Minimally invasive (13)C-breath test to examine phenylalanine metabolism in children with phenylketonuria.
BACKGROUND
Phenylketonuria (PKU) is an autosomal recessive disorder caused by deficiency of hepatic phenylalanine hydroxylase (PAH) leading to increased levels of phenylalanine in the plasma. Phenylalanine levels and phenylalanine hydroxylase (PAH) activity monitoring are currently limited to conventional blood dot testing. 1-(13)C-phenylalanine, a stable isotope can be used to examine phenylalanine metabolism, as the conversion of phenylalanine to tyrosine occurs in vivo via PAH and subsequently releases the carboxyl labeled (13)C as (13)CO2 in breath.


OBJECTIVE
Our objective was to examine phenylalanine metabolism in children with PKU using a minimally-invasive 1-(13)C-phenylalanine breath test ((13)C-PBT).


METHODS
Nine children (7 M: 2 F, mean age 12.5 ± 2.87 y) with PKU participated in the study twice: once before and once after sapropterin supplementation. Children were provided 6 mg/kg oral dose of 1-(13)C-phenylalanine and breath samples were collected at 20 min intervals for a period of 2h. Rate of CO2 production was measured at 60 min post-oral dose using indirect calorimetry. The percentage of 1-(13)C-phenylalanine exhaled as (13)CO2 was measured over a 2h period. Prior to studying children with PKU, we tested the study protocol in healthy children (n = 6; 4M: 2F, mean age 10.2 ± 2.48 y) as proof of principle.


RESULTS
Production of a peak enrichment (Cmax) of (13)CO2 (% of dose) in all healthy children occurred at 20 min ranging from 17-29% of dose, with a subsequent return to ~5% by the end of 2h. Production of (13)CO2 from 1-(13)C-phenylalanine in all children with PKU prior to sapropterin treatment remained low. Following sapropterin supplementation for a week, production of (13)CO2 significantly increased in five children with a subsequent decline in blood phenylalanine levels, suggesting improved PAH activity. Sapropterin treatment was not effective in three children whose (13)CO2 production remained unchanged, and did not show a reduction in blood phenylalanine levels and improvement in dietary phenylalanine tolerance.


CONCLUSIONS
Our study shows that the (13)C-PBT can be a minimally invasive, safe and reliable measure to examine phenylalanine metabolism in children with phenylketonuria. The breath data are corroborated by blood phenylalanine levels in children who had increased responses in (13)CO2 production, as reviewed post-hoc from clinical charts.
Child & Family Research Institute, BC Children's Hospital, Vancouver, British Columbia, Canada; Department of Pediatrics, University of British Columbia, British Columbia, Canada.
Abrar
A
Turki
Child & Family Research Institute, BC Children's Hospital, Vancouver, British Columbia, Canada; Department of Pediatrics, University of British Columbia, British Columbia, Canada.
Gayathri
G
Murthy
Department of Pediatrics, University of British Columbia, British Columbia, Canada; Division of Biochemical Diseases, BC Children's Hospital, British Columbia, Canada.
Keiko
K
Ueda
Department of Pediatrics, University of British Columbia, British Columbia, Canada; Division of Biochemical Diseases, BC Children's Hospital, British Columbia, Canada.
Barbara
B
Cheng
Department of Pediatrics, University of British Columbia, British Columbia, Canada; Division of Biochemical Diseases, BC Children's Hospital, British Columbia, Canada.
Alette
A
Giezen
Child & Family Research Institute, BC Children's Hospital, Vancouver, British Columbia, Canada; Department of Pediatrics, University of British Columbia, British Columbia, Canada; Division of Biochemical Diseases, BC Children's Hospital, British Columbia, Canada.
Sylvia
S
Stockler-Ipsiroglu
Child & Family Research Institute, BC Children's Hospital, Vancouver, British Columbia, Canada; Department of Pediatrics, University of British Columbia, British Columbia, Canada; School of Population and Public Health, University of British Columbia, British Columbia, Canada. Electronic address: relango@cfri.ubc.ca.
Rajavel
R
Elango
D000293:Adolescent; D001708:Biopterins / Q000031:analogs & derivatives / Q000627:therapeutic use; D001944:Breath Tests / Q000379:methods*; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D002648:Child; D005260:Female; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D018832:Molecular Chaperones / Q000627:therapeutic use; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000378:metabolism; D010661:Phenylketonurias / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism*
D002247:Carbon Isotopes; D018832:Molecular Chaperones; D002245:Carbon Dioxide; D001708:Biopterins; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase; C003402:sapropterin
(13)C-phenylalanine; Breath test; Phenylalanine hydroxylase; Phenylketonuria; Sapropterin; Stable isotopes