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Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening
Author(s) -
Febriani Andi Dwi Bahagia,
Sakamoto Akiko,
Ono Hiroaki,
Sakura Nobuo,
Ueda Kazuhiro,
Yoshii Chiyoko,
Kubota Miho,
Yanagawa Junko
Publication year - 2004
Publication title -
pediatrics international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.49
H-Index - 63
eISSN - 1442-200X
pISSN - 1328-8067
DOI - 10.1111/j.1442-200x.2004.01825.x
Subject(s) - homocystinuria , medicine , homocysteine , newborn screening , cystathionine beta synthase , high performance liquid chromatography , dried blood spot , chromatography , methionine , coefficient of variation , dried blood , pediatrics , amino acid , biochemistry , chemistry
Background: The most widely used method for newborn screening for homocystinuria (HCU) is a semiquantitative bacterial inhibition assay for measuring methionine concentration in dried blood spots (DBS). Because this method has resulted in a number of missed cases due to many factors, we developed a high performance liquid chromatography (HPLC) method with fluorescence detection to measure total homocysteine (tHcy) in DBS which might be useful for newborn screening for HCU.Methods: One disk of DBS 3 mm in diameter was sonicated in 10 min. The extract was reduced with dithioerythritol and was derivatized with 4‐aminosulfonyl‐7fluoro‐2,1,3‐benzoxadiazole before injection into HPLC.Results: This method showed good linearity ( r = 0.996), precision (coefficient of variation range 2.7−5%), and excellent correlation coefficient between DBS and serum tHcy, both in control ( r = 0.932) and patient samples ( r = 0.952). By this method, the mean tHcy concentration in DBS of preterm newborns, full‐term newborns, and adults was 1.4 ± 1.0, 2.5 ± 1.6, and 4.9 ± 1.5 µmol/L, respectively. The mean tHcy DBS concentration in two cases of cystathionine‐β‐synthase deficiency and one case of 5,10‐methylentetrahydrofolate reductase deficiency was 22.7 ± 2.88, 29.3 ± 1.90, and 41.3 µmol/L, respectively.Conclusions: The present method, which is rapid, user friendly and reliable, seems applicable to newborn screening of HCU in place of methionine measurement.