Exercise Stress Testing in Children with Metabolic or Neuromuscular Disorders

The role of exercise as a diagnostic or therapeutic tool in patients with a metabolic disease (MD) or neuromuscular disorder (NMD) is relatively underresearched. In this paper we describe the metabolic profiles during exercise in 13 children (9 boys, 4 girls, age 5–15 yrs) with a diagnosed MD or NMD. Graded cardiopulmonary exercise tests and/or a 90-min prolonged submaximal exercise test were performed. During exercise, respiratory gas-exchange and heart rate were monitored; blood and urine samples were collected for biochemical analysis at set time points. Several characteristics in our patient group were observed, which reflected the differences in pathophysiology of the various disorders. Metabolic profiles during exercises CPET and PXT seem helpful in the evaluation of patients with a MD or NMD

Exercise Stress Testing in Children with Metabolic or Neuromuscular Disorders

The role of exercise as a diagnostic or therapeutic tool in patients with a metabolic disease (MD) or neuromuscular disorder (NMD) is relatively underresearched. In this paper we describe the metabolic profiles during exercise in 13 children (9 boys, 4 girls, age 5–15 yrs) with a diagnosed MD or NMD. Graded cardiopulmonary exercise tests and/or a 90-min prolonged submaximal exercise test were performed. During exercise, respiratory gas-exchange and heart rate were monitored; blood and urine samples were collected for biochemical analysis at set time points. Several characteristics in our patient group were observed, which reflected the differences in pathophysiology of the various disorders. Metabolic profiles during exercises CPET and PXT seem helpful in the evaluation of patients with a MD or NMD

Fatal outcome due to deficiency of subunit 6 of the conserved oligomeric Golgi complex leading to a new type of congenital disorders of glycosylation

Deficiency of subunit 6 of the conserved oligomeric Golgi (COG6) complex causes a new combined N- and O-glycosylation deficiency of the congenital disorders of glycosylation, designated as CDG-IIL (COG6-CDG). The index patient presented with a severe neurologic disease characterized by vitamin K deficiency, vomiting, intractable focal seizures, intracranial bleedings and fatal outcome in early infancy. Analysis of oligosaccharides from serum transferrin by HPLC and mass spectrometry revealed the loss of galactose and sialic acid residues, whereas import and transfer of these sugar residues into Golgi-enriched vesicles or onto proteins, respectively, were normal to slightly reduced. Western blot examinations combined with gel filtration chromatography studies in patient-derived skin fibroblasts showed a severely reduced expression of the mentioned subunit and the occurrence of COG complex fragments at the expense of the integral COG complex. Sequencing of COG6-cDNA and COG6 gene resulted in a homozygous mutation (c.G1646T), leading to amino acid exchange p.G549V in the COG6 protein. Retroviral complementation of the patients' fibroblasts with the wild-type COG6-cDNA led to normalization of the COG complex-depending retrograde protein transport after Brefeldin A treatment, demonstrated by immunofluorescence analysis

A 2D-DIGE Approach To Identify Proteins Involved in Inside-Out Control of Integrins

Leukocyte integrins are functionally regulated by "inside-out" signaling, meaning that stimulus-induced signaling pathways act on the intracellular integrin tail and induce activation of the receptor at the outside. Both a change in conformation (affinity) and in clustering (avidity/valency) of the receptors has been described to occur. This inside-out signaling is essential for adequate migration of leukocytes to inflammatory sites; however, the exact underlying mechanism is not known. We used two variants of a mouse acute lymphocytic leukemia cell line (L1210), a suspension (L1210-S) and an adherent (L1210-A) variant that were characterized by nonactivated and activated integrins (beta(1), beta(2) and beta(3)), respectively. L1210-S and L1210-A cells were compared on protein expression profiles by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE). We found 86 protein spots that were more than 1.25-fold different between L1210-A and L1210-S. Only 4 protein spots were more than 2.5-fold different. We identified 29 proteins by mass spectrometry among which were gelsolin, L-plastin, and Rho GTPase dissociation inhibitor 2. These proteins were upregulated in the L1210-A cells versus L1210-S, which was verified by Western blot analysis. Overexpression of gelsolin in U937 resulted in increased high affinity integrin expression and cell adhesion. Comparison of functionally different cell lines from similar origin by 2D-DIGE might be a successful approach to identify regulatory proteins involved in integrin inside-out control

Enhanced apoB48 metabolism in lipoprotein lipase X447 homozygotes

RATIONALE: Lipoprotein lipase (LPL) X447 homozygotes are characterized by enhanced conversion of TRL apoB100. Here, we set out to investigate whether this LPL variant is also associated with enhanced apoB48 clearance. Therefore, we evaluated apoB48 kinetics in X447 homozygotes in the fed state by infusion of isotope L-[1-(13)C]-valine and subsequent compartmental modeling. METHODS AND RESULTS: ApoB48 metabolism was assessed in five X447 homozygotes (X/X genotype) and five S447 homozygotes (S/S genotype). Subjects were continuously fed and received infusion of stable isotope L-[1-(13)C]-valine. Results were analyzed by SAAM II modeling. Fasting (2.4-fold, p=0.02) as well as non-fasting (1.6-fold, p=0.09) apoB48 concentration was increased in the X447 homozygotes compared to S447 homozygotes. In addition, the X447 homozygotes exhibited a 1.7-fold higher apoB48 poolsize (p=0.04). Interestingly, apoB48 fractional catabolic rate (FCR) was 1.9-fold higher (p=0.007) and apoB48 synthesis was more than two-fold higher (p=0.006) in the X447 homozygotes compared to S447 homozygotes. CONCLUSION: In the present study, we show that X447 homozygotes exhibit enhanced apoB48 clearance. Previously, these homozygotes were shown to present with enhanced apoB100 TRL conversion. Combined, this LPLS447X gain of function variant affects apoB48 as well as apoB100 TRL metabolism

Endogenous cholesterol synthesis is associated with VLDL-2 apoB-100 production in healthy humans

Subjects with high plasma cholesterol levels exhibit a high production of VLDL apolipoprotein B-100 (apoB-100), suggesting that cholesterol is a mediator for VLDL production. The objective of the study was to examine whether endogenous cholesterol synthesis, reflected by the lathosterol-cholesterol ratio (L-C ratio), affects the secretory rates of different VLDL subfractions. Ten healthy subjects were studied after overnight fasting. During a 10 h primed, constant infusion of 13C-valine (15 micromol/kg/h), enrichment was determined in apoB-100 from ultracentrifugally isolated VLDL-1 and VLDL-2 by gas chromatography mass spectrometry. The synthesis rates of VLDL-1 apoB-100 and VLDL-2 apoB-100, catabolism, and transfer were estimated by compartmental analysis. Mean VLDL-1 apoB-100 pool size was 90 +/- 15 mg, and mean VLDL-2 apoB-100 pool size was 111 +/- 14 mg. Absolute synthesis rate of VLDL-1 apoB-100 was 649 +/- 127 mg/day and 353 +/- 59 mg/day for VLDL-2 apoB-100. There was a strong association between the absolute synthesis rate of VLDL-2 apoB-100 and L-C ratio (r 2 = 0.61, P <0.01). In contrast, no correlation was observed between L-C ratio and absolute synthesis rate of VLDL-1 apoB-100 (r 2 = 0.302, P = 0.09). In conclusion, these data provide additional support for an independent regulation of VLDL-1 apoB-100 and VLDL-2 apoB-100 production. Endogenous cholesterol synthesis is correlated only with the VLDL-2 apoB-100 productio

Dried blood spot analysis: an easy and reliable tool to monitor the biochemical effect of hematopoietic stem cell transplantation in Hurler Syndrome patients

Hurler syndrome (HS), the most severe phenotype in the spectrum of mucopolysaccharidosis type I, is caused by a deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA). At present, hematopoietic stem cell transplantation (HSCT) is the only treatment able to prevent disease progression in the central nervous system, and therefore considered the treatment of choice in HS patients. Because IDUA enzyme activities after HSCT have been suggested to influence the prognosis of HS patients, monitoring these activities after HSCT remains highly important. The use of dried blood spots (DBS) for enzyme analysis can be a useful alternative to the conventional leukocyte assay. Importantly, this method allows for convenient worldwide shipment, and can therefore be applied to monitor patients from larger areas of the world, or during large-scale international studies. Furthermore, this method requires only a minimal amount of blood. From 13 HS patients receiving HSCT, 36 paired whole blood and DBS samples were analyzed to assess leukocyte and DBS IDUA activities, respectively. To correct for potential interfering factors, simultaneous assay of the alpha-Galactosidase-A (AGA) activity was performed in the DBS samples and an IDUA/AGA ratio was calculated. A strong linear correlation was demonstrated between the DBS IDUA/AGA ratio and the leukocyte IDUA activity (r2 = .875, P < .001). This correlation was applicable to all enzyme activities, including the activities measured early after HSCT as well as heterozygous activities because of mixed chimerism or the use of a carrier donor. These results demonstrate that the DBS method is reliable to monitor the biochemical effect of HSCT in HS patients