The identification of molecular biomarkers is critical for diagnosing and treating patients and for establishing a fundamental understanding of the pathophysiology and underlying biochemistry of inborn errors of metabolism. Currently, liquid chromatography/high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy are the principle methods used for biomarker research and for structural elucidation of small molecules in patient body fluids. While both are powerful techniques, several limitations exist that often make the identification of unknown compounds challenging. Here, we describe how infrared ion spectroscopy has the potential to be a valuable orthogonal technique that provides highly-specific molecular structure information while maintaining ultra-high sensitivity. Here, we characterize and distinguish two well-known biomarkers of inborn errors of metabolism, glutaric acid for glutaric aciduria and ethylmalonic acid for short-chain acyl-CoA dehydrogenase deficiency, using infrared ion spectroscopy. In contrast to tandem mass spectra, in which ion fragments can hardly be predicted, we show that the prediction of an IR spectrum allows reference-free identification in the case that standard compounds are either commercially or synthetically unavailable. Finally, we illustrate how functional group information can be obtained from an IR spectrum for an unknown and how this is valuable information to, for example, narrow down a list of candidate structures resulting from a database query. Early diagnosis in inborn errors of metabolism is crucial for enabling treatment and depends on the identification of biomarkers specific for the disorder. Infrared ion spectroscopy has the potential to play a pivotal role in the identification of challenging biomarkers.

Transmitochondrial cell lines were isolated by fusing mtDNA-less rho degrees 206 cells with enucleated fibroblasts derived from four members of a pedigree carrying in their muscle varying proportions of the mutation at position 3243 in the tRNA(Leu(UUR)) gene associated with the MELAS encephalomyopathy. The mitochondrial transformants derived from an asymptomatic individual were all homoplasmic for wild-type mtDNA. The proportion of wild-type transformants derived from clinically affected members of the pedigree appeared to decrease in correspondence with an increase in severity of the clinical symptoms of the cell donor. Furthermore, the average proportion of wild-type mtDNA in the transformants derived from each member of the pedigree was very similar to that found in mtDNA from the fibroblasts of that individual, suggesting that the distribution of genotypes in the transformants reflected fairly closely that in the fibroblasts. The genotype and phenotype of ten transformants derived from one severely affected individual were investigated during continuous culture up to 17-24 weeks after the transformation step. Six heteroplasmic clones showed a progressive increase in the proportion of mutant mtDNA, whereas the mitochondrial genotype remained constant in four clones apparently homoplasmic for wild-type mtDNA or nearly homoplasmic for mutant mtDNA. An analysis of the rate of repopulation of rho degrees 206 cells with fibroblast-derived mtDNA revealed a large variability among different transformants, with the full re-establishment of the control ratio of mtDNA to nuclear DNA being observed between approximately 6 weeks and more than 22 weeks after the transformation step. An increase in rate of O2 consumption generally accompanied the increase in mtDNA copy number of the transformants, pointing to the important role of the mtDNA copy number in determining the phenotype of a cell. The observation that a very small amount of wild-type mtDNA (2 to 5% of the control level), coexisting with strongly predominant mutant mtDNA, conferred upon the transformants a substantial respiratory capacity (50% or more) and the evidence of proportionality between O2 consumption rate and mtDNA copy number, which occurred at widely different mutant to wild-type mtDNA ratios, strongly suggest a contribution of the mutant mtDNA to the cell respiratory competence.

A 2-month-old boy died of a lethal infantile mitochondrial disease with severe lactic acidosis and involvement of the CNS. Histochemical analysis of skeletal muscle showed that cytochrome c oxidase staining was lacking in all muscle fibers but was present in arterioles. Ragged red fibers were not seen, but some fibers showed excessive staining for succinate dehydrogenase. Biochemical analysis revealed a combined complex I and IV deficiency in skeletal muscle but only a complex I deficiency in his fibroblasts. Two-dimensional native SDS electrophoresis confirmed these enzymatic findings at the protein level. Analysis of mitochondrial translation products in fibroblasts revealed no abnormalities, and analysis of mitochondrial DNA in muscle showed no depletion, large-scale deletions, or frequently occurring point mutations. We conclude that this disease must have been the result of either a nuclear DNA mutation in a gene controlling the expression or assembly of both complex I and the muscle-specific isoform of complex IV or, alternatively, a heteroplasmic point mutation in a mitochondrial tRNA, which codon is used more often by mtDNA encoded subunits of complex I than by mtDNA encoded subunits of complex IV. A different degree of heteroplasmy in skeletal muscle and fibroblasts would then explain the curious heterogeneous tissue expression of defects in this patient.NEUROLOGY 1996;47: 243-248

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