Tatiana Yuzyuk1, Aiping Liu2, Amanda Thomas3, JoDell E Wilson4, Irene De Biase3, Nicola Longo3, Marzia Pasquali3. 1. Department of Pathology, University of Utah, Salt Lake City, UT, USA; ARUP Laboratories, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA. Electronic address: tatiana.n.yuzyuk@aruplab.com. 2. ARUP Laboratories, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA. 3. Department of Pathology, University of Utah, Salt Lake City, UT, USA; ARUP Laboratories, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA. 4. Department of Pathology, University of Utah, Salt Lake City, UT, USA; ARUP Laboratories, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA; Quest Diagnostics Nichols Institute, Chantilly, Virginia, USA.
Abstract
OBJECTIVES: Elevated levels of pipecolic acid (PA), α-aminoadipic semialdehyde (AASA) and its cyclic form Δ1-piperideine-6-carboxylate (P6C) are characteristic of pyridoxine dependent epilepsy (PDE), a rare disorder of inborn error of metabolism. Recent studies showed the effectiveness of dietary therapy in PDE patients and emphasized the importance of the assessment of these metabolites for monitoring treatment efficacy. The objective of this study was to develop a robust and sensitive method for simultaneous quantification of AASA-P6C and PA in plasma and urine. DESIGN AND METHODS: Plasma and urine samples were derivatized with 3N HCl in n-butanol (v/v) and injected onto ACQUITY BEH-C18 column. A gradient of water/methanol containing 0.1% formic acid was used for the chromatographic separation of AASA, P6C and PA. The analytes' concentrations were calculated using their calibration curves and the sum of AASA and P6C (AASA-P6C) was calculated. To evaluate the clinical utility of this test, samples from unaffected controls and patients with confirmed PDE were analyzed. RESULTS: The performance characteristics of the assay as well as sample stability and interferences were determined. The intra- and inter- assay CVs were ≤2.9% and ≤10.9% for AASA-P6C, and ≤3.3% and ≤12.6% for PA, respectively. Reference ranges for AASA-P6C and PA in plasma and urine were established. Comparison of values obtained from unaffected controls and PDE patients showed high clinical sensitivity and specificity of the assay. CONCLUSIONS: This novel method for the simultaneous quantification of AASA-P6C and PA in plasma and urine can be used in a clinical laboratory setting for the diagnosis and monitoring of patients with PDE.
OBJECTIVES: Elevated levels of pipecolic acid (PA), α-aminoadipic semialdehyde (AASA) and its cyclic form Δ1-piperideine-6-carboxylate (P6C) are characteristic of pyridoxine dependent epilepsy (PDE), a rare disorder of inborn error of metabolism. Recent studies showed the effectiveness of dietary therapy in PDE patients and emphasized the importance of the assessment of these metabolites for monitoring treatment efficacy. The objective of this study was to develop a robust and sensitive method for simultaneous quantification of AASA-P6C and PA in plasma and urine. DESIGN AND METHODS: Plasma and urine samples were derivatized with 3N HCl in n-butanol (v/v) and injected onto ACQUITY BEH-C18 column. A gradient of water/methanol containing 0.1% formic acid was used for the chromatographic separation of AASA, P6C and PA. The analytes' concentrations were calculated using their calibration curves and the sum of AASA and P6C (AASA-P6C) was calculated. To evaluate the clinical utility of this test, samples from unaffected controls and patients with confirmed PDE were analyzed. RESULTS: The performance characteristics of the assay as well as sample stability and interferences were determined. The intra- and inter- assay CVs were ≤2.9% and ≤10.9% for AASA-P6C, and ≤3.3% and ≤12.6% for PA, respectively. Reference ranges for AASA-P6C and PA in plasma and urine were established. Comparison of values obtained from unaffected controls and PDE patients showed high clinical sensitivity and specificity of the assay. CONCLUSIONS: This novel method for the simultaneous quantification of AASA-P6C and PA in plasma and urine can be used in a clinical laboratory setting for the diagnosis and monitoring of patients with PDE.
Authors: Udo Fh Engelke; Rianne E van Outersterp; Jona Merx; Fred Amg van Geenen; Arno van Rooij; Giel Berden; Marleen Cdg Huigen; Leo Aj Kluijtmans; Tessa Ma Peters; Hilal H Al-Shekaili; Blair R Leavitt; Erik de Vrieze; Sanne Broekman; Erwin van Wijk; Laura A Tseng; Purva Kulkarni; Floris Pjt Rutjes; Jasmin Mecinović; Eduard A Struys; Laura A Jansen; Sidney M Gospe; Saadet Mercimek-Andrews; Keith Hyland; Michèl Aap Willemsen; Levinus A Bok; Clara Dm van Karnebeek; Ron A Wevers; Thomas J Boltje; Jos Oomens; Jonathan Martens; Karlien Lm Coene Journal: J Clin Invest Date: 2021-08-02 Impact factor: 14.808