title: Clinical relevance of the discrepancy in phenylalanine concentrations analyzed using tandem mass spectrometry compared with ion-exchange chromatography in phenylketonuria.
abstract: BACKGROUND
  Metabolic control of phenylketonuria (PKU) and compliance with the low-phenylalanine (phe) diet are frequently assessed by measuring blood phe concentrations in dried blood spots (DBS) collected by patients instead of plasma phe concentrations.
  
  
  OBJECTIVE
  Our objective was to investigate the difference in blood phe concentrations in DBS collected by subjects and analyzed using either a validated newborn screening tandem mass spectrometry (MS/MS) protocol or ion-exchange chromatography (IEC) compared to plasma phe concentrations obtained simultaneously and analyzed using IEC.
  
  
  METHODS
  Three to four fasting blood samples were obtained from 29 subjects with PKU, ages 15-49 years. Capillary blood was spotted on filter paper by each subject and the DBS analyzed using both MS/MS and IEC. Plasma was isolated from venous blood and analyzed using IEC.
  
  
  RESULTS
  Blood phe concentrations in DBS analyzed using MS/MS are 28% ± 1% (n = 110, p < 0.0001) lower than plasma phe concentrations analyzed using IEC resulting in a blood phe concentration of 514 ± 23 μmol/L and a plasma phe concentration of 731 ± 32 μmol/L (mean ± SEM). This discrepancy is larger when plasma phe is > 600 μmol/L. Due to the large variability across subjects of 13.2%, a calibration factor to adjust blood phe concentrations is not recommended. Analysis of DBS using IEC reduced the discrepancy to 15 ± 2% lower phe concentrations compared to plasma analyzed using IEC (n = 38, p = 0.0001). This suggests that a major contributor to the discrepancy in phe concentrations is the analytical method.
  
  
  CONCLUSIONS
  Use of DBS analyzed using MS/MS to monitor blood phe concentrations in individuals with PKU yields significantly lower phe levels compared to plasma phe levels analyzed using IEC. Optimization of current testing methodologies for measuring phe in DBS, along with patient education regarding the appropriate technique for spotting blood on filter paper is needed to improve the accuracy of using DBS to measure phe concentrations in PKU management.
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: Bridget M
authors.initials: BM
authors.lastname: Stroup
authors.affiliation: Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: Patrice K
authors.initials: PK
authors.lastname: Held
authors.affiliation: Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: Phillip
authors.initials: P
authors.lastname: Williams
authors.affiliation: Departments of Plant Pathology and Statistics, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: Murray K
authors.initials: MK
authors.lastname: Clayton
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: Sangita G
authors.initials: SG
authors.lastname: Murali
authors.affiliation: Department of Pediatrics, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Rice
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: Denise M
authors.initials: DM
authors.lastname: Ney
keywords: AAA, amino acid analyzer; Amino acid analyzer; Bland–Altman; DBS, dried blood spot; IEC, ion-exchange chromatography; MS/MS, tandem mass spectrometry; Newborn screening; PKU, phenylketonuria; Phenylalanine analytical methods; pah, phenylalanine hydroxylase; phe, phenylalanine; tyr, tyrosine