Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots.
BACKGROUND
Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS).


METHODS
We extracted 3/16-inch DBS punches with 300 microL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 microL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155-137) and 13C5-SUAC (m/z 160-142). Analysis time was 1.2 min.


RESULTS
SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13-81 micromol/L), with Tyr concentrations ranging from 65 to 293 micromol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 micromol/L.


CONCLUSIONS
The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.
Turgeon
Coleman
C
Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Magera
Mark J
MJ
Allard
Pierre
P
Tortorelli
Silvia
S
Gavrilov
Dimitar
D
Oglesbee
Devin
D
Raymond
Kimiyo
K
Rinaldo
Piero
P
Matern
Dietrich
D
D000596:Amino Acids / Q000097:blood*; D001800:Blood Specimen Collection; D002331:Carnitine / Q000031:analogs & derivatives* / Q000097:blood; D017022:Flow Injection Analysis; D006537:Heptanoates / Q000097:blood*; D006801:Humans; D007231:Infant, Newborn; D015997:Neonatal Screening / Q000379:methods*; D012189:Retrospective Studies; D053719:Tandem Mass Spectrometry; D020176:Tyrosinemias / Q000175:diagnosis*
D000596:Amino Acids; D006537:Heptanoates; C116917:acylcarnitine; C020804:succinylacetone; D002331:Carnitine