An innovative, time- and cost-saving method for the quantification of asymmetric dimethylarginine in serum by HPLC without evaporation

A new time‐ and energy‐saving method for the determination of asymmetric dimethylarginine in human serum is presented. Here, a newly developed eluent was used in the sample cleanup of the solid‐phase extraction whose composition makes an evaporation step redundant. After derivatization, asymmetric dimethylarginine was quantified by high‐performance liquid chromatography with fluorescent detection. The conditions of the solid‐phase extraction lead to a relative recovery of asymmetric dimethylarginine of 101%. A concentration of 25 ng/mL was found as the limit of quantification and the batch was highly linear from 25 to 800 ng/mL with the correlation coefficient R2 = 0,9999. Intra‐assay coefficients of variation <2.1% and inter‐assay coefficients of variation <3.1% indicate a high precision. Since no evaporation is necessary compared to previously published methods, this newly presented method does not only save time, but also is a cost‐ and energy‐saving alternative for the routine quantification of asymmetric dimethylarginine in serum

An innovative, time‐ and cost‐saving method for the quantification of asymmetric dimethylarginine in serum by high‐performance liquid chromatography without evaporation

A new time- and energy-saving method for the determination of asymmetric dimethylarginine in human serum is presented. Here, a newly developed eluent was used in the sample cleanup of the solid-phase extraction whose composition makes an evaporation step redundant. After derivatization, asymmetric dimethylarginine was quantified by high-performance liquid chromatography with fluorescent detection. The conditions of the solid-phase extraction lead to a relative recovery of asymmetric dimethylarginine of 101%. A concentration of 25 ng/mL was found as the limit of quantification and the batch was highly linear from 25 to 800 ng/mL with the correlation coefficient R2 = 0,9999. Intra-assay coefficients of variation <2.1% and inter-assay coefficients of variation <3.1% indicate a high precision. Since no evaporation is necessary compared to previously published methods, this newly presented method does not only save time, but also is a cost- and energy-saving alternative for the routine quantification of asymmetric dimethylarginine in serum

Novel Functional Features of cGMP Substrate Proteins IRAG1 and IRAG2

The inositol triphosphate-associated proteins IRAG1 and IRAG2 are cGMP kinase substrate proteins that regulate intracellular Ca2+. Previously, IRAG1 was discovered as a 125 kDa membrane protein at the endoplasmic reticulum, which is associated with the intracellular Ca2+ channel IP3R-I and the PKGIβ and inhibits IP3R-I upon PKGIβ-mediated phosphorylation. IRAG2 is a 75 kDa membrane protein homolog of IRAG1 and was recently also determined as a PKGI substrate. Several (patho-)physiological functions of IRAG1 and IRAG2 were meanwhile elucidated in a variety of human and murine tissues, e.g., of IRAG1 in various smooth muscles, heart, platelets, and other blood cells, of IRAG2 in the pancreas, heart, platelets, and taste cells. Hence, lack of IRAG1 or IRAG2 leads to diverse phenotypes in these organs, e.g., smooth muscle and platelet disorders or secretory deficiency, respectively. This review aims to highlight the recent research regarding these two regulatory proteins to envision their molecular and (patho-)physiological tasks and to unravel their functional interplay as possible (patho-)physiological counterparts

Loss of PKGIß/IRAG1 Signaling Causes Anemia-Associated Splenomegaly

Inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1) is a substrate protein of the NO/cGMP-signaling pathway and forms a ternary complex with the cGMP-dependent protein kinase Iβ (PKGIβ) and the inositol triphosphate receptor I (IP3R-I). Functional studies about IRAG1 exhibited that IRAG1 is specifically phosphorylated by the PKGIβ, regulating cGMP-mediated IP3-dependent Ca2+-release. IRAG1 is widely distributed in murine tissues, e.g., in large amounts in smooth muscle-containing tissues and platelets, but also in lower amounts, e.g., in the spleen. The NO/cGMP/PKGI signaling pathway is important in several organ systems. A loss of PKGI causes gastrointestinal disorders, anemia and splenomegaly. Due to the similar tissue distribution of the PKGIβ to IRAG1, we investigated the pathophysiological functions of IRAG1 in this context. Global IRAG1-KO mice developed gastrointestinal bleeding, anemia-associated splenomegaly and iron deficiency. Additionally, Irag1-deficiency altered the protein levels of some cGMP/PKGI signaling proteins—particularly a strong decrease in the PKGIβ—in the colon, spleen and stomach but did not change mRNA-expression of the corresponding genes. The present work showed that a loss of IRAG1 and the PKGIβ/IRAG1 signaling has a crucial function in the development of gastrointestinal disorders and anemia-associated splenomegaly. Furthermore, global Irag1-deficient mice are possible in vivo model to investigate PKGIβ protein functions

IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP3 receptors /IP3R) via its coiled-coil domain and modulates Ca2+ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca2+ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP3 receptors and its impact on intracellular Ca2+ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP3R and altered Ca2+ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca2+, suggesting that IRAG2 leads to activation of IP3R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca2+ signaling, which regulates exocrine pancreatic function