Useful second‐tier tests in expanded newborn screening of isovaleric acidemia and methylmalonic aciduria

Abstract

Common use of pivalate-generating antibiotics in newborns in Japan and low cutoff value of C5-acylcarnitine (C5) to detect mild forms of isovaleric acidemia (IVA) led to 1,065 positive results from IVA screening among 146,000 newborns tested by tandem mass spectrometry over the last 3 years. Using our method to determine isovalerylglycine (IVG) levels in dried blood spots (DBS) as a second-tier test with IVG cutoff value of 0.5 nmol/ml in DBS, one patient with severe IVA was identified, and no recall of the second DBS was needed. Retrospective analysis revealed that most patients with moderate to severe forms of IVA have decreased free-carnitine levels shortly after birth and higher levels of IVG than those of C5, which suggests that this method is useful in evaluating the severity of IVA. Another second-tier test, to measure methylmalonic acid (MMA) levels in DBS by gas chromatography/mass spectrometry (GC/MS), has been developed to overcome difficulties in screening methylmalonic aciduria (MMAU) and propionic acidemia. Methanol extract from DBS was dried and derivatized using N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide. GC/MS was performed using splitless injection, electron-impact ionization, and selected ion monitoring for data recording. MMAU patients had much higher DBS concentrations of MMA (24.2-321.9 nmol/ml) than control newborns (0.34 ± 0.11 nmol/ml). MMA measurement in DBS was thought to provide useful information about the severity of MMAU, as MMAU patients with high levels of MMA had decreased levels of free carnitine and mildly increased levels of propionylcarnitine.

Clinical validation of cutoff target ranges in newborn screening of metabolic disorders by tandem mass spectrometry: A worldwide collaborative project

“…When applicable, this statistical revision will take into consideration as a determining factor the growing number of available second tier tests. [37][38][39][40][41][42] These tests are performed on the same specimen submitted for the primary screening, with no additional patient contact, targeting informative analytes, which are not included in the primary screening.…”

N-carbamylglutamate treatment for acute neonatal hyperammonemia in isovaleric acidemia

“…More recently, a third group of individuals with mild biochemical abnormalities who can be asymptomatic have been identified through newborn screening of blood spots by tandem mass spectrometry. IVA is sometimes complicated by episodes of hyperammonemia [7][8][9][10].…”

Aspects of Newborn Screening in Isovaleric Acidemia

“…In order to differentiate SBCADD from IVA and to exclude interference from antibiotics, urine acylglycine analysis and/or quantitative organic acid analysis are performed. Furthermore, several strategies for second-tier testing in dried blood spots have been developed, including stable isotope dilution MS/MS analysis to determine isovalerylglycine [63,64] and ultra-performance liquid chromatography (UPLC)-MS/MS analysis of C5-carnitines [59,65].…”