Restricting dietary magnesium accelerates ectopic connective tissue mineralization in a mouse model of pseudoxanthoma elasticum (Abcc6(-/-) ).

Ectopic mineralization, linked to a number of diseases, is a major cause of morbidity and mortality in humans. Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder characterized by calcium phosphate deposition in various tissues. The mineral content of diet has been suggested to modify the disease severity in PXE. The aim of this study is to explore the role of diet with reduced magnesium in modifying tissue mineralization in a mouse model of PXE. Abcc6(-/-) mice were placed on either standard rodent diet (control) or an experimental diet low in magnesium at weaning (4 weeks) and examined for mineralization in the skin and internal organs at the ages of 1.5, 2 or 6 months by computerized morphometric analysis of histopathological sections and by chemical assay of calcium and phosphate. Abcc6(-/-) mice on experimental diet demonstrated an accelerated, early-onset mineralization of connective tissues, as compared to control mice. Wild-type or heterozygous mice on experimental diet did not show evidence of mineralization up to 6 months of age. All mice on experimental diet showed decreased urinary calcium, increased urinary phosphate and elevated parathyroid serum levels. However, no difference in bone density at 6 months of age was noted. Our findings indicate that the mineral content, particularly magnesium, can modify the extent and the onset of mineralization in Abcc6(-/-) mice and suggest that dietary magnesium levels may contribute to the phenotypic variability of PXE. The control of mineralization by dietary magnesium may have broader implications in general population in the context of vascular mineralization.

Magnesium carbonate-containing phosphate binder prevents connective tissue mineralization in Abcc6(-/-) mice-potential for treatment of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by ectopic mineralization of connective tissues primarily in the skin, eyes, and the cardiovascular system. PXE is caused by mutations in the ABCC6 gene. While PXE is associated with considerable morbidity and mortality, there is currently no effective or specific treatment. In this study, we tested oral phosphate binders for treatment of a mouse model of PXE which we have developed by targeted ablation of the corresponding mouse gene (Abcc6(-/-)). This "knock-out" (KO) mouse model recapitulates features of PXE and demonstrates mineralization of a number of tissues, including the connective tissue capsule surrounding vibrissae in the muzzle skin which serves as an early biomarker of the mineralization process. Treatment of these mice with a magnesium carbonate-enriched diet (magnesium concentration being 5-fold higher than in the control diet) completely prevented mineralization of the vibrissae up to 6 months of age, as demonstrated by computerized morphometric analysis of histopathology as well as by calcium and phosphate chemical assays. The magnesium carbonate-enriched diet also prevented the progression of mineralization when the mice were placed on that experimental diet at 3 months of age and followed up to 6 months of age. Treatment with magnesium carbonate was associated with a slight increase in the serum concentration of magnesium, with no effect on serum calcium and phosphorus levels. In contrast, concentration of calcium in the urine was increased over 10-fold while the concentration of phosphorus was markedly decreased, being essentially undetectable after long-term (> 4 month) treatment. No significant changes were noted in the serum parathyroid hormone levels. Computerized axial tomography scan of bones in mice placed on magnesium carbonate-enriched diet showed no differences in the bone density compared to mice on the control diet, and chemical assays showed a small increase in the calcium and phosphate content of the femurs by chemical assay, in comparison to mice on control diet. Similar experiments with another experimental diet supplemented with lanthanum carbonate did not interfere with the mineralization process in Abcc6(-/-) mice. These results suggest that magnesium carbonate may offer a potential treatment modality for PXE, a currently intractable disease, as well as for other conditions characterized by ectopic mineralization of connective tissues.

Comparison of 1D and 2D NMR spectroscopy for metabolic profiling.

High-resolution, liquid state nuclear magnetic resonance (NMR) spectroscopy is a popular platform for metabolic profiling because the technique is nondestructive, quantitative, reproducible, and the spectra contain a wealth of biochemical information. Because of the large dynamic range of metabolite concentrations in biofluids, statistical analyses of one-dimensional (1D) proton NMR data tend to be biased toward selecting changes in more abundant metabolites. Although two-dimensional (2D) proton-proton experiments can alleviate spectral crowding, they have been mainly used for structural determination. In this study, 2D total correlation spectroscopy NMR was used to compare the global metabolic profiles of urine obtained from wild-type and Abcc6-knockout mice. The 2D data were compared to an improved 1D experiment in which signal contributions from macromolecules and the urea peak have been spectroscopically removed for more accurate quantitation of low-abundance metabolites. Although statistical models from both 1D and 2D data could differentiate samples acquired from the two groups of mice, only the 2D spectra allowed the characterization of statistically relevant changes in the low-abundance metabolites. While acquisition of the 2D data require more time, the data obtained resulted in a more meaningful and comprehensive metabolic profile, aided in metabolite identifications, and minimized ambiguities in peak assignments.

High levels of desmosines in urine and plasma of patients with pseudoxanthoma elasticum.

BACKGROUND: Pseudoxanthoma elasticum (PXE), a rare heritable disorder caused by mutations of the ABCC6 gene, is characterized by fragmentation and mineralization of elastic fibres. We determined the extent of degradation of elastin by measuring and comparing the amount of desmosines in plasma and urine of PXE patients, healthy carriers and normal subjects. METHODS: Using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) we measured the amount of desmosines in the urine of 46 individuals (14 PXE patients, 17 healthy carriers and 15 controls) and in the plasma of 56 subjects (18 PXE patients, 23 healthy carriers and 15 controls). Pseudoxanthoma elasticum patients and carriers were identified by clinical, structural and molecular biology analyses. RESULTS: The urinary excretion of desmosines was two-fold higher in PXE patients than in controls (P < 0.01); the values for healthy carriers were intermediate between those of PXE patients and controls. A very similar trend between patients and their relatives was observed for plasma desmosines. There was a significant correlation between the amount of the desmosines in plasma and urine. Moreover, a positive correlation was observed between urinary desmosine content and age of the patients as well as between urinary desmosine content and severity of clinical manifestations. CONCLUSIONS: Both the urinary and plasma desmosine concentrations indicate that elastin degradation is higher in PXE patients and, to a lesser extent, in healthy carriers than in normal subjects. Data seem to indicate that the amount of elastin breakdown products correlates with the age of patients as well as with the severity of the disease.

Anomalous structure of urinary glycosaminoglycans in patients with pseudoxanthoma elasticum.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a hereditary connective tissue disease in which proteoglycans have altered properties. We investigated whether altered proteoglycan metabolism occurs in vivo and may be reflected in the urine of PXE individuals by analyzing the excreted polysaccharides. METHODS: We measured sulfated glycosaminoglycans in the urine of 10 PXE-affected patients, 12 healthy carriers, and 20 healthy controls by agarose gel electrophoresis. Chondroitin sulfate and heparan sulfate disaccharides were also quantified by treatment with specific lyases and separation of products by chromatography. RESULTS: Total polysaccharides were 34% lower in the urine of PXE-affected patients and 17% lower in healthy carriers than in the control group. Chondroitin sulfate was significantly (P <0.01) decreased, and heparan sulfate was significantly increased. The ratio of chondroitin sulfate to heparan sulfate was 2.7 for PXE-affected patients, 2.3 for healthy carriers, and 10.7 for controls. In PXE-affected individuals and carriers, chondroitin sulfate contained more 4-sulfated disaccharide, less 6-sulfated disaccharide, and decreased nonsulfated disaccharide. Heparan sulfate from PXE-affected individuals and healthy carriers produced significantly less N-sulfated disaccharide and more disaccharide sulfated at the C-6 position with no significant abnormality of the nonsulfated disaccharide percentage and sulfates:disaccharide ratio. CONCLUSIONS: The urinary data support the concept that the inherited defect of the ABCC6/MRP6 transporter in PXE alters metabolism of key polysaccharides. Structural analysis of urinary sulfated polyanions may be useful in the diagnosis of PXE.

Plasma and urinary endothelin-1 titers and plasma von Willebrand activity in Pseudoxanthoma elasticum.

We found high endothelin-1 and von Willebrand factor plasma titers not only in two individuals (daughter and father) affected with Pseudoxanthoma elasticum but also in a young unaffected relative. These findings raise the possibility that these molecules could be the first biochemical fingerprints of this, still not clinically evident, rare inherited disorder of elastic tissue.

Glycosaminoglycans of skin and urine in pseudoxanthoma elasticum: evidence for chondroitin 6-sulfate alteration.

Abnormally elevated hyaluronic acid and dermatan sulfate were isolated from lesional skin of a patient with severe pseudoxanthoma elasticum. These glycosaminoglycans (estimated as uronic acid) surpassed the normal controls by 33.6 and 4.8 magnitudes, respectively. Urine chondroitin 6-sulfate of the same patient moved faster by electrophoresis on cellulose acetate than its counterpart isolated from urine of another four patients or from the normal controls. Hyaluronic acid and chondroitin 6-sulfate exceeded their counterparts in normal urine by 2- to 10- and 4- to 18-folds, respectively. These increments correlated with the pseudoxanthoma elasticum severity in three of the five patients studied. The data showed: the first conclusive evidence that dermatan sulfate increased in lesional skin of a patient, with severe pseudoxanthoma elasticum, who also had considerably augmented HA, alteration of the same patient's urine chondroitin 6-sulfate, and diversified urine glycosaminoglycans in pseudoxanthoma elasticum.