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Diphosphoinositide and triphosphoinositide in animal tissues. Extraction, estimation and changes post mortem.

ABSTRACT: 1. A method is presented for the determination of the di- and tri-phosphoinositide in animal tissues. 2. The polyphosphoinositides are quantitatively extracted into chloroform-methanol-hydrochloric acid solvent after a preliminary chloroform-methanol (1:1, v/v) extraction to remove the bulk of the other phospholipids. On washing this extract with n-hydrochloric acid the polyphosphoinositides pass completely into the lower chloroform-rich phase. Their concentrations in the lower phase are determined by chromatography on formaldehyde-treated paper or chromatography and ionophoresis of the acid hydrolysis products. 3. When guinea-pig brain is extracted by the method of Folch (1942), considerable hydrolysis of the triphosphoinositide and accumulation of diphosphoinositide occurs during the initial acetone extraction. 4. The tri- and di-phosphoinositide contents of rat and guinea-pig brain decline substantially within a few minutes after death. 5. The concentrations of tri- and di-phosphoinositide in rat brain are not changed by insulin-hypoglycaemia or electrical stimulation. 6. Examination of frozen rat tissues showed that the brain contained the highest concentration of polyphosphoinositides. Much smaller amounts are present in kidney, and only trace quantities in liver and lung. None could be detected in spleen, heart and skeletal muscle.

SUBMITTER: Dawson RM

PROVIDER: S-EPMC1207197 | biostudies-other | 1965 Sep

REPOSITORIES: biostudies-other

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Project description:BackgroundReactive microglia and recruited peripheral macrophages contribute to the pathogenesis of Alzheimer's dementia (AD). Monocytes, macrophages and microglia all express the marker colony-stimulating factor 1 receptor (CSF1R). 4-Cyano-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)-1H-pyrrole-2-carboxamide (1) is a high-affinity antagonist for CSF1R. We report the radiosynthesis of both [3H]1 and [11C]1. The PET imaging properties of [11C]1 in mice and baboon were investigated. [3H]1 was studied in Bmax measurement in post-mortem autoradiography in the frontal cortex, inferior parietal cortex and hippocampus from donors diagnosed with AD and age-matched controls. In vitro binding affinity of 1 was measured commercially. Nor-methyl-1 precursor was radiolabeled with [11C]iodomethane or [3H]iodomethane to produce [11C]1 and [3H]1, respectively. Ex vivo brain biodistribution of [11C]1 was compared in normal mice versus lipopolysaccharide-administered (LPS) murine model of neuroinflammation. Dynamic PET imaging was performed in a healthy male Papio anubis baboon. Post-mortem autoradiography with [3H]1 was performed in frozen sections using a standard saturation binding technique.ResultsCompound 1 exhibits a high in vitro CSF1R binding affinity (0.59 nM). [11C]1 was synthesized with high yield. [3H]1 was synthesized similarly (commercially). Biodistribution of [11C]1 in healthy mice demonstrated moderate brain uptake. In LPS-treated mice the brain uptake of [11C]1 was ~ 50% specific for CSF1R. PET/CT [11C]1 study in baboon revealed low brain uptake (0.36 SUV) of [11C]1. Autoradiography with [3H]1 gave significantly elevated Bmax values in AD frontal cortex versus control (47.78 ± 26.80 fmol/mg vs. 12.80 ± 5.30 fmol/mg, respectively, P = 0.023) and elevated, but not significantly different binding in AD hippocampus grey matter and inferior parietal cortex (IPC) white matter.ConclusionsCompound 1 exhibits a high in vitro CSF1R binding affinity. [11C]1 specifically labels CSF1R in the mouse neuroinflammation, but lacks the ability to efficiently cross the blood-brain barrier in baboon PET. [3H]1 specifically labels CSF1R in post-mortem human brain. The binding of [3H]1 is significantly higher in the post-mortem frontal cortex of AD versus control subjects.

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Diphosphoinositide and triphosphoinositide in animal tissues. Extraction, estimation and changes post mortem.

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