Determination of total homocysteine in dried blood spots using high performance liquid chromatography for homocystinuria newborn screening.
BACKGROUND
The most widely used method for newborn screening for homocystinuria (HCU) is a semi-quantitative 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 micro mol/L, respectively. The mean tHcy DBS concentration in two cases of cystathionine-beta-synthase deficiency and one case of 5,10-methylentetrahydrofolate reductase deficiency was 22.7 +/- 2.88, 29.3 +/- 1.90, and 41.3 micro mol/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.
Febriani
Andi Dwi Bahagia
AD
Department of Pediatrics, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan. afebriani@hotmail.com
Sakamoto
Akiko
A
Ono
Hiroaki
H
Sakura
Nobuo
N
Ueda
Kazuhiro
K
Yoshii
Chiyoko
C
Kubota
Miho
M
Yanagawa
Junko
J
D000704:Analysis of Variance; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D006710:Homocysteine / Q000097:blood*; D006712:Homocystinuria / Q000097:blood / Q000517:prevention & control*; D006801:Humans; D007231:Infant, Newborn; D016014:Linear Models; D015997:Neonatal Screening / Q000379:methods*; D015203:Reproducibility of Results
D006710:Homocysteine