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A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch

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Sci. Signal.  22 Aug 2017:
Vol. 10, Issue 493, eaal5241
DOI: 10.1126/scisignal.aal5241
  • Fig. 1 Proposed molecular pathways: precursor fatty acids and expression of genes coding for enzymes involved in biosynthesis of mediators in pain circuit tissues.

    (A) Proposed biosynthetic pathways include lipase-mediated release of esterified linoleic acid (LA), lipoxygenase-mediated peroxidation, and hydroperoxide isomerization. (B) The abundance of precursor fatty acids in rat tissues (n = 4 rats for each tissue). (C and D) Quantitative biosynthetic gene expression in human (C) (n = 8, 8, 4, and 3 for the skin, tibial nerve, DRG, and dorsal cord, respectively) and rat (D) (n = 3, 4, 8, and 6 rats for hind paw, sciatic nerve, DRG, and dorsal cord, respectively) pain circuit tissues. Box plots and error bars in (B) to (D) indicate means and SEM. AA, arachidonic acid; DHA, docosahexaenoic acid; PLA2, phospholipase A2; Alox, lipoxygenase; Cyp, cytochrome P-450 epoxygenase; HpODE, hydroperoxyoctadecadienoate; sFPKM, standard fragments per kilobase of transcript per million mapped reads.

  • Fig. 2 Free hydroxy-epoxy- and keto-epoxy-octadecenoates are increased in human psoriatic skin lesions in participants reporting itch.

    (A) Expression of genes coding the phospholipases, ALOX12B and CYP2S1 enzymes in human psoriatic skin lesions compared to nonlesional psoriatic skin (n = 10 participants per group). (B) Concentrations of free hydroxy-epoxy- and keto-epoxy-octadecenoates in psoriatic lesions compared to control human skin. Statistical analysis was performed using the Kruskal-Wallis test [n = 7, 3, and 5 participant specimens for control skin, psoriasis lesion (no itch), and psoriasis lesion (itch), respectively].

  • Fig. 3 Regioselective augmentation of CGRP release from adult rat DRG neurons.

    (A to C) Ex vivo CGRP release measured from adult rat DRG neuronal cultures in response to low pH (A and B) or capsaicin (C) [n = 9 wells (A) or 12 wells (B and C), each from three separate harvests]. (D) The shared 3-hydroxy-Z-pentenyl-E-epoxide moiety that is unique to these two lipids is the proposed pharmacophore mediating the effects of 11H-12,13E-LA and 11H-9,10E-LA. *P < 0.05 using analysis of variance (ANOVA) with Tukey’s post hoc test.

  • Fig. 4 Pain- and itch-related rodent behavior responses after intradermal injections.

    (A and B) C-fiber withdrawal latency responses (A) and Aδ fiber stimulation responses after injection of mediators or vehicle control (B) (30 μg per injection; n = 12, 11, and 10 rats for vehicle, 11H-12,13E-LA, and PGE2, respectively). (C) Scratching bouts after injection of mediators (100 μg per mediator; n = 8, 7, 6, and 8 mice for vehicle, 9K-12,13E-LA, 13K-9,10E-LA, and the mixture, respectively). (D and E) Time course of scratching responses (D) and cumulative scratching bouts (E) evoked by the various mediators. (F and G) Time course (F) and scratching responses and cumulative scratching bouts (G) evoked by histamine (50 μg; n = 6 mice each for histamine and control). (H) Dose response for scratching bouts after injection of 9K-12,13E-LA (n = 6 mice for each dose). (I) Cumulative scratching bouts after injection of 9K-12,13E-LA in wild-type compared to c-Kit mutant mice (n = 5 and 8 for wild-type and c-Kit mutant mice, respectively). C-fiber withdrawal responses were compared using one-way ANOVA followed by Dunnett’s multiple comparisons test. Aδ pain ratings and itch responses were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Time course itch-related scratching data (D to G) were analyzed by two-way repeated-measures ANOVA followed by Dunnett’s multiple comparison test to compare each treatment at each time point. *P < 0.05, **P < 0.01.

  • Fig. 5 Association between diet-induced changes in plasma linoleic acid derivatives and pain-related end points in the CDH trial.

    (A) Changes in plasma concentrations of individual and total hydroxy-epoxy-octadecenoates (H-E-LA) after decreased dietary intake of linoleic acid for 12 weeks in patients with CDH (n = 44 participants). Red dashed lines indicate the limit of quantitation; P values based on Wilcoxon matched-pairs signed-rank test. (B to D) Associations between diet-induced reductions in plasma hydroxy-epoxy-octadecenoate concentrations and headache hours per day (B) (n = 40 participants), headache days per month (C) (n = 44 participants), and headache impact (D) (n = 44 participants). Graphs include the headache outcomes (y axes) compared to the mediator concentrations at week 12 (x axis) based on a Poisson regression model controlling for each outcome and mediator concentration at baseline. 95% confidence intervals are shown in blue.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/493/eaal5241/DC1

    Fig. S1. Proton NMR data for authentic standards.

    Fig. S2. Identification of hydroxy-epoxy-octadecenoate and keto-epoxy-octadecenoates.

    Fig. S3. Basal and low pH–evoked CGRP release in response to lipid mediators.

    Table S1. Rat tissue concentrations of hydroxy-epoxy- and keto-epoxy-octadecenoates.

    Table S2. Concentrations of hydroxy-epoxy- and keto-epoxy-octadecenoates in control skin and psoriatic skin.

    Table S3. Percentage of total hydroxy-epoxy- and keto-epoxy-octadecenoates as free acids in control skin and psoriatic skin.

    Table S4. Serum concentrations of free hydroxy-epoxy- and keto-epoxy-octadecenoates in control and psoriatic patients.

    Table S5. Association between diet-induced changes in plasma linoleic acid derivatives and pain-related end points in the CDH trial.

    Table S6. Mass spectrometry parameters established for the sMRM mode.

    Table S7. Demographic and clinical characteristics of psoriatic patients and controls.

    Table S8. Baseline demographic and clinical characteristics of patients with CDH.

  • Supplementary Materials for:

    A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch

    Christopher E. Ramsden,* Anthony F. Domenichiello, Zhi-Xin Yuan, Matthew R. Sapio, Gregory S. Keyes, Santosh K. Mishra, Jacklyn R. Gross, Sharon Majchrzak-Hong, Daisy Zamora, Mark S. Horowitz, John M. Davis, Alexander V. Sorokin, Amit Dey, Danielle M. LaPaglia, Joshua J. Wheeler, Michael R. Vasko, Nehal N. Mehta, Andrew J. Mannes, Michael J. Iadarola

    *Corresponding author. Email: chris.ramsden{at}nih.gov

    This PDF file includes:

    • Fig. S1. Proton NMR data for authentic standards.
    • Fig. S2. Identification of hydroxy-epoxy-octadecenoate and keto-epoxy-octadecenoates.
    • Fig. S3. Basal and low pH–evoked CGRP release in response to lipid mediators.
    • Table S1. Rat tissue concentrations of hydroxy-epoxy- and keto-epoxy-octadecenoates.
    • Table S2. Concentrations of hydroxy-epoxy- and keto-epoxy-octadecenoates in control skin and psoriatic skin.
    • Table S3. Percentage of total hydroxy-epoxy- and keto-epoxy-octadecenoates as free acids in control skin and psoriatic skin.
    • Table S4. Serum concentrations of free hydroxy-epoxy- and keto-epoxy-octadecenoates in control and psoriatic patients.
    • Table S5. Association between diet-induced changes in plasma linoleic acid derivatives and pain-related end points in the CDH trial.
    • Table S6. Mass spectrometry parameters established for the sMRM mode.
    • Table S7. Demographic and clinical characteristics of psoriatic patients and controls.
    • Table S8. Baseline demographic and clinical characteristics of patients with CDH.

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    Citation: C. E. Ramsden, A. F. Domenichiello, Z.-X. Yuan, M. R. Sapio, G. S. Keyes, S. K. Mishra, J. R. Gross, S. Majchrzak-Hong, D. Zamora, M. S. Horowitz, J. M. Davis, A. V. Sorokin, A. Dey, D. M. LaPaglia, J. J. Wheeler, M. R. Vasko, N. N. Mehta, A. J. Mannes, M. J. Iadarola, A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch. Sci. Signal. 10, eaal5241 (2017).

    © 2017 American Association for the Advancement of Science

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