Isotope-dilution UPLC-MS/MS determination of cell-secreted bioactive lipids

Meyer, Anne-Louise; Thompson, John W.; Wang, Yiwen; Koseoglu, Secil; Dalluge, Joseph J.; Haynes, Christy L.

doi:10.1039/c3an00875d

Cited by 3 publications

(7 citation statements)

References 36 publications

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“…Secreted platelet-activating factor (PAF) was measured using a previously published UPLC–MS/MS method. 24 Because of the low recovery of PAF in Tyrode’s buffer, PAF secretion was measured from platelets incubated in phosphate buffered saline (free of Ca 2+ and Mg 2+ , containing 1 g/L glucose). A BCA protein assay was used for normalization of the results to prevent any differences in the data due to pelleting differences between control and phospholipid-incubated platelets.…”

Section: Methodsmentioning

confidence: 99%

Analytical Characterization of the Role of Phospholipids in Platelet Adhesion and Secretion

et al. 2014

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The cellular phospholipid membrane plays an important role in cell function and cell–cell communication, but its biocomplexity and dynamic nature presents a challenge for examining cellular uptake of phospholipids and the resultant effects on cell function. Platelets, small anuclear circulating cell bodies that influence a wide variety of physiological functions through their dynamic secretory and adhesion behavior, present an ideal platform for exploring the effects of exogenous phospholipids on membrane phospholipid content and cell function. In this work, a broad range of platelet functions are quantitatively assessed by leveraging a variety of analytical chemistry techniques, including ultraperformance liquid chromatography–tandem electrospray ionization mass spectrometry (UPLC–MS/MS), vasculature-mimicking microfluidic analysis, and single cell carbon-fiber microelectrode amperometry (CFMA). The relative enrichments of phosphatidylserine (PS) and phosphatidylethanolamine (PE) were characterized with UPLC–MS/MS, and the effects of the enrichment of these two phospholipids on both platelet secretory behavior and adhesion were examined. Results show that, in fact, both PS and PE influence platelet adhesion and secretion. PS was enriched dramatically and decreased platelet adhesion as well as secretion from δ-, α-, and lysosomal granules. PE enrichment was moderate and increased secretion from platelet lysosomes. These insights illuminate the critical connection between membrane phospholipid character and platelet behavior, and both the methods and results presented herein are likely translatable to other mammalian cell systems.

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Section: Methodsmentioning

confidence: 99%

Analytical Characterization of the Role of Phospholipids in Platelet Adhesion and Secretion

et al. 2014

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“…13, 14 Briefly, 50 µL supernatant samples were collected in a 96 well plate and stored overnight at −80°C. When thawed, supernatants were incubated for 60 min at 37°C with 100 µL 1 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide in 0.1 M citrate buffer at pH 4.5.…”

Section: Methodsmentioning

confidence: 99%

“…UPLC–MS/MS analysis was performed using a previously published method . Briefly, supernatants from MPMCs were collected, spiked with 25 ppb of each of the internal standards LTC4- d 5 , LTD4- d 5 , LTE4- d 5 , PGD2- d 9 , and PAF- d 4 and concentrated in a centrifugal evaporator until the remaining volume was approximately 50 μL.…”

Section: Methodsmentioning

confidence: 99%

“…To determine if incubation with 5-HT induced MPMC degranulation, supernatant content of the granule-stored enzyme β-hexosaminidase (β-Hex) was assessed as described previously. , Briefly, 50-μL supernatant samples were collected in a 96-well plate and stored overnight at −80 °C. When thawed, supernatants were incubated for 60 min at 37 °C with 100 μL of 1 mM 4-nitrophenyl N -acetyl-β- d -glucosaminide in 0.1 M citrate buffer at pH 4.5.…”

Section: Methodsmentioning

confidence: 99%

“…UPLC− MS/MS analysis was performed using a previously published method. 13 Briefly, supernatants from MPMCs were collected, spiked with 25 ppb of each of the internal standards LTC4-d 5 , LTD4-d 5 , LTE4-d 5 , PGD2-d 9 , and PAF-d 4 and concentrated in a centrifugal evaporator until the remaining volume was approximately 50 μL. After concentrating, salts were precipitated by the addition of 100 μL of icecold ethanol containing 0.1% formic acid (FA) and 200 μL mobile phase B (90/10 ACN/IPA containing 0.1% FA).…”

Section: ■ Methodsmentioning

confidence: 99%

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Time- and Concentration-Dependent Effects of Exogenous Serotonin and Inflammatory Cytokines on Mast Cell Function

et al. 2013

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Mast cells play a significant role in both the innate and adaptive immune response; however, the tissue-bound nature of mast cells presents an experimental roadblock to performing physiologically relevant mast cell experiments. In this work, a heterogeneous cell culture containing primary culture murine peritoneal mast cells (MPMCs) was studied to characterize the time-dependence of mast cell response to allergen stimulation, and the time- and concentration-dependence of the ability of the heterogeneous MPMC culture to uptake and degranulate exogenous serotonin using high performance liquid chromatography (HPLC) coupled to an electrochemical detector. Additionally, because mast cells play a central role in asthma, MPMCs were exposed to CXCL10 and CCL5, two important asthma-related inflammatory cytokines that have recently been shown to induce mast cell degranulation. MPMC response to both allergen exposure and cytokine exposure was evaluated for 5-HT secretion and bioactive lipid formation using ultraperformance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer (UPLC-MS/MS). In this work, MPMC response was shown to be highly regulated and responsive to subtle alterations in a complex environment through time and concentration dependent degranulation and bioactive lipid formation. These results highlight the importance of selecting an appropriate mast cell model when studying mast cell involvement in allergic response and inflammation.

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Stereochemistry- and concentration-dependent effects of phosphatidylserine enrichment on platelet function

Meyer

Gruba

Kim

et al. 2017

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Platelets are small (1-2μm in diameter), circulating anuclear cell fragments with important roles in hemostasis and thrombosis that provide an excellent platform for studying the role of membrane components in cellular communication. Platelets use several forms of communication including exocytosis of three distinct granule populations, formation of bioactive lipid mediators, and shape change (allowing for adhesion). This work explores the role of stereochemistry and concentration of exogenous phosphatidylserine (PS) on platelet exocytosis and adhesion. PS, most commonly found in the phosphatidyl-l-serine (l-PS) form, is exposed on the outer leaflet of the cell membrane after the platelet is activated. Knowledge about the impact of exogenous phosphatidylserine on cell-to-cell communication is limited (particularly concentration and stereochemistry effects). This study found that platelets incubated in l-PS or phosphatidyl-d-serine (d-PS) are enriched to the same extent with their respective incubated PS. All levels of l-PS enrichment also showed an increase in platelet cholesterol, but only the 50μM d-PS incubation showed an increase in cholesterol. The uptake of d-PS induced the secretion of granules and manufactured platelet activating factor (PAF) in otherwise unstimulated platelets. The uptake of l-PS had a greater impact on platelet stimulation by decreasing both the amount of δ-granule secretion and the amount of PAF that was manufactured.

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Isotope-dilution UPLC-MS/MS determination of cell-secreted bioactive lipids

Cited by 3 publications

References 36 publications

Analytical Characterization of the Role of Phospholipids in Platelet Adhesion and Secretion

Analytical Characterization of the Role of Phospholipids in Platelet Adhesion and Secretion

Time- and Concentration-Dependent Effects of Exogenous Serotonin and Inflammatory Cytokines on Mast Cell Function

Stereochemistry- and concentration-dependent effects of phosphatidylserine enrichment on platelet function

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