title: Development of an assay to simultaneously measure orotic acid, amino acids, and acylcarnitines in dried blood spots.
abstract: BACKGROUND
  Orotic aciduria in the presence of hyperammonemia is a key indicator for a defect in the urea cycle, specifically ornithine transcarbamylase (OTC) deficiency. Current newborn screening (NBS) protocols can detect several defects of the urea cycle, but screening for OTC deficiency remains a challenge due to the lack of a suitable assay. The purpose of this study was to develop a high-throughput assay to measure orotic acid in dried blood spot (DBS) specimens as an indicator for urea cycle dysfunction, which can be readily incorporated into routine NBS.
  
  
  METHODS
  Orotic acid was extracted from DBS punches and analyzed using flow-injection analysis tandem mass spectrometry (FIA-MS/MS) with negative-mode ionization, requiring <2 min/sample run time. This method was then multiplexed into a conventional newborn screening assay for analysis of amino acids, acylcarnitines, and orotic acid.
  
  
  RESULTS
  We describe 2 assays which can quantify orotic acid in DBS: a stand-alone method and a combined method for analysis of orotic acid, amino acids, and acylcarnitines. Both methods demonstrated orotic acid recovery of 75-85% at multiple levels of enrichment. Precision was also comparable to traditional FIA-MS/MS methods. Analysis of residual presumptively normal NBS specimens demonstrated a 5:1 signal to noise ratio and the average concentration of orotic acid was approximately 1.2 μmol/l. The concentration of amino acids and acylcarnitines as measured by the combined method showed no significant differences when compared to the conventional newborn screening assay. In addition, retrospective analysis of confirmed patients and presumptively normal newborn screening specimens suggests potential for the methods to identify patients with OTC deficiency, as well as other urea cycle defects.
  
  
  CONCLUSIONS
  The assays described here quantify orotic acid in DBS using a simple extraction and FIA-MS/MS analysis procedures that can be implemented into current NBS protocols.
authors.affiliation: Wisconsin State Laboratory of Hygiene, 465 Henry Mall, Madison, WI 53706, United States; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States. Electronic address: patrice.held@slh.wisc.edu.
authors.forename: Patrice K
authors.initials: PK
authors.lastname: Held
authors.affiliation: Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Atlanta, GA 30341, United States.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Haynes
authors.affiliation: Newborn Screening and Molecular Biology Branch, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Atlanta, GA 30341, United States.
authors.forename: Víctor R
authors.initials: VR
authors.lastname: De Jesús
authors.affiliation: Wisconsin State Laboratory of Hygiene, 465 Henry Mall, Madison, WI 53706, United States; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States.
authors.forename: Mei W
authors.initials: MW
authors.lastname: Baker
mesh_terms: D000596:Amino Acids / Q000097:blood*; D002331:Carnitine / Q000031:analogs & derivatives* / Q000097:blood; D059788:Dried Blood Spot Testing / Q000379:methods*; D005260:Female; D017022:Flow Injection Analysis; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D009963:Orotic Acid / Q000097:blood*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D056806:Urea Cycle Disorders, Inborn / Q000097:blood / Q000175:diagnosis
chemical_list: D000596:Amino Acids; C116917:acylcarnitine; D009963:Orotic Acid; D002331:Carnitine
keywords: Newborn screening; Ornithine transcarbamylase deficiency; Orotic acid; Urea cycle defects