Cytidine Diphosphate-Ribitol Analysis for Diagnostics and Treatment Monitoring of Cytidine Diphosphate-l-Ribitol Pyrophosphorylase A Muscular Dystrophy.

BACKGROUND: Many muscular dystrophies currently remain untreatable. Recently, dietary ribitol has been suggested as a treatment for cytidine diphosphate (CDP)-l-ribitol pyrophosphorylase A (CRPPA, ISPD), fukutin (FKTN), and fukutin-related protein (FKRP) myopathy, by raising CDP-ribitol concentrations. Thus, to facilitate fast diagnosis, treatment development, and treatment monitoring, sensitive detection of CDP-ribitol is required. METHODS: An LC-MS method was optimized for CDP-ribitol in human and mice cells and tissues. RESULTS: CDP-ribitol, the product of CRPPA, was detected in all major human and mouse tissues. Moreover, CDP-ribitol concentrations were reduced in fibroblasts and skeletal muscle biopsies from patients with CRPPA myopathy, showing that CDP-ribitol could serve as a diagnostic marker to identify patients with CRPPA with severe Walker-Warburg syndrome and mild limb-girdle muscular dystrophy (LGMD) phenotypes. A screen for potentially therapeutic monosaccharides revealed that ribose, in addition to ribitol, restored CDP-ribitol concentrations and the associated O-glycosylation defect of α-dystroglycan. As the effect occurred in a mutation-dependent manner, we established a CDP-ribitol blood test to facilitate diagnosis and predict individualized treatment response. Ex vivo incubation of blood cells with ribose or ribitol restored CDP-ribitol concentrations in a patient with CRPPA LGMD. CONCLUSIONS: Sensitive detection of CDP-ribitol with LC-MS allows fast diagnosis of patients with severe and mild CRPPA myopathy. Ribose offers a readily testable dietary therapy for CRPPA myopathy, with possible applicability for patients with FKRP and FKTN myopathy. Evaluation of CDP-ribitol in blood is a promising tool for the evaluation and monitoring of dietary therapies for CRPPA myopathy in a patient-specific manner.

Lithium potentiates agonist formation of [3H]CDP-diacylglycerol in human platelets.

Thrombin stimulated a rapid formation of [3H]CDP-diacylglycerol in platelets prelabelled with [3H]cytidine. This response was increased in the presence of LiCl after a delay of 5 min; potentiation could be prevented by myo-inositol. Since Li+ inhibits the liberation of inositol from inositol phosphates, the conversion of diacylglycerol to phosphatidylinositol via CDP-diacylglycerol may be dependent on the regeneration of inositol from this pathway.

Altered metabolism of poly(ADP-ribose) in the peripheral blood lymphocytes of patients with systemic lupus erythematosus.

A method was developed to measure poly(ADP-ribose) metabolism in peripheral blood lymphocytes. The technique involved the isolation of lymphocytes on Ficoll gradients, followed by lysis with 5M NaCl. The synthesis and degradation of poly(ADP-ribose) in this crude lysate, measured by the incorporation of 3H-labeled NAD into acid-precipitable counts, was compared in 18 patients with systemic lupus erythematosus (SLE), 10 patients with rheumatoid arthritis, and in 10 control patients without rheumatoid arthritis. Patients with SLE showed a 70% decrease in poly(ADP-ribose) synthesis (P less than 0.001); this decreased synthesis persisted even with the addition of histones or DNase. We present possible explanations of the role of poly(ADP-ribose) in SLE.

Tumor promoter phorbol-12-myristate-13-acetate induces poly ADP-ribosylation in human monocytes.

The tumor promoter phorbol-12-myristate-13-acetate (PMA) induces rapid poly ADP-ribosylation and a drop in cellular NAD concentration in human monocytes. The antioxidants CuZn-superoxide dismutase, catalase, glutathione peroxidase and butylated-hydroxytoluene inhibit the reaction indicating that active oxygen species produced in the PMA-induced oxidative burst represent intermediates. The inhibitor of ADP-ribosyl-transferase, 3-amino-benzamide, inhibited poly ADP-ribosylation but did not prevent the drop in NAD-levels. PMA also causes the slow accumulation of DNA strand breaks in monocytes. The difference in the kinetics of poly ADP-ribosylation and DNA breakage argues against a simple relationship between the two reactions.

Unique acceptors for poly(ADP-ribose) in resting, proliferating and DNA-damaged human lymphocytes.

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that MNNG treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histones. In MNNG-treated cells, an increase in ADP-ribosylation of histone H1 was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96 000. MNNG treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to be due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.

Evidence that human platelet alpha-adrenergic receptors coupled to inhibition of adenylate cyclase are not associated with the subunit of adenylate cyclase ADP-ribosylated by cholera toxin.

Exposure of the alpha-adrenergic receptor of the human platelet to agonist prior to solubilization stabilizes a receptor complex of the alpha-adrenergic receptor with the GTP-binding protein(s) which modulates receptor affinity for agonists (Smith, S. K., and Limbird, L. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4026-4030). The soluble alpha-adrenergic receptor is characterized by retention of sensitivity to GTP and a faster rate of sedimentation in sucrose gradients than antagonist-occupied or unoccupied receptors. The present studies were undertaken to determine whether the alpha-adrenergic receptor, which is coupled to inhibition of adenylate cyclase, contains the same GTP-binding protein that is involved in activation of adenylate cyclase. The GTP-binding protein that is coupled to activation of adenylate cyclase was labeled with [32P]ADP-ribose using cholera toxin. Incorporation of [32]ADP-ribose into a Mr = 42,000 peptide in human platelet membranes was paralleled by an enhancement of GTP-sensitive catalytic activity in the membranes. However, cholera toxin treatment did not modify alpha-receptor-mediated inhibition of adenylate cyclase or interaction of the alpha-receptor with agonist agents. Moreover, sucrose gradient centrifugation revealed that the [32P]ADP-ribosylated Mr = 42,000 subunit of the stimulatory GTP-binding protein did not appear to associate with the agonist-alpha-receptor complex. These data suggest that the GTP-binding protein that mediates GTP activation of adenylate cyclase in the human platelet membrane is distinct from the GTP-binding protein that modulates alpha-adrenergic receptor affinity for agonist agents and which associates with the receptor in the presence of agonists.

Chemical and enzymic degradations of nucleoside mono- and diphosphate sugars. I. Determination of the degradation rate during the glycosyltransferase assays.

In incorporation experiments used for the determination of glycosyltransferase activities, we demonstrated that the nucleoside diphosphate sugars are decomposed in three different ways: 1, transfer of the monosaccharide to acceptor molecule, catalyzed by glycosyltransferases; 2, degradation of the glycosyl nucleotides by nucleotide pyrophosphatase into monosaccharide 1-phosphates which are further hydrolyzed into free monosaccharides by phosphatases; 3, chemical decomposition of UDP-D-[14C]Gal; UDP-D-[14C]Glc and UDP-D-[14C]GlcUA into 1,2-cyclic phosphate derivatives of the corresponding monosaccharide. All the breakdown products of the nucleoside mono- and diphosphate sugars which are obtained during the incorporation experiments may be separated by paper chromatography and their amounts may be determined. Galactosyltransferase assays on human and rat serum have shown that the three different ways of decomposition of the nucleoside diphosphate sugars are dependent mostly on the concentration of divalent cations (Mn2+, Mg2+). Inhibition of the nucleotide pyrophosphatase activity is obtained with low concentrations of UMP, but increasing concentrations of UMP inhibit also the galactosyltransferase activity and consequently enhance the formation of galactose 1,2-monophosphate. A partial elimination of the nucleotide pyrophosphatase activity was achieved by the addition of increasing concentrations of UDP-D-Gal. These results demonstrate that the determination of glycosyltransferase activities in tissues and in biological fluids is not possible without a concomitant determination of the nucleotide pyrophosphatase activity present in the assay.