Very Long Chain Fatty Acid Analysis of Dried Blood Spots on Filter Paper to Screen for Adrenoleukodystrophy

Adrenoleukodystrophy (ALD) is an X-linked recessive disorder characterized by progressive demyelination of cerebral white matter and adrenal insufficiency. ALD is the most common peroxisomal disease, afflicting 1 in 20 000 male newborns. Patients with childhood ALD manifest changes of character or behavior and a decline of intelligence and visual or motor dysfunction as the first symptoms during school age. Most of them lapse into a vegetative state and die within several years of onset (1). Patients with all clinical subtypes, including childhood ALD, adolescent ALD, adult ALD (cerebral type), adrenomyeloneuropathy, and Addison disease without neurological symptoms, have defects in the ALD gene (2). Defective activity of peroxisomal lignoceroyl-CoA ligase is considered to lead to the accumulation of very long chain fatty acid (VLCFA) in various tissues and fluids as a secondary phenomenon (3)(4). Dietary erucic acid (C22:1) therapy may help prevent neurological deterioration in presymptomatic boys (5)(6). Patients in the early or presymptomatic stages may be candidates for bone marrow transplantation (5)(7). Gene therapy may also become possible. Screening for ALD, especially in the presymptomatic stage, will be important when these therapeutic methods are established, as in the cases of phenylketonuria or maple syrup urine disease. ALD is usually diagnosed by plasma VLCFA analysis (1)(8), sometimes followed by measurement of β-oxidation activity in cultured skin fibroblasts (9) and mutation analysis of the ALD gene (2)(10)(11)(12)(13)(14)(15)(16). However, screening requires a simpler and more economical method to deal with a large number of subjects. Utilization of a dried blood spot on filter paper for VLCFA analysis was reported previously (17). Here, we describe an easy method of VLCFA analysis of dried blood spots on filter paper and discuss the possibility of screening for ALD. Sep-Pak® silica cartridges for solid-phase extraction (VAC/1cc, part no. 23595) were purchased from Waters. n-Hexane and methyl-tert-butyl ether (MTBE) were prepared to make solution A (n-hexane/MTBE, 96:4, by vol) and solution B (n-hexane/MTBE, 200:3, by vol). Sep-Pak cartridges were preactivated by 1 mL of solution A followed by 3 mL of n-hexane. A dried blood spot on Guthrie filter paper, equivalent to 100 μL of blood, was added to a tube containing 0.25 mL of distilled water and 2.5 mL of chloroform/methanol (1:1, by vol) and soaked. The sample was then left for 1 h at room temperature after being shaken for a few minutes. After centrifugation, the eluent containing lipids was placed in a fresh tube and 1.25 mL of chloroform and 0.75 mL of distilled water were added. These contents were shaken for 3 min at room temperature and centrifuged for 3 min. The lowest fraction, containing total lipids, was aspirated and concentrated by evaporation. The residue was heated with 1 mL of 50 mL/L concentrated HCl in methanol at 100 °C for 1 h (17). There was no significant difference in the efficiency of methanolysis for 1 h or 2 h at 100 °C. Fatty acid methyl ester was extracted with 2 mL of n-hexane and concentrated by evaporation. The residue was dissolved in 0.5 mL of solution B and passed through the cartridge followed by 2.5 mL of solution B. Extraction of VLCFA took <4 h. After concentration, the extract was dissolved in 100 μL (minimal quantity for the autosampler) of n-hexane containing 0.05 g/L butyl hydroxytoluene and analyzed on a Hewlett-Packard 5890A gas chromatograph equipped with a splitless capillary injection system, a flame ionization detector, a fused silica capillary column (25 m × 0.32 mm, Model HP-1) and an autosampler (Model HP7673). The injection volume was 2 μL, and the temperatures at the injection and detection ports were 250 °C and 285 °C, respectively. The column temperature was increased from 60 °C to 180 °C at 15 °C/min, to 250 °C at 4 °C/min, to 280 °C at 15 °C/min, and maintained at 280 °C for 5 min. Helium was the carrier gas. Peaks were identified by comparison of retention times with those of authentic standards and measured by area. The ratios of lignoceric acid (C24:0) to behenic acid (C22:0) in dried blood spots on filter paper are shown in Fig. 1 , top. The mean ± SD values were 1.6 ± 0.2 in the 21 controls between ages 1 week and 1 month, 1.3 ± 0.1 in the 19 control infants of age 1 year, 1.2 ± 0.1 in the 27 controls between ages 2 and 5 years, and 1.3 ± 0.1 in the 25 controls between ages 6 and 15 years. The ratio was >1.5 in all ALD patients (1.8 ± 0.2) (10 with childhood ALD and 2 with adrenomyeloneuropathy) and in 6 of 7 carrier mothers (1.6 ± 0.2) (Fig. 1 , bottom). The C24:0/C22:0 ratios did not change at room temperature within 10 days. There were no significant sex- or age-related differences in the ratios of C24:0/C22:0 in serum sphingomyelin (8). In our previous data, the ratio of C24:0/C22:0 in the control group was 0.6 ± 0.1, whereas that of ALD patients was 1.4 ± 0.2 (8). About one-half of the control newborns showed C24:0/C22:0 ratios higher than the cutoff point. There may be an age-dependent difference in fatty acid composition of erythrocytes. It would be difficult to screen ALD patients in the neonatal period with the use of Guthrie paper. All children between ages 2 and 5 years showed ratios <1.5. ALD patients and many of the carriers could be distinguished. Although our method is not good for the screening of children under age 2 years, patients are in the presymptomatic stage during that period. The ratio of hexacosanoic acid (C26:0) to C22:0 was not useful for diagnosis because C26:0 produced a very small peak, and the SD was very wide. Therefore, it is appropriate to screen children between ages 2 and 5 years with a cutoff value of 1.5. Blood sampling is feasible because the health check for infants is performed at ages 4, 10, 18, and 36 months in Japan. Compared with the previous VLCFA analysis of serum, our method with dried blood spots on filter paper has some merits. First, it requires only 100 μL of blood, and the separation of serum is not necessary. Second, it is easy to use, and specimens can be sent by mail. Third, the extraction of fatty acids with the use of disposable prepacked silica Sep-Pak columns (18) is easy and takes a short time, so we can analyze samples from a large number of subjects. The gas chromatograph took ∼35 min to analyze one sample and 10 min to be ready for the next. At that rate, 32 subjects/day, i.e., 11 680 subjects/year can be analyzed theoretically with one gas chromatograph. Taking into account routine checks and maintenance, ∼100 gas chromatographs would be enough to analyze all male infants in Japan, 600 000 every year, and the time to measure a sample would be shortened. Although the extraction of fatty acids by our method is more complicated than that of Nishio et al. (17), the analysis by gas chromatography is easier to deal with and more economical than the gas chromatography–mass spectrometry they used. Therefore, despite some shortcomings such as the age dependence and the false-positive rate at a younger age, our method is useful for the screening of ALD in children. With our method, treatment in the early and presymptomatic stages is possible. The ratios of lignoceric acid (C24:0) to behenic acid (C22:0) in the dried blood spots on filter paper (top) and chromatograph of a sample from a control (bottom left) and from a patient with adrenoleukodystrophy (bottom right). Top: ALD, patients with adrenoleukodystrophy; Carrier, heterozygous mothers; 1W-1M, controls between ages 1 week and 1 month; 1Y, controls age 1 year; 2Y-5Y, controls between ages 2 and 5 years; 6Y-15Y, controls between ages 6 and 15 years. Mean ± SD in each group is expressed by a bar. Bottom, the peaks of C22:0 and C24:0 were identified by the retention time.