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Technical Briefs |
Departments of1 Genetics and 2 Medical Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
aaddress correspondence to this author at: Department of Genetics, King Faisal Specialist Hospital and Research Center, PO Box 3354, Riyadh 11211, Saudi Arabia; fax 966-1-442-4546, e-mail rashed{at}kfshrc.edu.sa
Methylmalonic acidemias, a group of heterogeneous disorders, are characterized by accumulation of methylmalonic acid (MMA) and its byproducts in biological fluids (1)(2). Methylmalonic acidemia is now included in all tandem mass spectrometry (MS/MS)-based newborn screening programs (3)(4)(5). Detection is based on the finding of increased propionylcarnitine and/or increased propionylcarnitine-to-acetylcarnitine ratio in dried blood spots (DBS) by MS/MS. These markers, however, are not specific because they are increased in propionic acidemia and, possibly, in multiple carboxylase deficiency (3). In most programs, newborns or patients with initial positive results are recalled for a second blood spot, and a urine sample is collected for organic acid analysis to differentiate among the three disorders.
In the present study, we used the intramolecular-excimer fluorescence derivatization approach of Nohta and coworkers (6)(7) to form a fluorescent derivative of MMA. This would allow the detection of MMA in DBS samples from affected neonates, leading to a conclusive diagnosis with the remains of the DBS within a short time, often the same working day.
From a DBS, four 3.2-mm discs were punched and extracted into 250 µL of methanol containing 20 µmol/L malonic acid (MA) as internal calibrator by vortex-mixing for 30 s and standing at room temperature for 1 h. After evaporation and reconstitution of the residue in 50 µL of water, we successively added 25 µL of 0.5 mol/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma) in water, 25 µL of 400 g/L pyridine in dimethylsulfoxide, and 50 µL of 15 mmol/L 1-pyrenebutyric hydrazide (Fluka Chemie) in dimethylsulfoxide, tightly capped and vortex-mixed the vials, and left them to stand at room temperature for 5 h. The injection volume was 5 µL, but injections up to 20 µL were possible without adverse effects on the resolution. Moreover, in case of a limited DBS sample, two punches can be used with the proportionate reduction in reagent volumes described above.
The dilabeling of MMA was confirmed by MS analysis (Quattro micro API; Micromass) with purified derivatives obtained by fractional collection. Two abundant ions at m/z 687 and m/z 709 appeared in the spectrum, which correspond to [M+H]+ of pyrene-dilabeled MMA derivative and the [M+Na]+ adduct, respectively.
Chromatography was performed on a Waters Breeze HPLC System, a model 2475 multi 
fluorescence detector (Waters) and a C8 Symmetry column [3.9 x 150 mm (i.d.), 5 µm bead size; Waters]. Mobile phases A and B were a mixture of acetonitrile–water (2:1 by volume) and acetonitrile, respectively, at a flow rate of 1 mL/min. The gradient was as follows: 0–5 min, 100% A; 5–8 min, 100% A to 10% A; 8–10 min, 10% A; 10–11 min, 10% A to 100% A; and 11–15 min, 100% A.
The maximum fluorescence values for the target analytes were obtained at an emission wavelength of 475 nm (excitation at 345 nm), with no interference observed from the monomeric fluorescing compounds, which emit at 385 nm. Fig. 1
shows three chromatograms obtained for a calibrator solution of MMA and internal calibrator and for extracts of DBS samples from known patients with methylmalonic acidemia. As shown in Fig. 1
, a single, well-resolved peak was obtained for each of the pyrene derivatives of internal calibrator and MMA at 4.5 and 5.5 min, respectively. No interference was observed from other components coextracted from the DBS for more than 100 different DBS samples analyzed or in pooled blood samples from healthy adults. Furthermore, the dicarboxylic acid isomer succinic acid, a potentially interfering factor, eluted earlier in the chromatogram at 3.9 min, whereas the higher homolog ethylmalonic acid eluted at 6.1 min (data not shown).
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The usefulness of the proposed method in giving a conclusive diagnosis for methylmalonic acidemia was assessed by a blinded retrospective study on DBS samples (n = 100) from known patients with methylmalonic acidemia (n = 33) or propionic acidemia (n = 33). The remaining DBS samples (n = 34) belonged to healthy infants. These samples were randomized, and the study was carried out with the analysts blinded to sample details. DBS samples prepared from MMA-supplemented blood served as calibrators for quantification. All samples from patients with methylmalonic acidemia gave a substantial peak corresponding to the pyrene derivative of MMA. On the other hand, no peak at the retention time of MMA was detectable in samples from healthy infants or from patients with propionic acidemia.
As shown in Table 1
, three groups of specimens from confirmed methylmalonic acidemia cases were analyzed: asymptomatic newborn screening specimens; symptomatic neonate specimens submitted for metabolic screening; and specimens from known patients. All three groups showed substantially increased MMA regardless of the age of the sample.
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In this report, we present the first clinical application of the intramolecular-excimer fluorescence derivatization approach for the determination of dicarboxylic acids in DBS specimens. Our main objective was to develop a second-tier test that would permit definitive diagnosis of methylmalonic acidemia from the same DBS sample received for MS/MS-based newborn screening. Thus, a newborn screening specimen flagged as abnormal because of high propionylcarnitine can be tested immediately for methylmalonic acidemia. The data presented for asymptomatic and symptomatic neonates show that this is possible.
Our results indicate that this method can be diagnostic for methylmalonic acidemia in DBS samples >100 months old, which suggests that this compound is relatively stable for many years if the DBS is stored at room temperature under relatively dry conditions. The method may therefore find use in retrospective screening of archived DBS specimens for previously undiagnosed cases suspected with methylmalonic acidemia. In this case, MS/MS analysis may be less useful because of the limited stability of carnitine esters (3).
To our knowledge, this is the first time that methylmalonic acidemia was definitively diagnosed from a DBS. Unfortunately, the method did not allow for the detection of control values for MMA, which are quite low (0–0.4 µmol/L). However, the same method may be useful if applied to serum or plasma samples and may serve as an alternative test for MMA in cobalamin deficiencies.
Acknowledgments
We gratefully acknowledge Prince Salman Center for Disability Research for funding this research.
References
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