Serum Mg Isotopic Composition Reveals That Mg Dyshomeostasis Remains in Type 1 Diabetes despite the Resolution of Hypomagnesemia.

Hypomagnesemia was historically prevalent in individuals with type 1 diabetes mellitus (T1DM), but contemporary results indicate an incidence comparable to that in the general population, likely due to improved treatment in recent decades, resulting in better glycemic control. However, a recent study found a significant difference between the serum Mg isotopic composition of T1DM individuals and controls, indicating that disruptions to Mg homeostasis persist. Significant deviations were also found in samples taken one year apart. To investigate whether the temporal variability in serum Mg isotopic composition is linked to the transient impact of administered insulin, Mg isotope ratios were determined in serum from 15 T1DM individuals before and one hour after insulin injection/meal consumption using multi-collector inductively coupled plasma-mass spectrometry. Consistent with results of the previous study, significant difference in the serum Mg isotopic composition was found between T1DM individuals and 10 sex-matched controls. However, the average difference between pre- and post-insulin injection/meal T1DM samples of 0.05 ± 0.13‱ (1SD) was not significant. No difference was observed for controls before (-0.12 ± 0.16‱) and after the meal (-0.10 ± 0.13‱) either, suggesting a lack of a postprandial Mg isotopic response within one hour of food consumption, and that the timing of the most recent meal may not require controlling for when determining serum Mg isotopic composition.

Multicollector Inductively Coupled Plasma-Mass Spectrometry with 1013 Ω Faraday Cup Amplifiers for Ultrasensitive Mg Isotopic Analysis of Cerebrospinal Fluid Microsamples.

Magnesium isotopic analysis of cerebrospinal fluid (CSF) is a potentially interesting approach for studies on neurodegeneration. However, this type of analysis is challenging because of the invasiveness of the sampling and small sample volume. In this work, a novel analytical method was developed for ultrasensitive Mg isotopic analysis of CSF microsamples via multicollector inductively coupled plasma-mass spectrometry (MC-ICP-MS) using high-gain 1013 Ω Faraday cup amplifiers. The intermediate and internal errors on the δ26Mg value were improved up to fourfold using 1013 Ω resistors for the monitoring of both the 24Mg and 26Mg isotopes and up to twofold using a 1011 Ω resistor for the most abundant 24Mg isotope and a 1013 Ω resistor for the 26Mg isotope. Magnesium isotope ratios measured at a concentration level of 7-10 µg L-1 were in good agreement with those obtained using the conventional method at a concentration level of 150 µg L-1. The expanded uncertainty for the quality control CSF material obtained at the ultratrace level was ±0.16‰. Ultrasensitive Mg isotopic analysis was carried out for CSF from hydrocephalus patients using only 5 µL of sample. δMg values thus obtained were not significantly different from those obtained using the conventional method using a sample volume of 400 µL instead (p ≤ 0.05). The Mg isotopic composition of the CSF from hydrocephalus patients ranged between -0.65 and 0.30‰, with a mean δ26Mg value of -0.14 ± 0.27‰.

Effect of Endotoxemia Induced by Intraperitoneal Injection of Lipopolysaccharide on the Mg isotopic Composition of Biofluids and Tissues in Mice.

Endotoxemia induced in vivo in mice by intraperitoneal injection of lipopolysaccharide (LPS) leads to (neuro)inflammation and sepsis. Also the homeostasis of mineral elements can be altered through mechanisms that still are poorly understood. The isotopic composition of Mg and the concentrations of the minor elements Ca, K, Mg, Na, P, and S were determined in biological fluids and tissues of young (14-28 weeks) and aged (40-65 weeks) LPS-injected mice and age-matched controls to reveal potential effects of the LPS-induced infection. Blood plasma of young and aged LPS-injected mice showed a heavy Mg isotopic composition, as well as elevated Mg and P concentrations, compared to matched controls. The plasma Mg isotopic composition was correlated with the P concentration in aged mice. Also the liver Mg isotopic composition was strongly affected in the young and aged LPS-injected mice, while for aged mice, an additional effect on the urine Mg isotopic composition was established. These observations were hypothetically associated with liver inflammation and/or hepatotoxicity, and reduced urinary Mg excretion, respectively. Also a regional endotoxin-induced difference was observed in the brain Mg isotopic composition for the aged mice only, and was attributed to potential disruption of the blood-brain barrier.

Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding.

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. However, the mechanistic contribution of the individual TPC subunits to plant CME remains elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

High-precision isotopic analysis of Mg and Ca in biological samples using multi-collector ICP-mass spectrometry after their sequential chromatographic isolation - Application to the characterization of the body distribution of Mg and Ca isotopes in mice.

A sequential chromatographic separation procedure for subsequent high-precision isotopic analysis of Mg and Ca via multi-collector ICP-mass spectrometry (MC-ICP-MS) from a single aliquot of sample was developed and used for a variety of animal/human biofluids and tissues. The procedure consists of a one-stage Mg isolation protocol (for most of the sample types) and a three-stage isolation protocol for Ca. AG50W-X8 strong cation exchange resin was used for the isolation of Mg and Ca, while Sr-resin was used to additionally purify the Ca fraction from Sr. Potential effects on the Mg isotope ratio measurement results caused by the possible presence of concomitant matrix elements (Cu, Fe, Zn, Ca) were systematically evaluated. δ26Mg values were biased for a Fe/Mg ratio > 0.13 and a Ca/Mg ratio > 1.5, resulting in a shift towards a lighter Mg isotopic composition. It was shown that the Mg isotope ratio data for Mg standards, the isotopic reference materials ERM-AE143 and IRMM 009 and the biological samples investigated are located on a mass-dependent fractionation line. Biological reference materials and commercially available serum samples were analyzed for both their Mg and Ca isotope ratios. For some of the biomaterials analyzed, the Ca isotope ratio data as obtained using MC-ICP-MS were further validated via their determination using double-spike thermal ionization mass spectrometry (DS-TIMS). The expanded uncertainty for δ26Mg was ≤ 0.12‰ and for δ44/42Ca ≤ 0.29‰. Biological fluids and tissues of mice were analyzed to characterize the body distribution of the stable isotopes of Mg and Ca. The isotopic variability among the body compartments was about 1.5‰ for Mg and 1.0‰ for Ca. Among the tissues explored, muscle tissue shows the lightest Mg and Ca isotopic compositions and liver the heaviest Mg and Ca isotopic compositions, respectively.