The natural history of classic galactosemia: lessons from the GalNet registry.

BACKGROUND: Classic galactosemia is a rare inborn error of carbohydrate metabolism, caused by a severe deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). A galactose-restricted diet has proven to be very effective to treat the neonatal life-threatening manifestations and has been the cornerstone of treatment for this severe disease. However, burdensome complications occur despite a lifelong diet. For rare diseases, a patient disease specific registry is fundamental to monitor the lifespan pathology and to evaluate the safety and efficacy of potential therapies. In 2014, the international Galactosemias Network (GalNet) developed a web-based patient registry for this disease, the GalNet Registry. The aim was to delineate the natural history of classic galactosemia based on a large dataset of patients. METHODS: Observational data derived from 15 countries and 32 centers including 509 patients were acquired between December 2014 and July 2018. RESULTS: Most affected patients experienced neonatal manifestations (79.8%) and despite following a diet developed brain impairments (85.0%), primary ovarian insufficiency (79.7%) and a diminished bone mineral density (26.5%). Newborn screening, age at onset of dietary treatment, strictness of the galactose-restricted diet, p.Gln188Arg mutation and GALT enzyme activity influenced the clinical picture. Detection by newborn screening and commencement of diet in the first week of life were associated with a more favorable outcome. A homozygous p.Gln188Arg mutation, GALT enzyme activity of ≤ 1% and strict galactose restriction were associated with a less favorable outcome. CONCLUSION: This study describes the natural history of classic galactosemia based on the hitherto largest data set.

[Chest pain, dyspnea, discomfort with swallowing]. / Thoraxschmerzen, Dyspnoe und Schluckbeschwerden.

Achalasia is a rare neuromuscular disease of the gastrointestinal tract, often characterized by unspecific chest pain, dysphagia and regurgitation. Our case shows the slowly progression of the disease and its frequent relapse. All possible treatment options are only for palliation, but depending on the method with good (long-term) results. In end-stage disease resection of the oesophagus is a possible treatment.

Intima-media thickness and carotid resistive index: progression over 6 years and predictive value for cardiovascular events.

PURPOSE: Intima-media thickness (IMT) of the common carotid artery and the resistive index (RI) of the internal carotid artery correlate with the degree of atherosclerosis and are predictors of cardiovascular morbidity and mortality. Limited or no data are available about long-term predictive values and the progression of the two markers themselves. MATERIALS AND METHODS: 145 patients with at least one cardiovascular risk factor or clinically manifest atherosclerosis were included. At enrollment and after 36 and 74 months, duplex sonographic measurements of IMT CCA and RI ICA were performed. During follow-up, the occurrence of cardiovascular events (cardiovascular death, myocardial infarction, stroke) was assessed. RESULTS: At baseline, IMT was 0.79 +/- 0.16 mm and RI 0.66 +/- 0.08. Log-rank analysis showed a continuous increase in the risk of a cardiovascular event with an increasing range of IMT (p = 0.011) and RI (p = 0.006). IMT progression in patients with low versus high atherosclerotic burden (as defined by SMART score < or =7 points and > 7 points) differs significantly (32 +/- 83 microm versus 95 +/- 125 microm; p < 0.002). IMT progression was even more pronounced in patients suffering a cardiovascular event (141 +/- 105 microm versus 54 +/- 111 microm; p < 0.001). No significant RI ICA progression could be detected during follow-up in any group (patients with low vs. high atherosclerotic burden 0.00 +/- 0.06 versus 0.00 +/- 0.04; p = n. s.; patients with vs. without cardiovascular event 0.00 +/- 0.05 versus 0.01 +/- 0.03; p = n. s.). CONCLUSION: Our results confirm the predictive value for cardiovascular events of RI and IMT in long-term follow-up. In contrast to RI, IMT increases over six years, above all in patients suffering a cardiovascular event. The results suggest that IMT is suitable for cardiovascular risk prediction as well as for progression measurements, while RI cannot be recommended for progression measurements. The effect of drug therapy on RI needs further clarification.

Quantitative d-dimer levels and the extent of venous thromboembolism in CT angiography and lower limb ultrasonography.

BACKGROUND: We evaluated the diagnostic yield of multidetector-row CT angiography and determined the clot burden within pulmonary vasculature as a measure of pulmonary embolism (PE) severity at different d-dimer levels and pretest clinical probabilities. PATIENTS AND METHODS: 254 consecutive patients referred to CT pulmonary angiography for suspected PE after d-dimer testing were grouped into clinical probability classes using Wells' score, and the frequency of PE was determined. A score representing clot burden within pulmonary vasculature was calculated from the number of obstructed segmental arteries in CT scans in a partly differing group of 96 PE positive patients. RESULTS: The prevalence of PE increases with the d-dimer level (7% at d-dimer levels of 0.5-1 microg/ml, reaching 90% at d-dimer levels > 9 microg/ml; p < 0.001). D-dimer levels above 4 microg/ml are associated with a significantly higher clot burden in pulmonary arteries (median score 11 versus 5, and 53% versus 16% of patients in the subgroup with a score > 10 points; p < 0.001), and thrombus in a main pulmonary artery was detected more frequently (37% versus 9%, p = 0.003). Similar results were obtained for distal versus proximal deep venous thromboses, detected by ultrasonography of the lower limb in a separate group of 44 patients. CONCLUSIONS: High d-dimer levels are associated with an increased prevalence of CT radiographic findings indicating extended clinically severe PE or lower limb venous thrombosis.

Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium.

Isotope effects on the central carbon metabolism due to the addition of variable amounts of D2O (0 to 70%) were investigated with biosynthetically directed fractional 13C-labeling for Escherichia coli BL21(DE3) cells during exponential growth on a M9 minimal medium containing a mixture of 70% unlabeled and 30% uniformly 13C-labeled glucose as the sole carbon source. The resulting 13C-labeling patterns in the amino acids were analysed by two-dimensional [13C,1H]-correlation spectroscopy. With the aforementioned growth conditions, higher D2O contents resulted in an increase of the anaplerotic supply of the tricarboxylic acid cycle via carboxylation of phosphoenolpyruvate when compared to the influx of acetyl-CoA. Furthermore, the addition of D2O affected the C1 metabolic pathways that involve Ser and Gly. Otherwise the E. coli cells showed identical topologies of the active biosynthetic pathways in H2O and at elevated D2O contents, and the metabolic flux ratios characterizing glycolysis and the pentose phosphate pathway were not measurably affected by the addition of D2O. Cells that had been adapted for growth in D2O exhibited the same response to the presence of D2O in the nutrient medium as non-adapted cells. Implications of these data for the preparation of recombinant deuterated proteins for NMR studies are discussed.

Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism.

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica.

Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.

Bioreaction network topology and metabolic flux ratio analysis by biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy.

Biosynthetically directed fractional 13C labeling of the proteinogenic amino acids is achieved by feeding a mixture of uniformly 13C-labeled and unlabeled carbon source compounds into a bioreaction network. Analysis of the resulting labeling pattern enables both a comprehensive characterization of the network topology and the determination of metabolic flux ratios. Attractive features with regard to routine applications are (i) an inherently small demand for 13C-labeled source compounds and (ii) the high sensitivity of two-dimensional [13C,1H]-correlation nuclear magnetic resonance spectroscopy for analysis of 13C-labeling patterns. A user-friendly program, FCAL, is available to allow rapid data analysis. This novel approach, which recently also has been employed in conjunction with metabolic flux balancing to obtain reliable estimates of in vivo fluxes, enables efficient support of metabolic engineering and biotechnology process design.

Metabolic fluxes in riboflavin-producing Bacillus subtilis.

The pentose phosphate pathway and the pyruvate shunt were identified as major pathways of glucose catabolism in a recombinant, riboflavin-producing Bacillus subtilis strain. Reactions connecting the tricarboxylic acid cycle and glycolysis, catalyzed by the malic enzyme and phosphoenolpyruvate carboxykinase, consume up to 23% of the metabolized glucose. These are examples of important fluxes that can be accessed explicitly using a novel analysis based on synergistic application of flux balancing and recently introduced techniques of fractional 13C-labeling and two-dimensional nuclear magnetic resonance spectroscopy. The overall flux distribution also suggests that B. subtilis metabolism has an unusually high capacity for the reoxidation of NADPH. Under the conditions investigated, riboflavin formation in B. subtilis is limited by the fluxes through the biosynthetic rather than the central carbon pathways, which suggests a focus for future metabolic engineering of this system.