Progress Toward Newborn Screening for X-linked Adrenoleukodystrophy (X-ALD) via Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

摘要

X-ALD is the most common genetically inherited peroxisomal disorder with an expected incidence of X-ALD males as 1 in 22,000 births. Approximately 35-40percent of X-ALD males have the childhood cerebral phenotype that is rapidly progressive once there is onset of brain white matter demyelination. Dietary therapy with Lorenzo's oil has been shown to prevent the neurological disease if started at 1 year of age. Bone marrow transplantation is an effective therapy if done at the first sign of progressive neurological disease. If the disease is not identified at the early stages, there is no known therapy and death usually occurs during childhood. Another clinical phenotype of X-ALD is adrenomyeloneuropathy (AMN) in which the subjects survive four to five decades. Hormone therapy for adrenal disease is effective for both the childhood and the adult phenotypes. Previous studies employing GC-MS analysis of saturated very long chain fatty acids (SVLCFA) in plasma extracts from newborns with X-ALD have shown that characteristic abnormalities of lipid metabolism are evident at birth (1). Preliminary studies employing LC-MS/MS analysis of lyso-phosphatidylcholine (lyso-PC) molecular species in blood spots and plasma have shown that two lyso-PCs with SVLCFA, tetracosanoyl-lyso-PC (24:0-lyso-PC) and hexacosanoyl-lyso-PC (26:0-lyso-PC) are elevated 5- to 10-fold in X-ALD subjects (2). The levels of eicosanoyl-lyso-PC (20:0-lyso-PC) and docosanoyl-lyso-PC (22:0-lyso-PC) are not significantly elevated in X-ALD subjects when compared to those of normal subjects. These finding strongly suggest that abnormal lipid metabolism characterized by elevated levels of 24:0- and 26:0-lyso-PC may be exploited as a means for screening for X-ALD in newborns. The high incidence of mortality of the childhood cerebral phenotype and debilitating morbidity of the AMN phenotype provide compelling reasons for newborn screening for X-ALD to identify subjects that could benefit with proven therapies of X-ALD. Authentic standards employed were as follows: lyso-PCs with saturated fatty acids ranging in chain length from 16-26 carbon atoms; saturated 16- and 18-O-alkyl linkages; oleic acid (18:1) and linoleic acid (18:2). Samples of venous spotted on #903 grade (S&S) filer paper were dried and stored at -20 degrees C until extraction. Tetradeuterated analogs of 16:0-lyso-PAF (~(2)H_(4)-lyso-PAF) and 26:0-lyso-PC (~(2)H_(4)-26:0-lyso-PC) were added prior to extraction with methanol. The extracts were dissolved in 100 (mu)l of methanol for injection. The authentic standards of the lyso-PCs were employed for: (1) optimization of the mass spectrometric parameters for instrument sensitivity and (2) reversed-phase chromatography for resolution of lyso-PC analytes from extraneous compounds present in extracts of blood spots and in plasma. ~(2)H_(4)-Lyso-PAF served as the internal standard for lyso-PCs with ester and alkyl linkages of fatty acids 16- and 18-cabon atoms. ~(2)H_(4)-26:0-Lyso-PC served as the internal standard for lyso-PCs with SVLCFA ranging in chain length from 20-26 carbon atoms.