Analytical Method for Skin and Plasma Lipids

Skin was divided into dermis and epidermis (on ice, by scalpel, with the aid of visual inspection at ×40 magnification) as previously described (17 (link), 20 (link)). Skin (5–30 mg) and plasma samples (∼1 ml) were extracted using ice-cold methanol (15%, v/v), and 12-hydroxyeicosatetraenoic acid (HETE)-d₈, prostaglandin (PG)B₂-d₄, 8 (9)-epoxy eicosatrienoic acid-d₁₁, and 8,9-dihydroxyeicosatrienoic acid-d₁₁ (20 ng each/sample; Cayman Chemicals, Ann Arbor, MI, USA) were used as internal standards. The extracts were semipurified using solid-phase extraction cartridges (C18-E; Phenomenex, Macclesfield, United Kingdom) to eliminate matrix effects. Ultraperformance liquid chromatography with electrospray ionization and tandem mass spectrometry (UPLC/ESI-MS/MS) analysis was performed on an Acquity UPLC pump (Waters, Wilmslow, United Kingdom) coupled to an electrospray ionization triple quadrupole mass spectrometer (Xevo TQ-S; Waters) as previously described (17 (link), 31 (link), 32 (link)). A detailed list of the multiple reaction monitoring transitions and collision energy settings is provided in Supplemental Table S1. Results are expressed as picograms per milligram protein (skin) or picograms per milliliter (plasma).

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Kendall A.C., Pilkington S.M., Murphy S.A., Del Carratore F., Sunarwidhi A.L., Kiezel-Tsugunova M., Urquhart P., Watson R.E., Breitling R., Rhodes L.E, & Nicolaou A. (2019). Dynamics of the human skin mediator lipidome in response to dietary ω-3 fatty acid supplementation. The FASEB Journal, 33(11), 13014-13027.

Publication 2019

Acquity UPLC pump Xevo TQ-S

12 hydroxyeicosatetraenoic acid Acid Cayman Cold Dermis Epidermis Epoxy Liquid chromatography Methanol Ms ms Plasma Prostaglandin b2 Protein Skin Solid phase extraction

Corresponding Organization : University of Manchester

Other organizations : National Health Service, Salford Royal NHS Foundation Trust

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Variable analysis

independent variables

Skin was divided into dermis and epidermis

dependent variables

Levels of 12-hydroxyeicosatetraenoic acid (HETE)-d8, prostaglandin (PG)B2-d4, 8 (9)-epoxy eicosatrienoic acid-d11, and 8,9-dihydroxyeicosatrienoic acid-d11 in skin and plasma samples

control variables

Skin and plasma samples were extracted using ice-cold methanol (15%, v/v)
Internal standards (12-hydroxyeicosatetraenoic acid (HETE)-d8, prostaglandin (PG)B2-d4, 8 (9)-epoxy eicosatrienoic acid-d11, and 8,9-dihydroxyeicosatrienoic acid-d11) were used
Extracts were semipurified using solid-phase extraction cartridges (C18-E) to eliminate matrix effects
Ultraperformance liquid chromatography with electrospray ionization and tandem mass spectrometry (UPLC/ESI-MS/MS) analysis was performed

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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