A new method to detect homocysteine in dried blood spots using thin layer chromatography

Abstract

Introduction: Homocystine had been difficult to detect by conventional methods in the newborn period even in patients with homocystinuria because homocystine easily conjugates with plasma proteins. However, the method of Kang et al. [1] enabled us to measure the total amount of homocysteine (free and protein-bound) in blood and urine by using 2-mercaptoethanol. We applied their method to thin layer chromatography (TLC) on the specimens from patients with homocystinuria due to cystathionine β-synthase deficiency, including two newborn patients. Methods: Extract from dried blood discs was applied to a cellulose thin layer plate after treatment with 2-mercaptoethanol (2-ME). Homocysteine-2-ME compound was identified by liquid chromatograph atmospheric pressure chemical ionization mass spectrography. Results: Using this technique, we identified total homocysteine in the blood not only of 10- and 12-year-old children with homocystinuria, but also of two newborn homocystinurics on the 11th and 20th day after birth, respectively. However, both newborns on day 5 showed only a trace amount of total homocysteine. Homocysteine in the dried blood discs of the two newborns was detectable even after 4 and 6.5 years storage at −15 °C. Conclusions: (1) Homocystinuria can be diagnosed by the TLC described here in the newborn period, possibly later than 2 weeks after birth; (2) a newly identified homocysteine-2-ME compound is S-(2-hydroxyethylthio)homocysteine; and (3) the methionine test had priority over the homocysteine test as a primary screening in our survey.