Separation of choline- and ethanolamine-labeled metabolites by ion-exchange high-pressure liquid chromatography

Mazzola, G.; Kent, Claudia

doi:10.1016/0003-2697(84)90436-6

Cited by 10 publications

(3 citation statements)

References 24 publications

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“…Measurement of choline-containing metabolites A sample (250 ,ul) of the aqueous layer (2.5 ml) remaining after the lipid extraction was subjected to h.p.l.c. on an ion-exchange column (Aminex A-27) in an isocratic mode (Mazzola & Kent, 1984). The choline phosphate and CDP-choline fractions were recovered from the column in scintillation vials and assayed for radioactivity.…”

Section: Pulse-chase Studiesmentioning

confidence: 99%

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells

Post¹

1987

Biochemical Journal

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Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.

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Section: Pulse-chase Studiesmentioning

confidence: 99%

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells

Post¹

1987

Biochemical Journal

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“…Gro P Ins is a charged metabolite that tends to undergo ionic changes that challenge the use of chromatographic methods for the analysis of this phosphoinositide-derived metabolite in biological samples. In particular, quantitative analysis by reversed-phase chromatography typically suffers from poor retention and peak shape of a charged analyte, whereas anion-exchange chromatography is limited by low selectivity [ 10 , 11 ]. Few liquid chromatography-mass spectrometry (LC-MS) methods have been proposed to overcome these technical drawbacks without fully complying with the demand of simplicity and accuracy for a general use of the analytical procedures [ 12 – 14 ].…”

Section: Introductionmentioning

confidence: 99%

An Improved UPLC-MS/MS Platform for Quantitative Analysis of Glycerophosphoinositol in Mammalian Cells

et al. 2015

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The glycerophosphoinositols constitute a class of biologically active lipid-derived mediators whose intracellular levels are modulated during physiological and pathological cell processes. Comprehensive assessment of the role of these compounds expands beyond the cellular biology of lipids and includes rapid and unambiguous measurement in cells and tissues. Here we describe a sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the most abundant among these phosphoinositide derivatives in mammalian cells, the glycerophosphoinositol (GroPIns). The method has been developed in mouse Raw 264.7 macrophages with limits of quantitation at 3 ng/ml. Validation on the same cell line showed excellent response in terms of linear dynamic range (from 3 to 3,000 ng/ml), intra-day and inter-day precision (coefficient of variation ≤ 7.10%) and accuracy (between 98.1 and 109.0%) in the range 10-320 ng/ml. As proof of concept, a simplified analytical platform based on this method and external calibration was also tested on four stimulated and unstimulated cell lines, including Raw 264.7 macrophages, Jurkat T-cells, A375MM melanoma cells and rat basophilic leukemia RBL-2H3 cells. The results indicate a wide variation in GroPIns levels among different cell lines and stimulation conditions, although the measurements were always in line with the literature. No significant matrix effects were observed thus indicating that the here proposed method can be of general use for similar determinations in cells of different origin.

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“…Choline metabolites (choline, phosphorylcholine, glycerophosphorylcholine, and CDP-choline) in the water-soluble extracts (final vol 50 µL) were partially fractionated by HPLC, following the two-column procedure of Mazzola and Kent (1984). Pure standards and Dragendorff's reagent were used to identify all choline-containing compounds.…”

Section: Extraction Procedures and Analytical Methodsmentioning

confidence: 99%

Region-selective decline of in vivo lipid synthesis in the aged rat visual system

Alberghina

Viola

1989

Molecular and Chemical Neuropathology

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[14C]palmitate and [3H]choline were injected intravitreally and, at the same time, intraventricularly in Wistar male rats at 4, 10, and 24 mo of age. The precursor incorporation into lipids of the retina, optic nerve tract, superior colliculus, and lateral geniculate body was followed for 2 h. The specific radioactivity of precursors pool (choline, phosphorylcholine, and free fatty acids) showed a marked decrease in optic nerve tract and lateral geniculate body of aged rats, whereas in retinal tissue and superior colliculus no changes were observed as a function of age. In rats of the three age groups, whole retina and superior colliculus showed neither changes of choline incorporation into phosphatidylcholine and sphingomyelin nor alteration of palmitate incorporation into diacylglycerols, triacylglycerols, and major phospholipid classes as a function of age. In sharp contrast, the optic nerve tract and, to a lesser extent, the lateral geniculate body exhibited a significant age-related decline of either the incorporation of both precursors into all lipid classes or the specific radioactivities of endogenous precursor pools. We concluded that the visual pathway structures are metabolically affected in a different manner by aging. Particularly, the ability of the retina and superior colliculus to metabolize lipids appeared to be age invariant. The marked decline of lipid biosynthesis with age, for some visual structures, is consistent with the trend generally observed in metabolic turnover and function of other CNS regions.

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Separation of choline- and ethanolamine-labeled metabolites by ion-exchange high-pressure liquid chromatography

Cited by 10 publications

References 24 publications

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells

An Improved UPLC-MS/MS Platform for Quantitative Analysis of Glycerophosphoinositol in Mammalian Cells

Region-selective decline of in vivo lipid synthesis in the aged rat visual system

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