Evaluation of acylcarnitines with dicarboxylic acid residues as candidate markers for neurodegenerative syndromes

The role of acylcarnitines with dicarboxylic acid residues has been investigated for the first time using labelled internal standards. The profile of acylcarnitines and amino acids is widely used in the neonatal screening, since a very long time, for the early diagnosis of metabolic disorders. The determination is performed by tandem mass spectrometry with electrospray ionization (ESI) and the semi-quantification of the different metabolites is evaluated by comparison with a suitable set of standards labeled with stable isotopes (2H and 13C). Recently some researchers suggested that these metabolites could be also markers for neurodegenerative syndromes. In this frame the role of acylcarnitines with dicarboxylic acid residues looks particularly important]. Commercially available kits containing isotopically labeled amino acids and acylcarnitines do not contain reference standards for carnitines O-acetylated with dicarboxylic acids. As we were interested to investigate this aspect, we synthesized two of these compounds with C6 and C12 dicarboxylic acid residues and containing three deuterium atoms in the carnitine moiety. The target standards have been obtained by optimized Steglich esterification reactions as butylated esters both at the carnitine and acid residue carboxylic groups. The analysis of dried blood spots was performed by the neonatal screening method by FIA-MS/MS using a precursors ion scan of m/z 85 for acylcarnitines and a neutral loss scan of m/z 102 for aminoacids. The novel standards have been added to commercial standards of labeled acylcarnitines and used to screen about 60 samples of dried blood spots provided by volunteers both healthy and bearing some neurodegenerative diseases. In particular, 25 samples came from healthy volunteers and have benn used as control samples; 27 samples were from patients with different mitochondrial disorders and 8 were from patients affected by Amyotrophic Later Sclerosis (ALS). The preliminary results obtained look encouraging, as shown in the figure below, even if some further experiments are needed. In particular, we could notice a general alteration of the acyl-carnitine profile for both the mithocondrial disorders and ALS, but this alteration was particularly significative for the acylcarnitines with dicarboxylic acid residues in patients affected by ALS. At present a larger scale screening is in progress in order to confirm these preliminary data.