Galactocerebrosidase assay on dried-leukocytes impregnated in filter paper for the detection of Krabbe disease.

BACKGROUND: Krabbe disease (KD) is an inherited lysosomal storage disease (LSD) caused by the deficiency of galactocerebrosidase (GALC) and is characterized by a severe and progressive leukodystrophy with death frequently before one year of life in the classical early-onset form. As a consequence of the enzyme defect, globoid cells containing undigested galactosylceramide are observed and are characteristic of the disease. Hematopoietic stem cell transplantation is the current treatment for this disease, with some success in the classical cases if performed very early in life. Definitive diagnosis of KD is generally accessed by determination of GALC in leukocytes or fibroblasts. For the last few years, dried-blood filter paper (DBFP) samples have been increasingly used for lysosomal enzyme assays. Originally, some lysosomal enzymes could not be tested in DBFP samples using fluorometric assays, including GALC, heparan-sulfamidase and a few others. Recently, we reported successful results using dried-leukocytes filter paper (DLFP) samples for heparan sulfamidase and ß-galactosidase. Extending these studies, we present now a new GALC assay on these type of samples. METHODS: Adapted leukocyte fluorometric assay was used for the evaluation of GALC in DLFP samples. RESULTS: Our results using this method showed a clear discrimination between GALC levels observed in KD patients and healthy controls. CONCLUSIONS: The assay is robust and reliable and could be adopted by reference laboratories for diagnosis of LSDs. It is expected that the use of DLPF would make it possible to diagnose patients living in isolated areas, where liquid samples usually have to be transported over several days and sometimes across country borders before reaching reference laboratories.

Incorporation of sialyl groups into a brain unidentified compound with lipophilic properties.

Lactosylceramide (I), galactosylceramide (II) and lipids extracted from rat brain with methanol (III) were assayed as AcNeu acceptors from CMP-[3H] AcNeu. Rat brain microsomes in 3.2% Triton CF54 or the 100 000 g supernatant of these microsomes were used as enzyme source. For quantification of reaction products two methods were used: a) precipitation with a TCA-PTA reagent followed by extraction of the precipitate with C:M (2:1) ; b) extraction with C:M (2:1) and partition by Folch's method without salt; radioactivity was measured in the washed lower phase. No incorporation into II was detected. Using method a), incorporation into I and III was obtained. It was also found that 35% of the activity toward I and 65% of that toward III was solubilized. Using method b) of quantification, radioactivity was found in the lower phase when III was the acceptor. The product of sialylation of III was soluble in ether. Run in a column of Sephadex LH20 was eluted with chloroform in the same fraction in which a proteolipid fraction was eluted. The possibilities that the compound is either a proteolipid protein or a glycopeptide containing sialic acid are discussed.