepancyin tryptophan concentration between the 2 assays, combined with the increased urinary excretion of GABA, [beta]-alanine, [beta]-aminoisobutyric acid, and glutamine, triggered an inquiry into the potential causes. The patient had a neonatal-onset seizure disorder, hypotonia, a developmental delay, and MRI findings compatible with Leigh disease, but isolated hypertryptophanuria is not a finding for this disorder. In addition, the overall prognosis of patients with Leigh disease is poor. The disease represents the clinical and radiologic expression of a group of inherited disorders of energy metabolism, specifically disorders of oxidative phosphorylation or pyruvate oxidation. The ultimate cause of Leigh disease is a failure of ATP production or energy metabolism. Leigh syndrome is diagnosed on the basis of progressive neurologic regression, often provoked by an infection, along with characteristic MRI findings (T2 prolongation in bilateral basal ganglia, in particular the putamen, brainstem nuclei, periaqueductal gray matter, and central white matter). Proton magnetic resonance spectroscopymay show increased lactate in the most affected areas. This description provides context for our study, because the increases in excreted urinary amino acids obtained with the ninhydrin-based Hitachi L-8800 analyzer are not typical of this disorder.

NONPHYSIOLOGICAL HYPERTRYPTOPHANURIA

True hypertryptophanuria can be caused either by a defect in the conversion of tryptophan to kynurenine or by an abnormality in renal amino acid transport. Although patients with Hartnup or Tada disease may exhibit a marked hypertryptophanuria, the degree to which tryptophan was increased in this patient's urine excretion suggested a nonphysiological mechanism. An inherent limitation of the Hitachi L-8800 amino acid system--and platforms based on liquid chromatography and spectrophotometry in general--is that analyte identification is based on retention times within the chromatographic system deployed. Thus, quantification may be compromised by interfering analytes coeluting with the analyte of interest. In our system, such a metabolite would both possess a tryptophan-like chromatographic affinity and react with ninhydrin. The tryptophan concentration of 71 [micro]mol/g creatinine measured with the LC-MS/MS system was within the expected concentration interval for tryptophan and was more consistent with the patient's clinical status. This fact, along with the enhanced specificity afforded by mass detection with LC-MS/MS analysis, confirmed our suspicion of pseudohypertryptophanuria in our patient.

[FIGURE 1 OMITTED]

POTENTIAL INFLUENCE OF ANTIEPILEPTIC DRUGS ON AMINO ACID CONCENTRATIONS

To identify the source of this artificial tryptophan increase, we considered the 5 prescribed therapeutic agents (Fig. 2) as potential interferences. The antiepileptic drug (AED) vigabatrin, a [gamma]-vinyl analog of the neurotransmitter GABA, appeared the most likely interfering compound because of the presence of a free primary amine and a carboxylic acid within its chemical structure that ninhydrin might label analogously to amino acids. This drug, an irreversible inhibitor of the enzyme GABA aminotransaminase (1) recently cleared by the US Food and Drug Administration, is used to treat spasms in infants and epileptic seizures. This induced reduction in GABA catabolism increases brain GABA concentrations to provide the drug's antiepileptic effect, because GABA is an important inhibitory transmitter in the central nervous system. Vigabatrin is cleared primarily via renal excretion and does not undergo extensive metabolism upon administration. Furthermore, vigabatrin alters the amino acid concentrations in the urine of certain patients, with the induction of increases in GABA, [beta]-alanine, and [beta]-aminoisobutyric acid, as observed in our patient (1,2).

To investigate this interference scenario, we qualitatively assessed the urinary presence of vigabatrin in house by an LC-MS/MS procedure that uses an AcquityUltraPerformance LC[R] System (Waters) coupled to a Waters Micromass[R] Quattro Premier MS/MS instrument. Amino acids were separated through ion-pairing reversed-phase liquid chromatography, with the ion-pair reagent pentadecafluorooctanoic acid providing the enhanced separation selectivity. The analytical protocol followed the methodology of published strategies for underivatized amino acid analyses by LC-MS/MS (3-5). This analysis revealed a large vigabatrin peak in the optimized MS/MS chromatogram (precursor m/z > production ion m/z; 130.1 m/z > 70.1 m/z). The identity of this parent drugwas confirmed by a retention time identical to that of a pure vigabatrin solution. The chromatograms of several control urine samples analyzed by the identical procedure were devoid of metabolite peaks when they were examined with the vigabatrin-specific MS/MS transition.

[FIGURE 2 OMITTED]

The presumptive presence of this AED in the urine of our patient was further confirmed by adding increasing amounts of vigabatrin (i.e., 0, 500, 1500, and 7500 [micro]mol/g creatinine) into aliquots of a control urine sample before UV analysis by the automated liquid chromatography amino acid analyzer. In the control urine sample, endogenous tryptophan eluted at 76.81 min. For the samples with added vigabatrin, the relative retention times were 76.86, 76.54, and 76.37 min for native tryptophan, and 77.82, 77.67, and 77.62 min for vigabatrin. The vigabatrin peak appeared immediately after the tryptophan peak in each of these samples, a result consistent with the 77.22-min elution time of the unknown interfering peak in our patient's urine. This assignment is supported by the relative proximity of the elution time for the purported vigabatrin interferent to the structurally analogous GABA (i.e., 75.40 min). Of note is that although the AED concentrations tested in these in vitro experiments were not sufficiently high to completely obscure the tryptophan peak, it is clear that the potential for such a scenario would exist at higher in vivo urine concentrations. Additionally, the limited sample volume precluded quantitative assessment of GABA, [beta]-alanine, and [beta]-aminoisobutyric acid by LC-MS/MS. Thus, our assignment of the increases in these amino acids to vigabatrin therapy was based on the literature (1, 2).

This case highlights vigabatrin's influence on urinary amino acid concentrations. In addition, it illustrates that knowledge of the ninhydrin-based chemical derivatization used within this amino acid analyzer provides the ability to distinguish potential interfering compounds according to their respective chemical structures and physicochemical properties. A careful review of both patient medications and the literature, especially with treatments that use recently approved therapeutics, should remain standard practice when laboratory results do not fit the patient's clinical picture. Given the widespread use of this assay for metabolic screening, particularly in pediatric settings, these tenets should reduce the risk of false chromatogram interpretations. Finally, as demonstrated through the incremental value gained, a wider acceptance and implementation of rapidly emerging quantitative LC-MS/MS amino acid protocols (6-9) may lead to a reduction in misdiagnoses of rare metabolic disorders.

QUESTIONS TO CONSIDER

1. What are 2 pathologic conditions that produce large increases in urine tryptophan?

2. What could potentially induce false increases in tryptophan concentrations when urine amino acids are quantified by ion-exchange liquid chromatographic separation and postcolumn ninhydrin derivatization before detection?

3. On the basis their respective chemical structures, do any of the prescribed AEDs react with ninhydrin and thus interfere with tryptophan measurement?

POINTS TO REMEMBER

* True hypertryptophanuria can be caused by a defect in the conversion of tryptophan to kynurenine (Hartnup disease) or by an abnormality in renal amino acid transport (e.g., Tada disease).

* Vigabatrin reacts with ninhydrin to interfere with urine amino acid quantification using a Hitachi L-8800 analyzer.

* Vigabatrin induces increases in urinary amino acids, particularly GABA, [beta]-alanine, and [beta]-aminoisobutyric acid.

* Knowledge of chemical structures and reaction mechanisms is critical for complete investigations of potentially interfering amino acid compounds.

* The literature describing potential interferences by therapeutic drugs should be consulted when non-physiological amino acid concentrations are obtained.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and(c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

References

(1.) Brandt NJ, Christensen E. [omega]-Aminoaciduria induced by [gamma]-vinyl-GABA. Lancet 1984;1(8374):450-1.

(2.) Lahat E, Ben-Zeev B, Zlotnik J, Sela BA. Aminoaciduria resulting from vigabatrin administration in children with epilepsy. Pediatr Neurol 1999;21:460-3.

(3.) Piraud M, Vianey-Saban C, Petritis K, Elfakir C, Steghens J-P, Morla A, Bouchu D. ESI-MS/MS analysis of underivatised amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionisation mode. Rapid Commun Mass Spectrom 2003;17:1297-311.

(4.) Piraud M, Vianey-Saban C, Petritis K, Elfakir C, Steghens J-P, Bouchu D. Ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometric analysis of 76 underivatized amino acids of biological interest: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Rapid Commun Mass Spectrom 2005;19:1587-602.

(5.) Piraud M, Vianey-Saban C, Bourdin C, Acquaviva-Bourdain C, Boyer S, Elfakir C, Bouchu D. 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;19:3287-97.

(6.) Zoppa M, Gallo L, Zacchello F, Giordano G. Method for the quantification of underivatized amino acids on dry blood spots from newborn screening by HPLC-ESI-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2006;831: 267-73.

(7.) Armstrong A, Jonscher K, Reisdorph NA. Analysis of underivatized amino acids in human plasma using ion-pairing reverse-phase liquid chromatography/time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2007;21:2717-26.

(8.) Dietzen DJ, Weindel AL, Carayannopoulus MO, Landt M, Normansell ET, Reimschisel TE, Smith CH. Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin derivatization. Rapid Commun Mass Spectrom 2008;22:3481-8.

(9.) Waterval WA, Scheijen JLJM, Ortmans-Ploemen MMJC, Habets-van der Poel CD, Bierau J. Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids is a reliable tool for the diagnosis and follow-up of patients with inborn errors of metabolism. Clin Chim Acta 2009;407:36-42.

Commentary

Tina M. Cowan *

Ptolemy et al. illustrate the long-recognized problem of coeluting drug peaks in cation-exchange amino acid analysis with ninhydrin detection. Such interference is often noted in the urine of patients on antibiotic therapy (ampicillin, amoxicillin), as well as other medications and radiopaque dyes (diatrizoate meglumine). Although anticonvulsants are less often observed, vigabatrin interference in patient urine samples has previously been described by Preece et al. (1) as a large peak eluting near tryptophan (specifically between ammonia and ornithine), undoubtedly representing the same peak as that seen here. Such interference is traditionally evaluated by comparing the ratio of the spectrophotometric absorbances at 570 nm and 440 nm to that from authentic amino acid calibrators. This calculation would have rapidly provided a clue that the peak represented an interferant rather than tryptophan. Additional support could have come by measuring the plasma tryptophan concentration, which is characteristically increased in primary disorders of tryptophan metabolism but, because of rapid renal drug clearance, is normal in cases of drug interference. Hartnup disease, a disorder of neutral amino acid transport, was less likely in this case, both on clinical grounds and because the overall Hartnup excretion pattern (including tryptophan, methionine, lysine, and glycine) was not present.

Interference from exogenous compounds can be overcome, as demonstrated here, by using the more specific approach of mass spectrometry. It is important to note, however, that increases in amino acids can be caused, either directly or indirectly, by a variety of therapies, including valproate (leading to increased glycine), methotrexate (homocysteine), arginine hydrochloride (arginine), and certain preparations of intravenous immunoglobulin (glycine). Analysis by mass spectrometry clearly would not circumvent the interpretive problems introduced by such preparations. Therefore, despite the increasing use of mass spectrometry by clinical laboratories, amino acid results should still be interpreted in the context of clinical, medication, and diet history whenever possible.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Reference

(1.) Preece MA, Sewell IJ, Taylor JA, Green A. Vigabatrin--interference with urinary amino acid analysis. Clin Chim Acta 1993;218:113-6.

Department of Pathology, Stanford University, Palo Alto, CA.

* Address correspondence to the author at: Stanford University, 3375 Hillview Ave., Palo Alto, CA 94304. Fax 650-724-1567; e-mail tcowan@stanford.edu.

Received December 16, 2010; accepted December 28, 2010.

DOI: 10.1373/clinchem.2010.160333

Commentary

Michael J. Bennett *

For several decades, the major method for the measurement of body fluid amino acids has been ion-exchange chromatographic separation with postcolumn ninhydrin derivatization and dual-wavelength spectrophotometric detection. Ninhydrin reacts with primary amines, including most amino acids, and some secondary amines. Identification is based on peak retention time and relative dual spectrophotometric signals. A number ofdrugs, including some commonly used antibiotics, have long been known to also react with ninhydrin and to interfere with amino acid measurement, particularly in urine (1).

This report highlights a case of a very large interference in the chromatographic region around tryptophan in a patient's urine that might have had inappropriate diagnostic implications had the investigation into the true nature of the ninhydrin-positive peak not been undertaken. Vigabatrin is one of the latest generation of anticonvulsant medications that interferes with the endogenous metabolism of [gamma]-aminobutyric acid aminotransferase. It is also a primary amine that is excreted unchanged in the urine. In the ion-exchange chromatography used in this report, the elution of urinary vigabatrin was sufficiently close to that of tryptophan to appear as a tryptophan peak and to initially indicate hypertryptophanuria. Such a finding was not consistent with the clinical phenotype of the patient, who was under investigation for a probable mitochondrial energy disorder that may have been associated with a generalized aminoaciduria. The subsequent differential diagnosis for tryptophanuria would have included Hartnup condition, a disorder caused by a mutation in the SLC6A19 transporter, in which urinary neutral amino acids are increased because of a failure of tubular reabsorption (2). Tryptophanuria associated with dwarfism is seen in the exceedingly rare defect initially described by Tada et al. and postulated to be a defect in the kynurenine pathway (3).

The true identity of the interfering compound was made via Ultra Performance Liquid Chromatography (UPLC) separation and peak identification by tandem mass spectrometry, which also demonstrated a nonpathologic urinary tryptophan concentration. The important point that this case report makes is the advantage of methods with positive identification that use mass spectrometry over methods that rely on retention time alone. This point can be applied to all separation procedures with non-mass spectrometric detection, including all HPLC, UPLC, capillary electrophoresis, and gas chromatography methods that rely on other detector types.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: M.J. Bennett, Clinical Chemistry, AACC.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

References

(1.) Slocum RH, Cummings JG. Amino acid analysis of physiological samples. In: Hommes FA, ed. Techniques in diagnostic human biochemical genetics. New York: Wiley-Liss; 1991. p 87-126.

(2.) Kleta R, Romeo E, Ristic Z, Ohura T, Stuart C, Arcos-Burgos M, et al. Mutations in SLC6A19, encoding BOAT1, cause Hartnup disorder. Nat Genet 2004;36: 999-1002.

(3.) Tada K, Ito H, Wada Y, Arakawa T. Congenital tryptophanuria with dwarfism ("H" disease-like clinical feature without indicanuria and generalized aminoaciduria):-a probably new inborn error of tryptophan metabolism. Tohoku J Exp Med 1963;80:118-34.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA.

* Address correspondence to the author at: Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, 5NW58, 34th St. and Civic Center Blvd., Philadelphia, PA 19104. Fax 215-590-1998; e-mail bennettmi@email.chop.edu.

Received December 7, 2010; accepted December 14, 2010.

DOI: 10.1373/clinchem.2010.160341

Adam S. Ptolemy, [1-2] * Yijun Li, [3] Tamara Sanderson, [1] Omar Khwaja, [4] Gerard T. Berry, [3] and Mark Kellogg [1]

[1] Department of Laboratory Medicine; [3] Manton Center for Orphan Disease Research, Division of Genetics, Department of Pediatrics; and [4] Department of Neurology, Children's Hospital Boston, Harvard Medical School, Boston, MA; and [2] current affiliation: Department of Laboratory Science, Covance Central Laboratory Services Inc., Indianapolis, IN.

[5] Nonstandard abbreviations: GABA, [gamma]-aminobutyric acid; LC-MS/MS, liquid chromatography-tandem mass spectrometry; AED, antiepileptic drug.

* Address correspondence to this author at: Department of Laboratory Science, Covance Central Laboratory Services Inc., 8211 SciCor Drive, Indianapolis, IN 46214. Fax 317-372-7990; e-mail: adam.ptolemy@covance.com.

Received February 5, 2010; accepted June 14, 2010.

DOI: 10.1373/clinchem.2010.144899
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Title Annotation:Clinical Case Study
Author:Ptolemy, Adam S.; Li, Yijun; Sanderson, Tamara; Khwaja, Omar; Berry, Gerard T.; Kellogg, Mark
Publication:Clinical Chemistry
Article Type:Clinical report
Geographic Code:1USA
Date:Apr 1, 2011
Words:3154
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