Specific GAG ratios in the diagnosis of mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) screening is tedious and still performed by analysis of total glycosaminoglycans (GAG) using 1,9-dimethylmethylene blue (DMB) photometric assay, although false positive and negative tests have been reported. Analysis of differentiated GAGs have been pursued classically by gel electrophoresis or more recently by quantitative LC-MS assays. Secondary elevations of GAGs have been reported in urinary tract infections (UTI). In this manuscript, we describe the diagnostic accuracy of urinary GAG measurements by LC-MS for MPS typing in 68 untreated MPS and mucolipidosis (ML) patients, 183 controls and 153 UTI samples. We report age-dependent reference values and cut-offs for chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS) and specific GAG ratios. The use of HS/DS ratio in combination to GAG concentrations normalized to creatinine improves the diagnostic accuracy in MPS type I, II, VI and VII. In total 15 samples classified to the wrong MPS type could be correctly assigned using HS/DS ratio. Increased KS/HS ratio in addition to increased KS improves discrimination of MPS type IV by excluding false positives. Some samples of UTI patients showed elevation of specific GAGs, mainly CS, KS and KS/HS ratio and could be misclassified as MPS type IV. Finally, DMB photometric assay performed in MPS and ML samples reveal four false negative tests (sensitivity of 94%). In conclusion, specific GAG ratios in complement to quantitative GAG values obtained by LC-MS enhance discrimination of MPS types. Exclusion of patients with UTI improve diagnostic accuracy in MPS IV but not in other types.

Complex I, V, and MDH2 deficient human skin fibroblasts reveal distinct metabolic signatures by 1 H HR-MAS NMR.

In this study, we investigated the metabolic signatures of different mitochondrial defects (two different complex I and complex V, and the one MDH2 defect) in human skin fibroblasts (HSF). We hypothesized that using a selective culture medium would cause defect specific adaptation of the metabolome and further our understanding of the biochemical implications for the studied defects. All cells were cultivated under galactose stress condition and compared to glucose-based cell culture condition. We investigated the bioenergetic profile using Seahorse XFe96 cell analyzer and assessed the extracellular metabolic footprints and the intracellular metabolic fingerprints using NMR. The galactose-based culture condition forced a bioenergetic switch from a glycolytic to an oxidative state in all cell lines which improved overall separation of controls from the different defect groups. The extracellular metabolome was discriminative for separating controls from defects but not the specific defects, whereas the intracellular metabolome suggests CI and CV changes and revealed clear MDH2 defect-specific changes in metabolites associated with the TCA cycle, malate aspartate shuttle, and the choline metabolism, which are pronounced under galactose condition.

Triheptanoin - Novel therapeutic approach for the ultra-rare disease mitochondrial malate dehydrogenase deficiency.

Mitochondrial malate dehydrogenase (MDH2) deficiency (MDH2D) is an ultra-rare disease with only three patients described in literature to date. MDH2D leads to an interruption of the tricarboxylic acid (TCA) cycle and malate-aspartate shuttle (MAS) and results in severe early onset encephalopathy. Affected infants suffer from psychomotor delay, muscular hypotonia and frequent seizures. Laboratory findings are unspecific, including elevated lactate in blood and cerebrospinal fluid. Brain magnetic resonance imaging reveals delayed myelination and brain atrophy. Currently there is no curative therapy to treat this devastating disease. Here, we present a female patient diagnosed with MDH2D after a stroke-like episode at 18 months. Trio-whole exome sequencing revealed compound heterozygous missense variants in the MDH2 gene: c.398C>T, p.(Pro133Leu) and c.445delinsACA, p.(Pro149Hisfs*22). MDH2 activity assay and oxygraphic analysis in patient's fibroblasts confirmed the variants were pathogenic. At the age of 36 months, a drug trial with triheptanoin was initiated and well tolerated. The patient's neurologic and biochemical phenotype improved and she had no further metabolic decompensations during the treatment period suggesting a beneficial effect of triheptanoin on MDH2D. Further preclinical and clinical studies are required to evaluate triheptanoin treatment for MDH2D and other TCA cycle and MAS defects.

Live monitoring of cellular metabolism and mitochondrial respiration in 3D cell culture system using NMR spectroscopy.

BACKGROUND: Because of the interplay between mitochondrial respiration and cellular metabolism, the simultaneous monitoring of both cellular processes provides important insights for the understanding of biological processes. NMR flow systems provide a unique window into the metabolome of cultured cells. Simplified bioreactor construction based on commercially available flow systems increase the practicability and reproducibility of bioreactor studies using standard NMR spectrometers. We therefore aim at establishing a reproducible NMR bioreactor system for metabolic 1H-NMR investigations of small molecules and concurrent oxygenation determination by 19F-NMR, with in depth description and validation by accompanying measures. METHODS: We demonstrate a detailed and standardized workflow for the preparation and transfer of collagen based 3D cell culture of high cell density for perfused investigation in a 5 mm NMR tube. Self-constructed gas mixing station enables 5% CO2 atmosphere for physiological pH in carbon based medium and is perfused by HPLC pump. RESULTS & DISCUSSION: Implemented perfused bioreactor allows detection of perfusion rate dependent metabolite content. We show interleaved dynamic profiling of 26 metabolites and mitochondrial respiration. During constant perfusion, sequential injection of rotenone/oligomycin and 2-deoxy-glucose indicated immediate activation and deactivation of glycolytic rate and full inhibition of oxygen consumption. We show sensitivity to detect substrate degradation rates of major mitochondrial fuel pathways and were able to simultaneously measure cellular oxygen consumption.

Selective galactose culture condition reveals distinct metabolic signatures in pyruvate dehydrogenase and complex I deficient human skin fibroblasts.

INTRODUCTION: A decline in mitochondrial function represents a key factor of a large number of inborn errors of metabolism, which lead to an extremely heterogeneous group of disorders. OBJECTIVES: To gain insight into the biochemical consequences of mitochondrial dysfunction, we performed a metabolic profiling study in human skin fibroblasts using galactose stress medium, which forces cells to rely on mitochondrial metabolism. METHODS: Fibroblasts from controls, complex I and pyruvate dehydrogenase (PDH) deficient patients were grown under glucose or galactose culture condition. We investigated extracellular flux using Seahorse XF24 cell analyzer and assessed metabolome fingerprints using NMR spectroscopy. RESULTS: Incubation of fibroblasts in galactose leads to an increase in oxygen consumption and decrease in extracellular acidification rate, confirming adaptation to a more aerobic metabolism. NMR allowed rapid profiling of 41 intracellular metabolites and revealed clear separation of mitochondrial defects from controls under galactose using partial least squares discriminant analysis. We found changes in classical markers of mitochondrial metabolic dysfunction, as well as unexpected markers of amino acid and choline metabolism. PDH deficient cell lines showed distinct upregulation of glutaminolytic metabolism and accumulation of branched-chain amino acids, while complex I deficient cell lines were characterized by increased levels in choline metabolites under galactose. CONCLUSION: Our results show the relevance of selective culture methods in discriminating normal from metabolic deficient cells. The study indicates that untargeted fingerprinting NMR profiles provide physiological insight on metabolic adaptations and can be used to distinguish cellular metabolic adaptations in PDH and complex I deficient fibroblasts.

Correction to: Selective galactose culture condition reveals distinct metabolic signatures in pyruvate dehydrogenase and complex I deficient human skin fibroblasts.

The original version of this article contained an error in Table 2. The text in the second header line should read "GAL supernatant" and "GAL Medium" instead of "GLC supernatant" and "GLC Medium". The corrected Table 2 is given below. The original article has been corrected.