Isotope dilution gas chromatography/mass spectrometry for platinum determination in urine

AbstractThe therapeutic importance of platinum (Pt) compounds, the growing accessibility of gas chromatography/mass spectrometry (GC/MS) systems in clinical laboratories, and the lack of a mass spectrometric method for the determination of Pt in biological samples motivated us to develop an isotope dilution GC/MS assay for Pt. The method is based on the use of lithium bis(trifluoroethyl)dithiocarbamate, Li(FDEDTC), as a chelating agent and enriched 192Pt for isotope dilution. Conditions were optimized for the precise and accurate determination of isotope ratios of Pt by using a 10-m DB-1 fused silica capillary column and a reverse-geometry double-focusing mass spectrometer with selected ion monitoring. An overall precision of 1% was obtained by combining within-run precision and between-run precision at the 10-ng level. No appreciable memory effect was observed when samples with different isotope ratios were analyzed sequentially. The method was validated by the quantitation of Pt in National Institute of Standards and Technology freeze-dried urine sample SRM 2670. A concentration value of 125 ± 6 μg/L (n = 6) was obtained by using four different sets of isotope ratios in the molecular ion and supports the National Institute of Standards and Technology recommended value of 120 ± ? μg/L. Limits-of-quantitation, estimated at 3 μg/L, are made possible by the high sensitivity of the method and the low blank value for t

Unilateral ureteral obstruction impairs renal antioxidant enzyme activation during sodium depletion

Unilateral ureteral obstruction impairs renal antioxidant enzyme activation during sodium depletion.BackgroundObstructive nephropathy leads to progressive renal tubular atrophy and interstitial fibrosis and is associated with sodium wasting and sodium depletion. Renal damage resulting from unilateral ureteral obstruction (UUO) may be aggravated by reactive oxygen species (ROS), which are produced by a variety of processes. Ideally, deleterious effects of ROS are attenuated by antioxidant enzymes, including the superoxide dismutases, glutathione peroxidases, catalase, and glutathione-S-transferases. The general paradigm is that tissue damage occurs when ROS production is greater than the protective capacity of the antioxidant enzymes.MethodsThis study was designed to investigate the response of renal antioxidant enzymes to UUO and sodium depletion. Adult, male Sprague-Dawley rats received normal-sodium or sodium-depleted diets and were subjected to UUO or sham operation. Obstructed (UUO), intact opposite, or sham-operated kidneys were harvested after 14days, and antioxidant enzyme activities were measured in kidney homogenates. Thiobarbituric acid reactive substances were measured in these homogenates at 3 and 14days after UUO or sham operation as an index of ROS production.ResultsRenal interstitial area, a measure of fibrosis, was increased by UUO and was doubled in sodium-depleted animals. Sodium depletion increased manganese superoxide dismutase, glutathione peroxidases, and glutathione-S-transferase activities in sham-operated kidneys but not in UUO kidneys. Relative to intact opposite kidneys, UUO kidneys had reduced activities of catalase, manganese superoxide dismutase, and glutathione-S-transferase in normal-sodium animals and all antioxidant enzymes tested in sodium-depleted animals. Renal thiobarbituric acid reactive substances were increased by three days of UUO and were increased further by 14days of sodium depletion.ConclusionIn summary, sodium depletion increased several renal antioxidant enzymes, consistent with a stress response to increased ROS production. Further, UUO not only reduced antioxidant enzyme activities but also inhibited increases seen with sodium depletion. We conclude that suppression of renal antioxidant enzyme activities by UUO contributes to the progression of renal injury in obstructive nephropathy, a process exacerbated by sodium depletion

AT1 Receptor Induced Alterations in Histone H2A Reveal Novel Insights into GPCR Control of Chromatin Remodeling

Chronic activation of angiotensin II (AngII) type 1 receptor (AT1R), a prototypical G protein-coupled receptor (GPCR) induces gene regulatory stress which is responsible for phenotypic modulation of target cells. The AT1R-selective drugs reverse the gene regulatory stress in various cardiovascular diseases. However, the molecular mechanisms are not clear. We speculate that activation states of AT1R modify the composition of histone isoforms and post-translational modifications (PTM), thereby alter the structure-function dynamics of chromatin. We combined total histone isolation, FPLC separation, and mass spectrometry techniques to analyze histone H2A in HEK293 cells with and without AT1R activation. We have identified eight isoforms: H2AA, H2AG, H2AM, H2AO, H2AQ, Q96QV6, H2AC and H2AL. The isoforms, H2AA, H2AC and H2AQ were methylated and H2AC was phosphorylated. The relative abundance of specific H2A isoforms and PTMs were further analyzed in relationship to the activation states of AT1R by immunochemical studies. Within 2 hr, the isoforms, H2AA/O exchanged with H2AM. The monomethylated H2AC increased rapidly and the phosphorylated H2AC decreased, thus suggesting that enhanced H2AC methylation is coupled to Ser1p dephosphorylation. We show that H2A125Kme1 promotes interaction with the heterochromatin associated protein, HP1α. These specific changes in H2A are reversed by treatment with the AT1R specific inhibitor losartan. Our analysis provides a first step towards an awareness of histone code regulation by GPCRs

Disparate Central and Peripheral Effects of Circulating IGF-1 Deficiency on Tissue Mitochondrial Function

© 2019, The Author(s). Age-related decline in circulating levels of insulin-like growth factor (IGF)-1 is associated with reduced cognitive function, neuronal aging, and neurodegeneration. Decreased mitochondrial function along with increased reactive oxygen species (ROS) and accumulation of damaged macromolecules are hallmarks of cellular aging. Based on numerous studies indicating pleiotropic effects of IGF-1 during aging, we compared the central and peripheral effects of circulating IGF-1 deficiency on tissue mitochondrial function using an inducible liver IGF-1 knockout (LID). Circulating levels of IGF-1 (~ 75%) were depleted in adult male Igf1f/f mice via AAV-mediated knockdown of hepatic IGF-1 at 5 months of age. Cognitive function was evaluated at 18 months using the radial arm water maze and glucose and insulin tolerance assessed. Mitochondrial function was analyzed in hippocampus, muscle, and visceral fat tissues using high-resolution respirometry O2K as well as redox status and oxidative stress in the cortex. Peripherally, IGF-1 deficiency did not significantly impact muscle mass or mitochondrial function. Aged LID mice were insulin resistant and exhibited ~ 60% less adipose tissue but increased fat mitochondrial respiration (20%). The effects on fat metabolism were attributed to increases in growth hormone. Centrally, IGF-1 deficiency impaired hippocampal-dependent spatial acquisition as well as reversal learning in male mice. Hippocampal mitochondrial OXPHOS coupling efficiency and cortex ATP levels (~ 50%) were decreased and hippocampal oxidative stress (protein carbonylation and F2-isoprostanes) was increased. These data suggest that IGF-1 is critical for regulating mitochondrial function, redox status, and spatial learning in the central nervous system but has limited impact on peripheral (liver and muscle) metabolism with age. Therefore, IGF-1 deficiency with age may increase sensitivity to damage in the brain and propensity for cognitive deficits. Targeting mitochondrial function in the brain may be an avenue for therapy of age-related impairment of cognitive function. Regulation of mitochondrial function and redox status by IGF-1 is essential to maintain brain function and coordinate hippocampal-dependent spatial learning. While a decline in IGF-1 in the periphery may be beneficial to avert cancer progression, diminished central IGF-1 signaling may mediate, in part, age-related cognitive dysfunction and cognitive pathologies potentially by decreasing mitochondrial function

Metabolic and Stress Response Changes Precede Disease Onset in the Spinal Cord of Mutant SOD1 ALS Mice

Many Amyotrophic Lateral Sclerosis (ALS) patients experience hypermetabolism, or an increase in measured vs. calculated metabolic rate. The cause of hypermetabolism and the effects on neuronal metabolism in ALS are currently unknown, but the efficacy of dietary interventions shows promise for metabolism as an ALS therapeutic target. The goal of this study is to measure changes in metabolic pathways as a function of disease progression in spinal cords of the SOD1G93A mouse model of ALS. We conducted a comprehensive assessment of protein expression for metabolic pathways, antioxidants, chaperones, and proteases in lumbar spinal cord from male SOD1G93A mice at pre-onset, onset, and end-stages of the disease using targeted proteomic analysis. These results reveal that protein content of metabolic proteins including proteins involved in glycolysis, β-oxidation, and mitochondrial metabolism is altered in SOD1G93A mouse spinal cord well before disease onset. The changes in mitochondrial metabolism proteins are associated with decreased maximal respiration and glycolytic flux in SOD1G93A dermal fibroblasts and increased hydrogen peroxide and lipid hydroperoxide production in mitochondria from sciatic nerve and gastrocnemius muscle fibers at end stage of disease. Consistent with redox dysregulation, expression of the glutathione antioxidant system is decreased, and peroxiredoxins and catalase expression are increased. In addition, stress response proteases and chaperones, including those involved in the mitochondrial unfolded protein response (UPRmt), are induced before disease onset. In summary, we report that metabolic and stress response changes occur in SOD1G93A lumbar spinal cord before motor symptom onset, and are primarily caused by SOD1G93A expression and do not vary greatly as a function of disease course

HIV-1 Virological Synapse is not Simply a Copycat of the Immunological Synapse

The virological synapse (VS) is a tight adhesive junction between an HIV-infected cell and an uninfected target cell, across which virus can be efficiently transferred from cell to cell in the absence of cell-cell fusion. The VS has been postulated to resemble, in its morphology, the well-studied immunological synapse (IS). This review article discusses the structural similarities between IS and VS and the shared T cell receptor (TCR) signaling components that are found in the VS. However, the IS and the VS display distinct kinetics in disassembly and intracellular signaling events, possibly leading to different biological outcomes. Hence, HIV-1 exploits molecular components of IS and TCR signaling machinery to trigger unique changes in cellular morphology, migration, and activation that facilitate its transmission and cell-to-cell spread

Elevated phospholipid hydroperoxide glutathione peroxidase (GPX4) expression modulates oxylipin formation and inhibits age-related skeletal muscle atrophy and weakness

Our previous studies support a key role for mitochondrial lipid hydroperoxides as important contributors to denervation-related muscle atrophy, including muscle atrophy associated with aging. Phospholipid hydroperoxide glutathione peroxidase 4 (GPX4) is an essential antioxidant enzyme that directly reduces phospholipid hydroperoxides and we previously reported that denervation-induced muscle atrophy is blunted in a mouse model of GPX4 overexpression. Therefore, the goal of the present study was to determine whether GPX4 overexpression can reduce the age-related increase in mitochondrial hydroperoxides in skeletal muscle and ameliorate age-related muscle atrophy and weakness (sarcopenia). Male C57Bl6 WT and GPX4 transgenic (GPX4Tg) mice were studied at 3 to 5 and 23–29 months of age. Basal mitochondrial peroxide generation was reduced by 34% in muscle fibers from aged GPX4Tg compared to old WT mice. GPX4 overexpression also reduced levels of lipid peroxidation products: 4-HNE, MDA, and LOOHs by 38%, 32%, and 84% respectively in aged GPX4Tg mice compared to aged WT mice. Muscle mass was preserved in old GPX4 Tg mice by 11% and specific force generation was 21% higher in old GPX4Tg versus age matched male WT mice. Oxylipins from lipoxygenases (LOX) and cyclooxygenase (COX), as well as less abundant non-enzymatically generated isomers, were significantly reduced by GPX4 overexpression. The expression of cPLA2, 12/15-LOX and COX-2 were 1.9-, 10.5- and 3.4-fold greater in old versus young WT muscle respectively, and 12/15-LOX and COX-2 levels were reduced by 37% and 35%, respectively in muscle from old GPX4Tg mice. Our study suggests that lipid peroxidation products may play an important role in the development of sarcopenia, and their detoxification might be an effective intervention in preventing muscle atrophy