Lactobacillus acidophilus (LA) was selected as probiotic of choice in our present study due its common availability and established immunomodulatory properties. In the present study, we report for the first time that administration of LA in ovariectomized (ovx) mice enhances both trabecular and cortical bone microarchitecture along with increasing the mineral density and heterogeneity of bones. This effect of LA administration is due to its immunomodulatory effect on host immune system. LA thus skews the Treg-Th17 cell balance by inhibiting osteoclastogenic Th17 cells and promoting anti-osteoclastogenic Treg cells in ovx mice. LA administration also suppressed expression of osteoclastogenic factors  and increased expression of anti-osteoclastogenic factors . Taken together the present study for the first time clearly demonstrates the therapeutic potential of LA as an osteo-protective agent in enhancing bone health  in postmenopausal osteoporosis.Osteoporosis is one of the most important but often neglected bone disease associated with aging and postmenopausal condition leading to bone loss and fragility. Probiotics have been associated with various immunomodulatory properties and have the potential to ameliorate several inflammatory conditions including osteoporosis.  Lactobacillus acidophilus administration bone mass in post-menopausal osteoporotic mice by skewing Treg-Th17 balance. •Post-menopausal osteoporosis is one of most commonly occurring skeletal diseases leading to bone loss and fragility.•Probiotics possess various immunomodulatory properties and have the potential to ameliorate several inflammatory conditions including post-menopausal osteoporosis.•Lactobacillus acidophilus (LA) in ovx mice skews Treg-Th17 cell balance in vivo by inhibiting osteoclastogenic Th17 cells and promoting anti-osteoclastogenic Treg cells.We report for the first time that administration of a common probiotic •Also, LA administration suppresses expression of osteoclastogenic  and enhances expression of anti-osteoclastogenic  factors in ovx mice.•The present study for the first time clearly demonstrates the therapeutic potential of LA as a potent osteo-protective agent in enhancing bone health in post-menopausal osteoporosis. Also, the effect of LA on Treg-Th17 equilibrium is still not defined. LA is one of the most common probiotic strains available in our homes . In our present study, we report for the first time that administration of LA due to its immunomodulatory effect on Treg-Th17 cell balance (enhanced Treg and decreased Th17 cells) in vivo, suppresses expression of osteoclastogenic factors  and induces expression of anti-osteoclastogenic factors (IL-10 and IFN-γ) leading to enhanced bone mass. The present study highlights this interesting and novel field of bone biology (termed now as “Osteomicrobiology”) and will have immense clinical implications in near future (Regulatory T cells (Treg cells) inhibit osteoclastogenesis and are thus known protectors of bone health. Whereas Th17 cells induce osteoclastogenesis and are involved in enhanced bone loss in osteoporosis. Also, Treg cells inhibit the differentiation of Th17 cells and vice-versa . This der future . Adminis22.19 cfu/ml Lactobacillus acidophilus suspension) constituted in drinking water for a period of 6 weeks post-ovariectomy as per standardized protocols  of 8–10 weeks with an average body weight of 25 g were selected. Mice were housed in standard cages, maintained under specific pathogen-free conditions and fed sterilized food and autoclaved water ad libitum. Mice were either ovariectomized (ovx) or sham operated with intra-peritoneal injection of Ketamine (100–150 mg/kg) and Xylazine (5–16 mg/kg) through standard protocol and were grouped under three categories with 10 mice in each group . Ovx + LA group received daily oral dose of 200 μl  ATCC 4356 was provided by Dr. Rajesh K. Mondal from Department of Microbiology, Dr. Harisingh Gour Central University, Sagar (MP)-470003, India. Briefly, LA were inoculated in deMan, Rogosa, and Sharpe broth  and grown at 37 °C for 20 h, then resuspended in phosphate-buffered saline (PBS) before administering to the mice with 200 μl suspension (109 cfu/ml Lactobacillus acidophilus).2.3The following antibodies/kits were bought from BD Biosciences (USA): PerCP-Cy-5.5 Rat Anti-Mouse CD4-(RM4-5) (550954), APC-Rat-Anti-Mouse-CD8a-(53-6.7) (561093), PE-Rat-Anti-Mouse-CD45R/B220-(RA3-6B2) (553089) and mouse Cytometric Bead Array-(CBA) kit. The reagents Anti-Human/Mouse Rorγt-PE-(AFKJS-9) (12-6988), Anti-Mouse/Rat Foxp3-APC-(FJK-16s) (17-5773), Foxp3/Transcription factor staining buffer (0-5523-00), RBC lysis buffer (00-4300-54) were obtained from eBioscience (USA). Anti-Mouse-CD254-(TRANCE-RANKL)-PE (IK22/5) (510005), Anti-Mouse-CD8-PE/Cy7 (53-6.7) (100721), Anti-Mouse-IFN-γ-FITC (XMG 1.2) (505805) and Anti-Mouse-IL-17A-PE (TC11-8H10.1) (506903) were obtained from BioLegend (USA). Mouse/Rat Estradiol ELISA Kit  and all procedures were followed according to the manufacturer's instruction.2.4For SEM analysis, femur cortical bone samples were kept in 1% Triton for 48–72 h and later transferred to 1×PBS solution till final analysis is done. Bone slices were made and dried under incandescent bulb before SEM analysis and scanned in NOVA NANOSEM 450 microscope equipped with a tungsten filament gun operating at WD 10.6 mm and 20 kV. SEM images were digitally photographed at low (15×), intermediate (1000×), and high magnifications  to picture the best cortical structure. Images were later processed and analyzed using Adobe Photoshop 7.0. The data was further analyzed using MATLAB software .2.5Femur cortical bone samples were dried in dust free environment with 60 W lamps for 6 h followed by high vacuum drying and subsequently analyzed by Atomic Force Microscope , operating under the Acoustic AC mode (AAC or Tapping mode). This was assisted with cantilever (NSC 12(c) from MikroMasch, Silicon Nitride Tip) by NanoDrive™ version 8 software at a force constant of 0.6 N/m having resonant frequency at 94–136 kHz. The images were taken in air at room temperature at the scan speed of 1.5–2.2 lines/s. The data analysis was done using of Nanoscope analysis software. The data was further analyzed using MATLAB software .2.63) were used. For each analysis, the estimated BMD was determined based on linear correlation between the μCT attenuation coefficient and BMD  and lumbar vertebrae-V (trabecular) was performed using SkyScan 1076 scanner (SkyScan). Scanning was done at 70 kV, 100 mA using a 0.5-mm aluminum filter and exposure set to 590 ms. In total, 1800 projections were collected at a resolution of 6.93 μm/pixel. For purpose of carrying out reconstruction process NRecon software was used. Bone length was determined from the rendered 3D images in CTAn software. The distance from trochanter to edge of femoral condyles defined total femoral length. Tibial length calculated as distance from medial condyle to medial malleolus. Manual segmentation of 2D slice of sagittal images was done to isolate growth plates from the surrounding bone tissue in micro-CT images followed by reconstruction to render 3D images. It was from these images that growth plate height was measured using data viewer software. For trabecular region analysis, ROI was drawn at a total of 100 slices in secondary spongiosa at a distance of 1.5 mm from distal border of growth plates excluding the parts of cortical bone and primary spongiosa. In vivo measurement of LV5 trabecular was done by encompassing 50 continuous slices which start from the beginning of trabecular bone within spinal body . The CTA and BMD .2.7−1; scan speed 2.5 kHz, and 128 scans co-addition, in the KBr pellet form. With the aid of Savitzky-Golay algorithm noise was eliminated to obtain smooth spectra of samples. The samples were held in contact with a prism made of highly refractive material transmitting infrared rays which are made incident on the sample at angle that induces total reflection. Bones were kept in 1% Triton, washed after 24 h and dried under 60 W lamps for 6 h followed by high vacuum drying. Dry bones were made into powder form and mixed with KBr (potassium bromide) in 1:100 ratios and analyzed for FTIR.The FTIR analysis of femur cortical bones was done by using 8400S-Fourier Transform Infrared Spectrophotometer (SHIMADZU), working on resolution 4 cm2.8T cells in single-cell suspensions isolated from bone marrow and spleen were stained with PerCP-Cy5.5-anti-CD4 and PE/Cy-7-anti-CD8 or APC-anti-CD8. Cells were allowed to pellet down after centrifugation at 1800 rpm for 5 min followed by addition of 1 ml RBC lysis buffer. Cells were washed thoroughly with wash buffer and fixed-permeabilized with Foxp3 permeabilization buffer for 30 min on ice. Intracellular Foxp3, RANKL and Rorγt staining was performed using APC-conjugated anti-Foxp3, PE-conjugated anti-RANKL and PE-conjugated anti-Rorγt Abs in permeabilization buffer for 45 min on ice. Cells were washed thoroughly with permeabilization buffer and analyzed by flow cytometry.Intracellular staining of Tregs and Th17 cells was performed by stimulating cells for 5-6 h with PMA (50 ng/ml) and ionomycin (500 ng/ml). After 1 h, GolgiStop (1 μg/ml) was added in the cultures to block cytokine secretion. Cells were first surface stained with PerCP-Cy5.5-anti-CD4 and PE/Cy-7-anti-CD8 antibodies for 30 min on ice. Cells were washed thoroughly with wash buffer and further fixed-permeabilized with Foxp3 fixation-permeabilization buffer for 30 min on ice. Cells were washed thoroughly with Foxp3-permeabilization buffer and stained intracellularly with APC conjugated anti-Foxp3 and PE-conjugated anti-IFN-γ; FITC-conjugated anti-IL-10 and PE-conjugated IL-17A antibodies in permeabilization buffer for 30 min on ice . Cells w2.9Levels of IL-6, IL-10, IL-17A, IFN-γ and TNF-α in blood serum were assessed by fluorescent bead-based technology using CBA kit and all procedures were followed according to the manufacturer's instruction . Fluorescent signals were read and analyzed on a FACS Aria III flow cytometer (BD Biosciences) with the help of BD FCAP Array software (BD Biosciences).2.10t-test for paired or nonpaired data as appropriate. Analysis was performed using Sigma plot software . Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05  with respect to ovx mice group.The results were evaluated by using ANOVA with subsequent comparisons by Student 33.1th week post-ovariectomy), mice were sacrificed, and femur bone collected for SEM (Ovx status was routinely verified for every batch of ovariectomized mice by Estradiol (E2) estimation from the serum samples of sham and ovx groups using enzyme-linked immunosorbent assay as per manufacturer's instruction. Notably, we consistently observed significantly decreased levels of estradiol (E2) in ovx mice as compared to sham groups . To determine the effect of LA administration on bone health we were first interested in looking for different signs of bone remodelling by scanning the surface of bones by SEM/AFM. To determine the same, mice were divided in three group's viz. sham, ovx and ovx + LA and at the end of the experiment  is a multi-paradigm 3.2p < 0.01), trabecular thickness (Tb. Th) (p < 0.01), trabecular number (Tb. N) (p < 0.0001) and connectivity density (Conn. Den.) (p < 0.001) (p < 0.01) and trabecular pattern factor (Tb. Pf.) (p < 0.01)  for quantitating various bone morphometric and geometric parameters related to bone loss. Since the lumbar vertebrae-5 (LV-5) is considered as one of the best diagnostic parameters to study the effect of bone loss via μCT in osteoporosis, we too were interested in analysing the effect of LA intake on LV-5 vertebrae. Strikingly, μCT analysis clearly pointed towards a significant improvement of the LV-5 microarchitecture in LA administered group with respect to both sham and ovx group . We obse< 0.001) ; whereas < 0.01) . Togethe3.3p < 0.01), Tb. Th (p < 0.01), Tb. N (p < 0.01) and Conn. Den (p < 0.01) (p < 0.01) and Tb. Pf (p < 0.01) (p < 0.01), Tb. Th (p < 0.05), Tb. N (p < 0.01) and Conn. Dn (p < 0.01) along with decreased Tb. Sp (p < 0.01) and Tb. Pf (p < 0.01)  ; but dec < 0.01)  in compa < 0.01)  with sig < 0.01) . The ovx < 0.01)  in compa < 0.01) , with si3.4p < 0.01), femur trabecular (p < 0.01), tibia trabecular (p < 0.05), tibia cortical (p < 0.05) and femur cortical (p < 0.05) (p < 0.01), XST (p < 0.005) and c/p ratio (p < 0.05) in femur cortical bones with respect to ovx group  defines the quantity of mineralized tissue present (size and density) in bones and is usually considered for determining tendency of bones to undergo fracture. Thus, we were excited to next determine the effect of LA administration on BMD for both cortical and trabecular bones. Interestingly, both trabecular and cortical bones from femur/tibia/LV5 illustrated a significant increase in their respective BMDs. The BMDs of ovx + LA administered group was significantly enhanced as compared to ovx group in LV-5-trabecular ( < 0.05) A. The co < 0.05) , it thus < 0.05) . Thus, wvx group B. In sum3.5+ and Rorγt+ master-transcription factors for Treg and Th17 cells respectively. Mice were sacrificed after completion of 6 weeks of LA administration, and total lymphocyte populations derived from bone marrow and spleen were analyzed for percentage of CD4+Foxp3+Treg cells, CD8+Foxp3+Treg cells and CD4+Rorγt+Th17 cells by flow cytometry. Gating strategy for CD4+Foxp3+Treg cells, CD8+Foxp3+Treg cells and CD4+Rorγt+Th17 in bone marrow and spleen was performed as per representative figures in Supplementary Figs. S2 and S3 respectively. As compared with ovx mice, LA administration in ovx mice led to 1.5-fold increase in CD4+Foxp3+Treg cells in bone marrow (3.42 ± 2% in ovx to 8.20 ± 1% in LA) (p < 0.01) and one-fold increase in spleen (3.17 ± 2% in ovx to 7.38 ± 1.5% in LA) (p < 0.01) (+Foxp3+ Treg cells in bone marrow (0.3 ± 0.01% in ovx to 1.2 ± 0.02% in LA) (p < 0.01) and three-fold increase in spleen (1.83 ± 0.42% in ovx to 7.08 ± 0.86% in LA) (p < 0.01) (+Rorγt+Th17 cells in bone marrow (9.4 ± 0.63% in ovx to 2.11 ± 0.03% in LA) (p < 0.01) and >1.5-fold decrease in spleen (9.76 ± 0.56% in ovx to 6.21 ± 0.41% in LA) (p < 0.01)  A. Intere < 0.01) B as comp < 0.01) C with re+ and later analyzed for the expression of both Foxp3+IL-10+ (Treg) cells and Rorγt+IL-17+ (Th17) cells. We observed that LA administration in ovx mice led to >1.5-fold increase in Foxp3+IL-10+ cells in bone marrow (2.01 ± 0.42% in ovx to 7.34 ± 0.78% in LA) (p < 0.001) and an increase of close to two-fold in spleen (1.78 ± 0.45% in ovx to 4.32 ± 0.43% in LA) (p < 0.01) (+IL-17+ cells in bone marrow (6.11 ± 0.78% in ovx to 1.35 ± 0.12% in LA) (p < 0.01) and >1.5-fold in spleen (7.23 ± 0.45% in ovx to 3.87 ± 0.32% in LA) (p < 0.01) (+ Treg cells (both CD4 and CD8) and inhibiting Th17 cells in vivo.It is now well established that IL-10 cytokine possess anti-inflammatory properties and is responsible for regulatory functions of Tregs during inflammation . On the  < 0.01) D along w < 0.01) E. Collec3.6p < 0.01), TNF-α (p < 0.01) and IL-17A (p < 0.01) in comparison to ovx group (p < 0.05) and IL-10 (p < 0.01) which are known anti-osteoclastogenic factors/cytokines (+T cells in bone marrow (8.12 ± 0.75% in ovx to 4.01 ± 0.32% in LA) (p < 0.01) along with two-fold reduction in spleen (26.32 ± 1.22% in ovx to 13.75 ± 2.11% in LA) (p < 0.01) with respect to ovx mice (p < 0.01) and more than one-fold decrease in spleen (42.3 ± 2% in ovx to 26.1 ± 1% in LA) (p < 0.05) with respect to ovx mice  and Lactobacillus plantarum (NTU 102) fermented milk to ovx mice resulted in higher trabecular number , organic phase  and water . With th+T cells have established role in inflammatory bone loss (+ and CD8+) are associated with bone protective functions  and inhibits Th17 cells in bone marrow, a primary site of osteoclastogenesis. Taken together these results clearly demonstrate the immunomodulatory role of LA in enhancing bone health by inhibiting osteoclastogenic Th17 cells and promoting anti-osteoclastogenic Treg cells.Since, LA has been associated with various immunomodulatory properties ; we nextone loss . Also, tunctions , whereasunctions . Several regions . LA has  regions , along w regions . Among t regions  along wi regions . In the Postmenopausal bone loss is regarded as an inflammatory condition because of the causal role of the immune system  and the th women and every 5th men in the world  entirely protected ovx mice against bone loss and results in a net gain of bone mass in sex steroid deficient mice model. Consistent with these physiological findings, LA administration skews Treg-Th17 cell balance and dampens the levels of osteoclastogenic cytokines following sex steroid depletion. The development of osteoporosis can be influenced by various lifestyle factors, and managing the nutritional status is the biggest key preventive measure, particularly for aged population. Also, a recent article by Laird et al.  reports he world .


The dihydrobenzimidazol-2-one moiety is essentially planar with the prop-2-yn- 1-yl substituent rotated well out of this plane. In the crystal, C—H⋯π(ring) interactions and C—H⋯O hydrogen bonds form corrugated layers parallel to  interactions and C—HProp⋯ODhyr  hydrogen bonds form corrugated layers parallel to (10Bnz⋯ODhyr (Bnz = benzene) hydrogen bonds and head-to-tail, slipped, π-stacking [centroid-to-centroid distance = 3.7712 (7) Å] interactions between dihydrobenzimidazol-2-one moieties. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions to the crystal packing are from H⋯H (44.1%), H⋯C/C⋯H (33.5%) and O⋯H/H⋯O (13.4%) interactions. Hydrogen-bonding and van der Waals interactions are the dominant interactions in the crystal packing. Computational chemistry calculations indicate that in the crystal, C—H⋯O hydrogen-bond energies are 46.8 and 32.5 (for C—HProp⋯ODhyr) and 20.2 (for C—HBnz⋯ODhyr) kJ mol−1. Density functional theory (DFT) optimized structures at the B3LYP/6–311 G level are compared with the experimentally determined molecular structure in the solid state. The HOMO–LUMO behaviour was elucidated to determine the energy gap.In the title molecule, C N-ribosyl-dimethylbenzimidazole and it serves as the axial ligand for cobalt in vitamin B12 -one in the presence of tetra-n-butylammonium bromide as catalyst and potassium carbonate as base, to give the title compound, I in good yield. We report herein on its synthesis, the molecular and crystal structures along with the Hirshfeld surface analysis and the intermolecular interaction energies and the density functional theory (DFT) computational calculations carried out at the B3LYP/6–311 G level for comparison with the experimentally determined molecular structure in the solid state.Benzimidazole is an aromatic heterocyclic organic compound that plays an important role in medicinal chemistry and pharmacology. The most prominent benzimidazole moiety present in nature is In the title compound, the dihydrobenzimidazol-2-one moiety is planar to within 0.0160 (8) Å (r.m.s. deviation = 0.0082) with atom C7 deviating the most from the mean plane and a prop-2-yn-1-yl substituent rotated well out of this plane as shown by the C1—N2—C9—C10 torsion angle of 62.16 (13)° Fig. 1.Mthy⋯Cg1i interactions ; which are connected along the b-axis direction by C—HBnz⋯ODhyr hydrogen bonds (Bnz = benzene and Dhyr = dihydro) and along the a-axis direction at ca 90° to this and parallel to (10Prop⋯ODhyr hydrogen bonds (Table 1Cg2⋯Cg1ii = 3.7712 (7) Å, dihedral angle = 0.96 (6)°; symmetry code: (ii) 1 – x, 1 – y, 1 – z; Cg1 and Cg2 are the centroids of rings A and B (N1/N2/C1/C6/C7) and C—HProp⋯ODhyr (Prop = prop-2-yn-1-yl) hydrogen bonds  analysis . The C⋯C contacts, Fig. 7e, have an arrow-shaped distribution of points with the tip at de = di = 1.75 Å. The H⋯N/N⋯N contacts, contributing 2.9% to the overall crystal packing, are depicted in Fig. 7f as widely scattered points. Finally, the N⋯C/C⋯N interactions, contributing 2.4% to the overall crystal packing, are shown in Fig. 7g as tiny characteristic wings with the tips at de + di = 3.45 Å.The overall two-dimensional fingerprint plot, Fig. 7S Table 2; these adnorm plotted onto the surface are shown for the H⋯H, H⋯C/C⋯H and H⋯O/O⋯H interactions in Fig. 8a–c, respectively.The Hirshfeld surface representations with the function et al., 2015The Hirshfeld surface analysis confirms the importance of H-atom contacts in establishing the packing. The large number of H⋯H, H⋯C/C⋯H and H⋯ O/O⋯H interactions suggest that van der Waals interactions and hydrogen bonding play the major roles in the crystal packing  is the sum of electrostatic (Eele), polarization (Epol), dispersion (Edis) and exchange-repulsion (Erep) energies  were calculated to be −17.4 (Eele), −3.5 (Epol), −62.6 (Edis), 46.5 (Erep) and −46.8 (Etot) for C11—H11⋯O1, −12.4 (Eele), −1.9 (Epol), −41.6 (Edis), 29.6 (Erep) and −32.5 (Etot) for C9—H9B⋯O1 and −13.7 (Eele), −3.7 (Epol), −15.5 (Edis), 17.0 (Erep) and −20.2 (Etot) for C3—H3⋯O1.The intermolecular interaction energies were calculated using the CE–B3LYP/6–31G energy model available in via density functional theory (DFT) using the standard B3LYP functional and 6–311 G basis-set calculations . The significance of η and σ is for the evaluation of both the reactivity and stability. The electron transition from the HOMO to the LUMO energy level is shown in Fig. 9H-1,3-benzodiazol-2-one ring. The energy band gap [ΔE = ELUMO − EHOMO] of the molecule is about 5.4115 eV, and the frontier molecular orbital energies, EHOMO and ELUMO are −5.8885 and −0.4770 eV, respectively.The optimized structure of the title compound in the gas phase was generated theoretically et al., 2018abet al., 2012et al., 2015et al., 2013et al., 2016The syntheses of several N-substituted benzimidazol-2-one analogues have been reported  to 1.4632 (11) Å, and so are quite comparable. The exocyclic groups in I are in an anti-arrangement with the prop-2-yn-1-yl group rotated by 62.16 (13)° out of the plane of the bicyclic moiety (as measured by the C1—N2—C9—C10 torsion angle). In the other three, these substituents are also anti and in II the corresponding torsion angle is 73.46 (18)° while in III they are 82.58 (15) and 74.31 (14)°. In IV the torsion angles are 106.0 (3) and 113.4 (3)° indicating a rotation in the opposite direction from the first three.The closest examples to the title compound, H-benzimidazol-2(3H)-one (3.61 mmol), iodomethane (6.73 mmol) and potassium carbonate (6.24 mmol) in DMF (15 ml) was added a catalytic amount of tetra-n-butylammonium bromide (0.37 mmol). The mixture was stirred for 24 h. The solid material was removed by filtration and the solvent evaporated under vacuum. The solid product was purified by recrystallization from ethanol to afford colorless crystals (yield: in 82%).To a mixture of 1-(prop-2-ynyl)-1Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989019015779/lh5936sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019015779/lh5936Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019015779/lh5936Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019015779/lh5936Isup4.cmlSupporting information file. DOI: 1967468CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This is the second reported crystal structure of an adduct of a 2,3-disubstituted 1,3-thiazolidine-4-one ligand and triphenyltin chloride. The tin atom adopts a trigonal–bipyramidal coordination geometry with the O and Cl atoms in the axial sites. O)triphenyltin(IV), [Sn(C6H5)3Cl(C15H19NOS)], the tin(IV) atom exhibits a trigonal–bipyramidal coordination geometry with the three phenyl groups in equatorial positions and the chloride anion and ligand oxygen atom present at axial sites [O—Sn—Cl = 175.07 (14)°]. The thiazolidinone ring of the ligand adopts an envelope conformation with the S atom as the flap. The dihedral angles between the heterocycle ring plane  are 44.3 (9)° with respect to the pendant C-phenyl plane and 34.3 (11)° to the N-cyclohexyl ring . The C-phenyl and N-cyclohexyl ring are close to orthogonal to each other, with a dihedral angle of 81.1 (4)° between them. In the crystal, molecules are linked by weak C—H⋯Cl hydrogen bonds to generate [001] chains.In the centrosymmetric (racemic) title compound, chlorido(3-cyclohexhyl-2-phenyl-1,3-thiazolidin-4-one-κ Although Mössbauer spectroscopy was not used in our study, we see the same coordination properties with the title molecule in the X-ray structure. The Sn—O bond length was found to be 2.500 Å for the tin–diphenylthiazolidinone adduct, using Mössbauer techniques as well as the X-ray data, whereas, the X-ray data for the title compound yields an Sn—O bond length of 2.488 (4) Å. These values are almost the same and show no difference in having the presence of phenyl and a cyclohexyl group at C2 and N3 (C21 and N1 in our numbering scheme) versus a phenyl group at each location.The most closely related structure previously reported is that of 2,3-diphenyl-1,3-thiazolidin-4-one as a 1:1 adduct with triphenyltin chloride  Å, C—H⋯Cl = 140°] helps to consolidate the packing.The surface of the title compound is primarily hydrophobic due to four aromatic and one aliphatic ring resulting in intermolecular van der Waals interactions Fig. 2 between et al., 1995There is only one closely related structure previously reported and that is 2,3-diphenyl-1,3-thiazolidin-4-one as a 1:1 adduct with triphenyltin chloride (Smith N)-3-cyclohexyl-2-phenyl-1,3-thiazolidine-4-one has been previously reported  in a 50 ml round-bottom flask while stirring at room temperature for 3 h. Stirring was then stopped and the solution was allowed to stand for an additional 10 h. A precipitate was apparent, which was filtered and the filtrate was reduced under vacuum on a rotary evaporator, dried under vacuum to give an oily residue, which formed crystals when heated in ligroin. Recrystallization from ligroin solution yielded 0.0022 mol (97% yield) of the title 1:1 complex in the form of colorless blocks: m.p. 372–375 K (no literature reports).The 1:1 adduct with triphenyltin chloride was prepared by dissolving 0.0023 mol of Triphenyltinchloride-3-cyclohexyl-2-phenyl-1,3-thiazolidin-4-one: Yield (97%); m.p. 372–375 K, cm−1 1658.6 (C=O); 1H NMR (CDCl3): 7.78–7.27 , 5.66 , 3.89 , 3.85–3.78 , 3.58 , 1.79–0.91 ; 13C NMR: 171.77 (C4), 142.98, 137.78, 136.34 , 130.62, 129.32 , 129.07, 128.88, 128.52, 126.38, 62.83 (C2), 56.30, 33.23 (C5), 31.03, 30.12, 26.10, 25.42. C33H34OClSnNS.Rint = 0.0721. Upon refinement we observed positional disorder in almost a fourth of the structure (nine out of thirty-eight non-H atoms). As a result, some refinement parameters such as the ADP max/min ratio (8.2) for one of the atoms are slightly above optimal values but the atomic connectivity is clearly established. Crystal data, data collection and structure refinement details are summarized in Table 2Uiso(H) = 1.2Ueq (aromatic or methylene C) or 1.5Ueq(methyl C).In spite of our search for a better crystal we had to work with one that was not optimal, as is evident from the high value of 10.1107/S2056989019001592/hb7780sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019001592/hb7780Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019001592/hb7780Isup3.molSupporting information file. DOI: 1894217CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanoic acid (α-T-13′-COOH) can be synthesized from α-tocopherol in a human liver-on-chip, and is detected in human and mouse plasma at concentrations (8–49 nM) that inhibit 5-lipoxygenase in human leukocytes. α-T-13′-COOH accumulates in immune cells and inflamed murine exudates, selectively inhibits the biosynthesis of 5-lipoxygenase-derived lipid mediators in vitro and in vivo, and efficiently suppresses inflammation and bronchial hyper-reactivity in mouse models of peritonitis and asthma. Together, our data suggest that the immune regulatory and anti-inflammatory functions of α-tocopherol depend on its endogenous metabolite α-T-13′-COOH, potentially through inhibiting 5-lipoxygenase in immune cells.Systemic vitamin E metabolites have been proposed as signaling molecules, but their physiological role is unknown. Here we show, by library screening of potential human vitamin E metabolites, that long-chain ω-carboxylates are potent allosteric inhibitors of 5-lipoxygenase, a key enzyme in the biosynthesis of chemoattractant and vasoactive leukotrienes. 13-((2 Vitamin E metabolites are proposed to have signalling capacity, but how they may regulate immune responses is still unclear. Here the authors show that a vitamin E metabolite, α-T-13′-COOH, can inhibit 5-lipoxygenase and thereby suppress the synthesis of lipid mediators of immune activation and inflammatory responses. Vitamin E deficiency is causative for severe degenerative diseases, decline of the immune response, and potentially for atherosclerosis3. Supplementation of α-tocopherol  and other vitamin E forms above the recommended daily dose showed marked anti-inflammatory, anti-atherosclerotic, and anti-tumor efficacy in animal models2. However, human clinical intervention studies provided mixed results regarding efficacy and safety of α-T (1a)5. The interest in vitamin E was recently renewed based on placebo-controlled, randomized, multicenter trials with positive outcomes for non-alcoholic fatty liver disease (NAFLD)7 and Alzheimer disease8. Up to now, the molecular mechanism(s) and target(s) underlying the disease-modifying activities of vitamin E are still diffuse.Vitamin E, a mixture of eight natural vitamers  suppress inflammation by targeting 5-lipoxygenase (5-LO), which catalyzes the initial steps in the biosynthesis of potent immunomodulatory lipid mediators9  from phospholipids by cytosolic phospholipase A2 (cPLA2), AA is transferred by the membrane-bound 5-LO-activating protein (FLAP) to 5-LO that produces 5-hydro(pero)xy-eicosatetraenoic acid (5-H(P)ETE) and subsequently LTA4 , which mediate smooth muscle contraction and enhance vascular permeability19.We here provide evidence that physiologically formed metabolites of α-T , modulates the lipoxygenation of AA in leukocytes20. Other forms of vitamin E were described to inhibit cellular LT formation in vitro by suppressing Ca2+-influx, extracellular signal-regulated kinase (ERK)1/2 signaling and movement of 5-LO to FLAP22. However, the concentrations of the vitamin E forms required to suppress 5-LO product formation are not reached in human plasma23, and it has been speculated that endogenous vitamin E is biotransformed to active metabolites, as established for other fat-soluble vitamins24.5-LO has been discussed as potential target of vitamin E before2 26, another key enzyme of pro-inflammatory eicosanoid biosynthesis. Since these metabolites were detected neither in human nor rodent plasma unless vitamin E was supplemented at pharmacological doses28, the physiological role of LCMs and their mode of action remained enigmatic. Two LCMs derived from α-T (1a) were recently discovered that occur at low nanomolar concentrations in human plasma29 and inhibit pro-inflammatory signal transduction at supra-physiological LCM concentrations30. Here, we identify 13-((2R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanoic acid  as allosteric and selective high-affinity inhibitor of 5-LO, which is substantially produced in a human liver organoid and reaches plasma concentration in humans and mice at which LT formation in human leukocytes is efficiently inhibited  accumulates in leukocytes and at sites of inflammation and suppresses acute inflammation in murine peritonitis. Conclusively, our data strongly suggest that α-T-13′-COOH (4a) is a highly potent bioactive metabolite of α-T (1a), which essentially contributes to the anti-inflammatory properties of α-T (1a) by targeting 5-LO.Hepatic ω- and β-oxidation of vitamin E produces long-chain metabolites (LCMs), which differ in oxygenation , saturation, and length of the phytyl side chain as well as in methylation and phase II metabolism of the chromanol coreGarcinia amplexicaulis (Clusiaceae). This comprehensive library was complemented by semi-synthesized analogs (see Supplementary Note 1) and contains intermediates of human vitamin E metabolism. Screening of this library for inhibition of 5-LO revealed high-affinity inhibitors among ω-alcohols and ω-carboxylates, including T and TE metabolites of the α-, β-, γ-, and δ-series, with the ω-carboxylated δ-TE  being most potent (IC50 = 35 nM) , IC50 = 750 nM for human recombinant 5-LO). The proposed intermediate LCM of mammalian hepatic δ-TE (1h) metabolism δ-tocodienol (DE)-13′-COOH (4l) lacks the Δ11′-double bond of TEs  in inhibiting 5-LO  were equally active. Non-specific nuisance inhibition due to formation of detergent-sensitive colloid-like aggregates was excluded since the detergent Triton X-100 neither substantially affected 5-LO inhibition by δ-TE-13′-COOH (4h) nor by α-T (1a) .In order to thoroughly investigate metabolites of vitamin E forms, we first constructed a library based on an original set of ω-oxidized tocotrienols (TEs) isolated from TEs Fig. 31 and is1a) Fig. . The 5-L32 that occupy the AA-binding site and block substrate access or interfere with the active-site iron. An additional class of 5-LO inhibitors act at the C2-like domain, thereby preventing 5-LO translocation and binding to the nuclear membrane. As shown for δ-TE-13′-COOH (4h) as representative, LCMs inhibit 5-LO independent of the AA concentration  was amide-coupled to an amino-functionalized resin  inhibit 5-LO comparably to acidic LCMs  did not prevent 5-LO binding to the δ-TE-13′-COOH (4h) construct  concentrations, far above the effective concentrations that inhibit 5-LO , we combined molecular docking simulations with site-directed mutagenesis experiments. Docking scores are listed in Supplementary Table 4h) was predicted for a cavity between the regulatory C2-like and the catalytic domain of 5-LO, where hydrogen bonds to Arg101, Trp102 and Gln15 are potentially formed , but were divergent and inconclusive for less potent Ts as well as side chain-truncated 3′- or 5′-carboxylates  likely form hydrogen bonds at both ends of the cavity, namely between the farnesyl side chain and either Arg101, Gln141, or Val110 and the chromanol moiety and either Trp102, Asp170, or Gln15. While Trp102 is conserved in LO isoenzymes, Gln15 is unique for 5-LO, and Arg101 is present only in 5-LO, 12-LO-1, and 15-LO-1 but is replaced by glutamine in 12R-LO and 15-LO-2   in 5-LO rather precludes a redox-type mode of enzyme inhibition since the distance between the redox-active chromanol and the active site iron is too far for direct electron transfer, and also the existence of an electron transport chain is not obvious from the structure.The triple mutation of Trp102Ala/Trp13Ala/Trp75Ala or Arg101Asp replacement markedly altered the susceptibility of 5-LO towards δ-TE-13′-COOH (4h) Fig. . The Trp18. Whether δ-TE-13′-COOH (4h) competes with membrane phospholipids for 5-LO binding was investigated by phosphatidylcholine supplementation. Addition of phosphatidylcholine significantly impaired 5-LO inhibition by δ-TE-13′-COOH (4h) in contrast to phosphatidylethanolamine  targets a so far unexploited cavity that might represent a potential allosteric binding site for non-competitive 5-LO inhibitors.The proposed binding pocket for LCMs partially overlaps with the membrane binding site at the 5-LO C2-like domain that associates with phosphatidylcholineine Fig. , which d27 and liver28. To quantify vitamin E metabolites in plasma and tissues, we developed a sensitive ultraperformance liquid chromatography ESI tandem mass spectrometry (UPLC-MS/MS)-based quantitation method. LCMs were extracted from biological samples by successive addition of methanol and chloroform , separated by C18-RP UPLC and detected by ESI-MS/MS through multiple reaction monitoring in the negative ionization mode. The 16 LCMs were quantified based on external calibration (eight standards) in combination with δ-TE-13′-COOH (4h) as internal standard. The detection response of α-T-13′-COOH (4a) was linear between 1 and 250 nM  as major systemic vitamin E metabolite (8–49 nM)  or ω-alcohols, for which the method is less sensitive (Supplementary Table 4a) in plasma as human volunteers . Most remarkable, ω-carboxylates are strongly enriched in the peritoneal exudates at sites of inflammation, with α-T-13′-COOH (4a) reaching concentrations up to 200 nM at 30 min post zymosan injection  Fig. , b in acved Fig. . Other ωers Fig. . Moreoveion Fig. .1a; 50 mg/kg) to mice significantly increased α-LCM levels in the exudates when given before immune cells infiltrate into the peritoneal cavity  at a later time point was without effect on exudate LCMs  levels in plasma but not in exudates  pools with different metabolic fates. Together, α-T-13′-COOH (4a) is present in human and murine plasma, accumulates at sites of inflammation, and reaches concentrations in vivo at which 5-LO is effectively inhibited in vitro.Oral administration of α-T  in human liver, we used an in vitro organoid model of the human liver sinusoid. The liver-on-chip consists of two porous membranes serving as cell substrate and scaffold. The vascular layer resembles the endothelial lining of the liver sinusoid and comprises human umbilical vein endothelial cells (HUVEC) co-cultured with primary human monocyte-derived macrophages that mimic the functionality of immunomodulatory Kupffer cells  48 h after addition of α-T  as determined by UPLC-MS/MS  as one of the major metabolites  metabolites reaching significance for α-T-13′-COOH (4a)  to the 13′-carboxylate (4a) was below the detection limit in the absence of cells.The hepatic compartment is formed by two hepatic layers aligned to each other, which contain HepaRG cells differentiated to hepatocyte-like and biliary-like cells that are co-cultured with LX-2 cells (stellate cell surrogates)tes Fig. . Inhibit4a) Fig. . Autoxid4a) suppressed 5-LO product formation in human PMNL , and inhibited LTB4 formation in activated human blood at physiologically relevant in vivo concentrations (100 nM)  targets additional factors in cellular 5-LO product biosynthesis. First, we assessed the viability of peripheral blood mononuclear cells (PBMC) up to 24 h. Cell viability was not reduced by LCMs, except for the 12a′-CH2OH series, albeit at high concentrations (10 µM) only  less potent (one to three magnitudes) than isolated 5-LO, whereas the inhibitory activity of LCMs was either slightly decreased (δ- and β-series and α-TEs), hardly affected (γ-TEs) or even markedly increased (α-Ts) in leukocytes Table . Thus, αMNL Fig.  and monoMNL Fig.  more potnM) Fig. . The higly Table .Fig. 3Ac4a) and δ-TE-13′-COOH (4h) consistently inhibited 5-LO also when exogenous AA was supplied  neither affected intracellular Ca2+, an essential cofactor for cellular 5-LO activation  at submicromolar concentrations, intracellular Ca2+-influx was significantly reduced at ≥1 µM  selectively enriches in leukocytes. In fact, human PMNL preferentially accumulate α-T-13′-COOH (4a) over δ-TE-13′-COOH (4h), reaching micromolar intracellular concentrations when assuming a spherical shape of PMNL with an average diameter of 13 µm  in leukocytes thus provides an explanation for the superior inhibition of 5-LO compared to cell-free assays. Accordingly, the concentrations of α-T-13′-COOH (4a), α-T-11′-COOH (11a) and α-T-9′-COOH (12a) are substantially higher in peritoneal leukocytes  significantly impaired 12-LO (IC50 > 3 µM)  inhibited (i) isolated bovine COX-1 (IC50 > 10 µM)   (IC50 = 9.9 µM) -1 in microsomal preparations (IC50 = 8.8 µM)   potently inhibits 5-LO (high affinity target) but hardly other enzymes within eicosanoid biosynthesis (low affinity targets). The consequences on the cellular lipid mediator network were investigated in activated human monocytes by comprehensive profiling of eicosanoids and docosanoids using UPLC-MS/MS. α-T-13′-COOH (4a) inhibited the biosynthesis of 5-LO-derived products, i.e., LTB4, its isomers, and 5-hydroxy-eicosatetraenoic acid (5-HETE), but neither markedly suppressed the formation of COX-derived PGs, 12- and 15-LO products nor other lipid mediators  on the cellular lipid mediator profile is determined by 5-LO inhibition. A similar profile was observed for δ-TE-13′-COOH (4h)  Fig. . Other LµM) Fig.  and COX-µM) Fig. , (ii) huµM) Fig. , (iii) hµM) Fig. , and (ivµM) Fig. . In contµM) Fig.  and COX-tes Fig.  were notors Fig. . Thus, t4a) was investigated for zymosan-induced murine peritonitis in vivo. In this model, LTs and other lipid mediators critically contribute to initiation and progression of inflammation35  and α-T-13′-COOH (4a) were given i.p. to circumvent the hepatic first-pass metabolism to LCMs. Accordingly, the concentration of LCMs in plasma and exudate (3:1 distribution) was substantially increased only when α-T-13′-COOH (4a) was administered, whose degradation to short-chain metabolites was minute  reached substantially higher concentrations in plasma and exudate upon i.p. injection  was significantly less effective in reducing exudate levels of LTC4 -treated mice revealed a preferential drop of 5-LO products among various lipid mediators as shown by UPLC-MS/MS , which accumulates at sites of inflammation, possesses potent anti-inflammatory activity seemingly by suppressing LT formation. Interestingly, α-T-13′-COOH (4a) was more effective in reducing LT production and limiting inflammation than the clinically used 5-LO inhibitor zileuton at 30 min post zymosan injection  on cell infiltration vanished at the resolution phase (18 h)  strongly increased the circulating resolvin E3 in plasma  in systemic resolution.The resolution phase of murine peritonitis was studied at 18 h after zymosan injection, which is the latest time point before peritoneal cell numbers readjust to baseline  Fig. . Lipid m h) Fig. , with bo h) Fig. . Similarsma Fig.  while thsma Fig. , which m2, where LTs play a fundamental role in pathogenesis, e.g., by inducing brochoconstriction11. Thus, we investigated the effect of α-T-13′-COOH (4a) in an experimental model of asthma. Mice were sensitized to ovalbumin (OVA), and bronchi were harvested to assess airway hyper-reactivity  lowered pulmonary LTC4 to basal levels  in limiting asthmatic airway contraction.Vitamin E status has been associated with prevalence of asthma in humansity Fig. . Adminisels Fig.  and signels Fig. , which p4a) is endogenously formed in humans and mice, (ii) its biosynthesis is ascribed to hepatocytes in human liver organoids, (iii) its basal plasma concentration is relevant to regulate 5-LO activity, (iv) it strongly accumulates at sites of inflammation, (v) it selectively reduces 5-LO products in vitro and in vivo along with suppression of acute inflammation in murine peritonitis, and (vi) it reduces LT-mediated bronchial hyper-reactivity in an experimental model of asthma—one of the most relevant and validated LT-driven diseases18. It is tempting to speculate that α-T-13′-COOH (4a) regulates 5-LO activity in circulating immune cells depending on diet, metabolic turnover and inflammatory status.Here, we report on the discovery of a bioactive metabolite of vitamin E that is endogenously formed in humans and mice and may contribute to the anti-inflammatory properties of vitamin E as an allosteric high-affinity inhibitor of 5-LO  in inflamed exudates from murine acute peritonitis correlates with substantial uptake of the LCM by PMNL, which infiltrate into the peritoneal cavity and represent major sources for 5-LO-derived LTs35. PMNL preferentially take up α-LCM, as reflected by superior cell-based 5-LO inhibitory activities versus isolated 5-LO, and confirmed for α-T-13′-COOH (4a) by analyzing intracellular LCM concentrations. On the one hand, the higher lipophilicity of α-T-13′-COOH  compared to δ-TE-13′-COOH  might facilitate passive membrane transfer. On the other hand, selectivity for α-T-13′-COOH (4a) might depend on specific uptake, cellular sorting or retention mechanisms, which are unknown for LCMs so far. The hepatic cytosolic α-tocopherol transfer protein (α-TPP) binds to α-T (1a) with high affinity and facilitates the transfer into secreted plasma lipoproteins36. A similar mechanism might retain α-LCMs in PMNL and potentially other cell types.The accumulation of α-T-13′-COOH (4h) inhibit 5-LO but only α-T-13′-COOH , α-T-13′-CH2OH (2a)29 and chain-shortened ω-carboxylates of α-T  have been detected in humans and rodents. The physiological relevance of other LCMs remains speculative in light of comparable detection limits for long-chain ω-carboxylates. γ- and δ-LCMs might gain importance when natural vitamin E forms are supplemented. In fact, γ-carboxychromanols are present in plasma of mice (up to 150 nM) receiving γ-T (1c)/γ-TE (1g)31. Conjugated LCMs, such as O-sulfates, even reach higher plasma levels but our docking studies rather preclude efficient 5-LO inhibition, at least by targeting the proposed LCM binding site. Along these lines, O-sulfates also failed to inhibit COX-226.Multiple LCMs 37. Alternative mechanisms that regulate 5-LO product formation, i.e., the supply of AA, Ca2+-dependent nuclear translocation of 5-LO and interaction with FLAP, are seemingly not targeted by α-T-13′-COOH (4a). Moreover, LCMs do not compete with AA at the active site but may preferentially bind to an allosteric pocket that partially overlaps with the membrane binding region of 5-LO, as exemplarily shown for δ-TE-13′-COOH . Of interest, δ-TE-13′-COOH (4h) is a major secondary metabolite contained in the bitter nut G. kola—an anti-inflammatory, bronchodilatory and anti-infectious remedy in African traditional medicine38. The physiologically relevant LCM of δ-TE (1h), which has been proposed to be a tocodienol lacking the Δ11 double bond of TEs31, less potently inhibits 5-LO when compared with δ-TE-13′-COOH (4h) , i.e., COX-1, mPGES-1, and 12-LO, does not markedly influence eicosanoid and docosanoid profiles in vitro and in vivo. However, it is likely that the weak interaction of α-T-13′-COOH (4a) with these targets buffers the redirection of AA when 5-LO is blocked and thus contributes to eicosanoid network stability as recently discussed40. Most remarkably, α-TE (1e) has been reported to inhibit porcine 12-LO thereby blocking glutamate-induced cell death at nanomolar concentrations in neuronal cells41. We confirmed the inhibition of human 12-LO in PMNL by TEs and identified LCMs with superior inhibitory activity, though the physiological relevance of 12-LO as target is contrasted by the discrepancy between high effective concentrations (IC50 > 3 µM) and low plasma levels of TEs (≤0.35 µM)23 and LCMs (refs. 29 and this study) unless α-TE (1e) is supplemented42.Interference with low-affinity targets of α-T-13′-COOH  inhibits protein kinase (PK)Cα by binding to its regulatory domain and activates phosphatase type 2A, which de-phosphorylates and thus inactivates protein PKCα2, whereas γ-T (1c) induces PKCα activity and negatively correlates with lung function in humans2. Conclusively, the composition and local tissue concentration of vitamin E forms and their conversion to bioactive metabolites are important factors that might influence the clinical outcome of vitamin E supplementation studies.Multiple, partly opposing molecular mechanisms have been reported for vitamin E forms and LCMs. Besides their well-established anti-oxidative properties, Ts, TEs, and LCMs differentially regulate gene expression of inflammatory, lipid metabolic, and neuroactive proteins, and they act on enzymes with important roles in immunoregulation1a) intake in diseases with inflammatory component, such as atherosclerosis, lung inflammation and cancer, whereas clinical vitamin E intervention studies often produced disappointing results or even questioned the safety of pharmacological doses of vitamin E (≥400 IU/d)5. Along these lines, vitamin E deficiency increased LT biosynthesis/turnover in humans and rats44, whereas an excess of vitamin E produced contradictory results5. We hypothesize that individual differences in the capacities to biosynthesize LCMs essentially propagate such discrepancies, which become relevant at high doses of vitamin E and might have a strong impact on the clinical outcome.Individual differences in vitamin E form uptake, distribution and metabolism are likely smaller in animal studies with defined diet and genetic background than in humans participating in clinical trials. Accordingly, animal studies showed clear benefits for α-T  in rats28. Moreover, metabolic dysregulations that promote NAFLD led to reduced hepatic expression of CYP4F2, the initial enzyme in LCM biosynthesis, in a high fat murine model of steatosis46, and to lower excretion of vitamin E metabolites by patients with metabolic syndrome47. In consequence, the progression from healthy liver to non-alcoholic steatohepatitis is accompanied by enhanced 5-LO product levels48, and deletion of 5-LO has (i) anti-steatotic, anti-inflammatory, and anti-fibrotic effects in experimental models of liver disease49, (ii) protects from hepatic inflammatory injury, and (iii) improves insulin signaling at high-fat diet14. Based on these findings, we speculate that the reduced production of 5-LO-inhibitory LCMs promotes liver pathologies where LT biosynthesis is increased. Less understood is the role of vitamin E in the resolution of inflammation, which is driven by specialized pro-resolving mediators that are generated from eicosapentaenoic acid and docosahexaenoic acid by the concerted action of 5-, 12-, and/or 15-LO16. Thus, 5-LO-targeting LCMs might impede resolution, unless their local concentration at sites of inflammation is strictly regulated, which seems to be the case for α-T-13′-COOH (4a) that rapidly peaks and then depletes in inflamed exudates. This aspect has particular relevance for fish oil supplements where Ts, including α-T (1a) and γ-T (1c), are added as an anti-oxidant because their proposed health benefits have been ascribed to the 5-LO-dependent conversion of eicosapentaenoic acid and docosahexaenoic acid to specialized pro-resolving mediators . Interestingly, α-T-13′-COOH (4a) strongly increased systemic resolvin E3 levels during the resolution phase of peritonitis, which may suggest a pro-resolving function of the LCM.5-LO generates bioactive lipid mediators that are involved in inflammatory and allergic reactions as well as in autoimmune diseases, and in fact, vitamin E intake has long been associated with the regulation of related diseases, including asthma, cardiovascular diseases, and Alzheimer’s disease50. Vitamin E has been proposed to protect from membrane lipid peroxidation and ferroptotic cell death in glutathione peroxidase (Gpx)4-deficient T-lymphocytes, which expand and provide protection from acute lymphocytic choriomeningitis virus and Leishmania major parasite infections51. Recently, ferroptosis was ascribed to LO-dependent formation of oxidized arachidonic and adrenic phosphatidylethanolamines, and inhibition of ferroptotic death by α-T (1a) and TEs was demonstrated21. Accordingly, oral vitamin E partially reduced lipid peroxidation and oxidative stress through inhibition of 5-LO in PBMCs from hemodialysis patients52.Besides interference with LT formation, inhibition of 5-LO by LCMs might also modulate immune functions through suppressing ferroptosis4a) essentially contributes to the anti-inflammatory activity of α-T (1a) as endogenous, high-affinity inhibitor of 5-LO. Moreover, we provide mechanistic insights into the binding mode and inhibitory mechanism of 5-LO thereby suggesting a novel allosteric mode of enzyme inhibition. Our data strongly prompt for consideration of specific vitamin E metabolism when recommending dietary doses, designing clinical studies, and evaluating the therapeutic value of vitamin E in the context of personalized medicine.In conclusion, we here provide strong evidence that the vitamin E metabolite α-T-13′-COOH  was purchased from Enzo Life Sciences , β-T , γ-T , δ-T  were obtained from Sigma-Aldrich , and α- , β- , γ- , δ-TE , 3,4-dihydro-6-hydroxy-α,2,5,7,8-pentamethyl-2H-1-benzopyran-2-pentanoic acid , 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman , 2,7,8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman  and 2,8-dimethyl-2-(2′-carboxyethyl)-6-hydroxychroman  were from Cayman Chemicals . Vitamin E forms and metabolites were dissolved in DMSO, stored in the dark at −20 °C under argon, and freezing/thawing cycles were kept to a minimum. Zileuton was from Carbosynth ; sesamin was from Biopurify Phytochemicals .α-T  was isolated from G. kola, and α-T-13′-COOH , δ-T-13′-COOH , α-T-13′-CH2OH , and δ-T-13′-CH2OH  were semi-synthesized53. δ-TE-13′-CH2OH , γ-TE-12a′-CH2OH , δ-TE-12a′-CH2OH , δ-TE-12a’/13′-diCH2OH , γ-TE-12a′/13′-diCH2OH , the adduct -bi-O-amplexichromanol , and γ-TE-13′-COOH  were isolated from stem barks of G. amplexicaulis, an endemic tree from New Caledonia55. α-TE-13′-COOH  and β-TE-13′-COOH  were semi-synthesized from δ-TE-13′-COOH (4h)56. Chemical procedures for the multi-step semi-synthesis of α-TE-13′-CH2OH , β-TE-13′-CH2OH , δ-DE-13′-COOH , and δ-TE-13′-CONHBz  and the corresponding spectral data are described in Supplementary Note 1. The purity of the original semi-synthetic compounds α-TE-13′-CH2OH (2e), β-TE-13′-CH2OH (2f), α-TE-13′-COOH (4e), β-TE-13′-COOH (4f), δ-DE-13′-COOH (4l), and δ-TE-13′-CONHBz (7) has been determined using HPLC-ELSD analysis. The purity of all the other vitamin E derivatives mentioned in this section has been assessed by means of HPLC-DAD and -FL analysis.δ-TE-13′-COOH  from fasted (12 h) adult (18–65 years) male and female registered healthy volunteers, with informed consent. These subjects donated blood every 8–12 weeks, had no apparent infections, inflammatory conditions, or current allergic reactions  and had not taken antibiotics or anti-inflammatory drugs for at least 10 days prior to blood collection. Leukocytes were concentrated by centrifugation of the freshly withdrawn blood (4000 × g/20 min/20 °C) and centrifuged on lymphocyte separation medium . PMNL were recovered from the pellet after hypotonic lysis of erythrocytes. Platelets were obtained from the supernatant and washed with PBS pH 5.9/0.9% NaCl (1:1). Monocytes were isolated from the PBMC fraction by adherence to culture flasks  for 1.5 h at 37 °C and 5% CO2 in RPMI 1640 medium (Sigma-Aldrich) containing FCS , L-glutamine  and penicillin/streptomycin  (monocyte medium). The purity of the monocyte preparation was >85% as defined by forward- and side-light scatter properties and detection of the CD14 surface molecule by flow cytometry .Human blood (containing 3.13% sodium citrate) and leukocyte concentrates were provided by the Institute for Transfusion Medicine of the University Hospital Jena (Germany). Human PMNL, monocytes, PBMC, and platelets were freshly isolated from leukocyte concentrates2. Confluent cells were detached using trypsin/EDTA  and reseeded at 1 × 104 cm2. Cell lines were tested for mycoplasma. HEK-293 was reported to be a misidentified cell line, which was used because stably transfected cells were available as validated experimental cell-based model for studying the regulation of 5-LO58. None of the cell lines used in this study were authenticated.Human lung adenocarcinoma epithelial A549  and human embryonic kidney (HEK)-293 cells (ATCC) were grown in DMEM/high glucose  containing heat-inactivated FCS (10%) and penicillin/streptomycin at 37 °C and 5% COE. coli Bl21 (DE3) cells were transformed with pT3–5LO plasmid to express human recombinant 5-LO, which was purified by affinity chromatography on an ATP-agarose column59. In brief, E. coli was lysed in 50 mM triethanolamine/HCl pH 8.0 plus EDTA (5 mM), soybean trypsin inhibitor (60 µg/ml), phenylmethanesulphonyl fluoride (1 mM), dithiothreitol (1 mM), and lysozyme (1 mg/ml). After sonication (3 × 15 s), the homogenate was centrifuged at 10,000 × g for 15 min and the supernatant again at 40,000 × g for 70 min at 4 °C. The supernatant was applied to an ATP-agarose column (Sigma-Aldrich), and the column was washed with (i) PBS pH 7.4, 1 mM EDTA, (ii) 50 mM phosphate buffer pH 7.4, 0.5 M NaCl, 1 mM EDTA, and (iii) 50 mM phosphate buffer pH 7.4, 1 mM EDTA. 5-LO was eluted with 50 mM phosphate buffer pH 7.4, 1 mM EDTA, 20 mM ATP.2 (2 mM). After 10 min at 37 °C, an equal volume of ice-cold methanol was added and formed 5-LO products ETE) were extracted using Sep-Pak C18 35 cc Vac Cartridges . 5-LO products were separated by RP-HPLC on a Nova-Pak C18 Radial-Pak Column  under isocratic conditions (73% methanol/27% water/0.007% trifluoroacetic acid) at a flow rate of 1.2 ml/min and detected at 235 and 280 nm59. 5-LO product formation for vehicle control: 5 µM AA, 256 ± 68 ng; 20 µM AA, 818 ± 116 ng; 40 µM AA, 262 ± 33 ng; 80 µM AA, 208 ± 72 ng. Zileuton (IC50 = 0.84 ± 0.23 µM) and BWA4C  were used as reference 5-LO inhibitors. To investigate whether α-T (1a) and δ-TE-13′-COOH (4h) reversibly inhibit 5-LO, the purified enzyme was pre-incubated with the test compounds at 30 and 0.1 µM for 15 min and then 10-fold diluted to a final compound concentration of 3 and 0.01 µM, respectively. AA was added after incubation for 5 min on ice. Triton X-100  was added to the reaction buffer to recognize nuisance inhibition of 5-LO (5-LO product formation: 360 ± 63 ng). Whether δ-TE-13′-COOH (4h) targets the membrane binding site of 5-LO was addressed by supplementation of phosphatidylcholine from egg york  and the non-binding control phosphatidylethanolamine from bovine brain .Purified 5-LO (0.5 µg) in PBS pH 7.4 containing EDTA (1 mM) and ATP (1 mM) was pre-incubated with LCMs for 10 min at 4 °C, pre-warmed for 30 s at 37 °C and 5-LO product formation was started by addition of AA  plus CaCl6/six-well plate) were stimulated with lipopolysaccharide  for 24 h at 37 °C and 5% CO2 in monocyte medium. Cells were washed and medium was exchanged. Freshly isolated PMNL (1 × 107) and stably transfected HEK-293 (1 × 106) cells were suspended in PBS pH 7.4 containing 1 mg/ml glucose and 1 mM CaCl2. After pre-incubation with test compounds for 10 min at 37 °C, cells were treated with AA  and/or Ca2+-ionophore A23187  for 10 min at 37 °C (PMNL and HEK-293 cells) or 30 min at 37 °C and 5% CO2 (monocytes). The reaction was stopped with an equal volume of methanol (PMNL and HEK-293 cells) or on ice (monocytes). For PMNL and HEK-293 cells, major 5-LO products ETE), 12-HETE (12-LO product from platelets in PMNL preparations), and 15-HETE (15-LO product) were extracted and analyzed by RP-HPLC as described for 5-LO products. Cysteinyl-LTs and oxidation products of LTB4 were not determined. A23187- and A23187/AA-treated PMNL and A23187/AA-treated HEK-293 (5-LO_wt), HEK-293 (5-LO_3W) and HEK-293 (5-LO_Arg101Asp) cells produced 135 ± 21, 636 ± 112, 300 ± 59, 133 ± 29, 55 ± 9 ng 5-LO products, respectively. PMNL synthesized 29 ± 6 ng 12-HETE and 97 ± 23 ng 15-HETE when challenged with A23187/AA. 5-LO products from monocytes were analyzed by UPLC-MS/MS. The 5-LO reference inhibitors zileuton (3 µM) and BWA4C (0.5 µM) suppressed 5-LO product formation by 78–98%.Monocytes (5 × 104h) to Toyopearl AF-Amino-650 M beads 60, δ-TE-13′-COOH  or 4-phenoxy butyric acid (12 mg) as control were dissolved in methanol/water (98/2) at pH 5 and coupled to washed Toyopearl resin (500 µl) by addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (40 mg). After 48 h at room temperature under pH control, the resin was washed and stored in methanol/water (20/80).To covalently link δ-TE-13′-COOH (4h) and either AA or α-T (1a) to 5-LO was investigated using an excess of AA and α-T (1a) in pull-down buffer (100 µM each).For pull-down of 5-LO, the drained resin (25 µl) was blocked with binding buffer  containing 0.2 mg/ml milk powder and 0.01% Triton X-100 for 1 h at 4 °C. Purified human recombinant 5-LO (5 µg) was added and the mixture was incubated for 1 h. After repeated washing with binding buffer, 5-LO was eluted with 2× Laemmli buffer, heated for 5 min at 95 °C and subjected to SDS-PAGE and Western blotting. Competitive binding of δ-TE-13′-COOH , which was provided by Dr. Dieter Steinhilber  and produced in-house61. Immunoreactive bands were stained with IRDye 800CW Goat anti-Mouse IgG . Data from densitometric analysis were background-corrected. Uncropped blots are shown in Supplementary Fig. Equal aliquots of pull-downs were resolved on 10% SDS-PAGE gels, proteins were transferred onto Hybond ECL nitrocellulose membranes , and 5-LO was detected using an Odyssey infrared imager (LI-COR Biosciences)https://www.chemcomp.com/journal/sitefind.htm). Cavities that exceeded a volume of 110 Å3 were investigated.The search for alternative binding pockets was performed with MOE’s pocket finder  scoring function. The protein target structure was based on stable 5-LO . Four virtual mutations were inserted to return to the 5-LO wild-type sequence: E13W, H14F, G75W and S76L. The residues were exchanged and the structure was energetically minimized in Discovery Studio . Hydrogens were added with GOLD, using default settings. The binding site was defined in an 8 Å radius around C2 of residue I167. Protein–ligand interactions of the docking poses were analyzed using LigandScout 3.12 (www.inteligand.com/ligandscout).The molecular docking simulation was conducted with GOLD 5.2 . Scoring was calculated using the CHEMPLP 58. The pcDN3.1/neom (+) vectors for 5-LO (wt) and 3W mutant expression were generous gifts by Dr. Dieter Steinhilber. The 5-LO Arg101Asp coding sequence from the E. coli expression vector pt3_5-LO_Arg101Asp  was cloned into the vector pcDNA3.1/neom_(+)_5-LO_Arg101Asp. EcoRI and XbaI restriction sites were introduced by polymerase chain reaction. The sequences of 5-LO (wt) and the site-directed mutants in the expression constructs were confirmed by sequencing by Eurofins Genomics  and are shown in Supplementary Table 2 and subsequently selected by geneticin (400 µg/ml) for 2–4 weeks.To stably express 5-LO and its mutants, HEK-293 cells were transfected with pcDNA3.1/neom (+)_5-LO and respective 5-LO mutants using LipofectamineMale CD-1 mice  or female BALB/c mice  were housed in a controlled environment (21 ± 2 °C) and provided with standard rodent chow and water. Animals were allowed to acclimate for four days prior to experiments and were subjected to 12 h light/12 h dark schedule. Mice were randomly assigned for the experiments, which were conducted during the light phase. The experimental procedures, according to Italian (DL 26/2014) and European (n. 63/2010/UE) regulations on the protection of animals used for experimental and other scientific purposes, were approved by the Italian Ministry .4a), δ-TE-13′-COOH (4h), α-T (1a), zileuton or vehicle  were administered to CD-1 mice 30 min before injection of zymosan . Alternatively, α-T , zileuton  or vehicle (0.5 ml of 0.5% carboxymethylcellulose and 10% Tween 20) were given 1 h prior zymosan injection, in combination with sesamin , as indicated. Mice were killed by inhalation of CO2 after another 30 min for determination of LTC4 levels and vascular permeability and after 4 h for the analysis of LTB4 levels, lipidomic profiles and immune cell infiltration35. Cell infiltration and lipid mediator profiles during resolution were studied after 18 h. Peritoneal exudates were collected and cells counted after vital trypan blue staining. LTC4, LTB4, and PGE2 were analyzed in the exudate by enzyme immunoassays  according to manufacturer’s instructions. To assess vascular permeability, Evans blue dye  was injected into the tail vain immediately before the induction of peritonitis35. Exudates were centrifuged  and the absorbance of the supernatants was measured at 610 nm using a Beckman Coulter DU730 spectrophotometer. To investigate the effect of α-T (1a) on the accumulation of α-T-13′-COOH (4a) in infiltrated leukocytes after peritonitis has been established, vehicle or α-T  were given 1 h before and/or 4 h post zymosan injection, and mice were killed after another 1.5 h. Leukocytes were obtained from peritoneal exudates by centrifugation .α-T-13′-COOH  or vehicle  were administered 30 min before injection of OVA  which was adsorbed to aluminum hydroxide gel . Mice were sacrificed on day 21, and bronchi were collected. Bronchial rings of 1–2 mm length were cut and placed in organ baths mounted to isometric force transducers  and connected to a Powerlab 800 (AD Instruments). Rings were initially stretched until a resting tension of 0.5 g was reached and allowed to equilibrate for at least 30 min. In each experiment, bronchial rings were challenged with carbachol (10−6 M) until the response was reproducible. Bronchial reactivity was then assessed from a cumulative concentration–response curve to carbachol (1 × 10−8 − 3 × 10−5 M). To determine pulmonary LTC4 levels, lungs were isolated and homogenized in PBS pH 7.4 (100 mg tissue/ml) prior to detection of LTC4 by enzyme immunoassay .BALB/c mice were sensitized to OVA, and bronchial reactivity and pulmonary LTC64. A 12 µm thick polyethylene terephthalate (PET) membrane (TRAKETCH) with a pore diameter of 8 µm and a pore density of 1 × 105 pores/cm²  was integrated in the upper and lower part of the biochip by heat-sealing with the bulk material 2 in William’s Medium E  containing 10% FCS , 5 µg/ml insulin (Sigma-Aldrich), 2 mM glutamine (GIBCO), 0.5 µM hydrocortisone-hemisuccinate (Sigma-Aldrich), 100 U/ml penicillin (GIBCO) and 100 µg/ml streptomycin (GIBCO) (hepatocyte growth medium) for 14 days before differentiation. Medium was renewed every 3–4 days. HepaRG differentiation was induced by medium supplementation with 2% DMSO for 14 days. Differentiated cells were used up to 4 weeks.HepaRG cells  were cultivated at 37 °C and 5% CO65. Experiments were performed with HUVECs cultured in Endothelial Cell Medium (ECM)  up to passage 4. HUVECs were seeded at a density of 1.5 × 105 cells/cm2 and cultured up to 95% confluence before being sub-cultured.HUVECs were isolated from human umbilical cord veins (HUVEC)5 cells/cm2. Cells were grown up to 95% confluence before being sub-cultured.LX-2 stellate cells were kindly provided by Dr. Ralf Weiskirchen . LX-2 cells were cultured in Dulbecco’s Minimum Essential Medium (DMEM) (Biochrom) supplemented with 10% FCS, 1 mM sodium pyruvate (GIBCO) and penicillin/streptomycin and seeded at a density of 2.0 × 107 cells/cm2 in X-VIVO 15 medium  supplemented with 10% autologous human serum, 10 ng/ml human granulocyte macrophage colony-stimulating factor (GM-CSF)  and penicillin/streptomycin. After 1 h incubation in a humidified cell incubator at 5% CO2 and 37 °C, the cells were washed twice with X-VIVO 15 medium and subsequently used for liver organoid assembly. Typically, 1 × 105 monocytes per ml whole blood were obtained. Monocytes were harvested by treatment with 4 mg/ml lidocain (Sigma-Aldrich) supplemented with 5 mM EDTA (Sigma-Aldrich).PBMCs of three different healthy donors were isolated by Biocoll (Biochrom) density gradient centrifugation and seeded in 6-well dishes with a density of 1.0 × 105 differentiated HepaRG cells were seeded at the bottom of the upper membrane and on top of the lower membrane in hepatocyte growth medium66. Liver models were assembled by staggered seeding of vascular and hepatic cell layers. In each sterilized biochip, 3 × 105 HUVECs and 1 × 105 monocytes were mixed and seeded on top of the upper membrane in the upper chamber and pre-incubated 3–4 h to allow attachment. Subsequently, biochips were flipped upside down, and 2 × 105 HUVECs and 6.7 × 104 monocytes were mixed and seeded on the bottom of the membrane in the lower chamber.For the preparation of “hepatocyte only biochips”, 6 × 106 differentiated HepaRG cells and 3.3 × 104 LX-2 cells were seeded at the bottom of the upper membrane, cultured for 24 h and equally seeded on top of the lower membrane in hepatocyte growth medium two to four days prior to experimental use. Medium 199 supplemented with 10% FCS, 680 mM l-glutamine, 25 µg/ml heparin, 7.5 µg/ml endothelial mitogen, 5 µg/ml vitamin C, 10% autologous human serum, 100 U/ml penicillin, and 100 µg/ml streptomycin was used in the upper and lower biochip chamber and hepatocyte growth medium in the medium biochip chamber.HUVEC/monocytes were co-cultured for at least 5 days with a daily medium exchange in ECM for the first two days and after reaching confluence in M199 medium (GIBCO) supplemented with 10 ng/ml epidermal growth factor (Sigma-Aldrich), 90 µg/ml heparin (Sigma-Aldrich), 2.8 µM hydrocortisone, 7.5 µg/ml endothelial mitogen , 5 µg/ml ascorbic acid (Sigma-Aldrich), 10 ng/ml GM-CSF to induce macrophage differentiation, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% autologous human serum from monocyte donors. Subsequently, 1 × 101a; 100 µM) or sesamin (3 µM) were added. Biochips were incubated for the indicated time, medium was collected, and the system was washed with methanol (500 µl). Medium and methanol fractions were combined, and LCMs were extracted and analyzed by UPLC-MS/MS.After rinsing the chambers of the biochip with medium as indicated above, vehicle (DMSO), α-T  for 10 min, primed with LPS (1 µg/ml) for 30 min and stimulated with N-formyl-methionyl-leucyl-phenylalanine  for 15 min at 37 °C.LPS-activated monocytes were treated with A23187 (2.5 µM) and AA (20 µM) to induce lipid mediator biosynthesis as described for LO product formation. Human blood was pre-incubated with vehicle (DMSO) or α-T-13′-COOH , human blood (2 ml), blood plasma (0.3 ml) and peritoneal exudates (1 ml) using Sep-Pak C18 35 cc Vac Cartridges (Waters)67. Aqueous samples were diluted in PBS pH 7.4 (530 µl), acidified with 1 M HCl (30 µl), supplemented with the internal standard PGB1  and added to the columns. After washing of the columns with water twice , lipid mediators were eluted with methanol (300 µl) and analyzed by UPLC-MS/MS.Lipid mediators were extracted from pelleted cells  and d8-5S-HETE, d4-LTB4, d5-lipoxin A4, d5-resolvin D2, d4-PGE2  and d8-AA  were added as internal standards. Samples were incubated at −20 °C for 1 h to precipitate proteins and then centrifuged . The supernatant was transferred to acidified water (8 ml) to adjust pH = 3.5 and loaded onto solid phase cartridges , which were equilibrated with methanol (6 ml) and water (2 ml). Columns were washed with water (6 ml) and hexane (6 ml), and lipid mediators were eluted with methyl formiate (6 ml). Eluates were evaporated to dryness and dissolved in methanol/water (50/50) for UPLC-MS/MS analysis.To extract lipid mediators of the resolution phase of peritonitis from mouse plasma and peritoneal exudates68. After evaporation of the organic layer, extracted LCMs were dissolved in 100 µl methanol and diluted. δ-TE-13′-COOH  was added as internal standard.Lipids, including LCMs, were extracted from PMNL, murine peritoneal exudates or murine plasma by successive addition of PBS pH 7.4, methanol, chloroform and saline TM UPLC system (Waters) and detected by multiple reaction monitoring using a QTRAP 5500 Mass Spectrometer  equipped with an electrospray ionization source67. Chromatography was conducted at a flow rate of 0.8 ml/min and a column temperature of 45 °C using acetonitrile plus 0.07% formic acid as mobile phase A and water/acetonitrile (90/10) plus 0.07% formic acid as mobile phase B. After isocratic separation at A/B = 30/70 for 2 min, a linear gradient to A/B = 70/30 was applied within 5 min. Multiple reaction monitoring in the negative ion mode was used to detect diagnostic ion fragments67. Data were normalized on the internal standard PGB1 and are given as relative intensities. The analytic method was optimized to compare lipid mediator profiles between samples and not for absolute quantification.Eicosanoids, docosanoids and free fatty acids were separated on an Acquity UPLC BEH C18 column  using an Acquity17. The amounts of lipid mediators and fatty acids were calculated based on 6 internal and 45 external standards17.For the resolution phase of peritonitis, lipid mediators were separated on an Acquity UPLC BEH C18 column  at 0.3 ml/min and 50 °C. The mobile phase was acidified with 0.01% acetic acid and consisted of methanol/water (42/58) that was ramped to methanol/water (86/14) over 12.5 min followed by isocratic elution with methanol/water (98/2) for 3 min. Diagnostic ion fragments of lipid mediators were detected in the negative ion mode using scheduled multiple reaction monitoring (window: 60 s)69 and are listed in Supplementary Table 4h), LCM concentrations were calculated from external calibration curves as specified in Supplementary Table LCMs were separated on an Acquity UPLC BEH C18 column  at a flow rate of 0.8 ml/min at 45 °C: 0 to 4.5 min, gradient from 50% mobile phase A /50% mobile phase B  to 100% B; 4.5 to 5.5 min, isocratic at 100% B. Multiple reaction monitoring was used to detect LCMs in the negative ion mode. The precursor-to-product ion transitions were selected according to Jiang et al.Automatic peak integration was performed with Analyst 1.6 software (Sciex) using IntelliQuan default settings.2, and cell viability was determined using a MTT assay67. PBMC in monocyte medium were incubated with MTT  in darkness for 4 h at 37 °C and 5% CO2. The formazan product was solubilized with SDS (10% in 20 mM HCl), and absorbance was measured at 570 nm using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific). Formazan formation was inhibited by staurosporine  and abolished by lysis of cells with Triton X-100 (1%).Freshly isolated human PBMC were incubated with vitamin E derivatives for 24 h at 37 °C and 5% CO35. Ascorbic acid , used as control, scavenged DPPH radicals by 82.4 ± 1.9%.Compounds were incubated with 2,2-diphenyl-1-picrylhydrazyl  in ethanol for 30 min. Absorbance was measured at 520 nm using a Multiskan Spectrum Microplate Reader (Thermo Fisher Scientific)6) were incubated with Fura-2/AM  for 45 min at 37 °C, washed, resuspended in PBS pH 7.4 plus 1 mg/ml glucose and transferred to fluorescene 96-well microplates (Greiner). After pre-incubation with compounds for 10 min, 1 mM Ca2+ was added, and cells were stimulated with fMLP (1 µM) for another 10 min. Intracellular Ca2+ concentrations were determined using a thermally (37 °C) controlled NOVOstar fluorescence microplate reader 17. The maximal fluorescence signal was recorded after lysis of PBMC with Triton X-100, and the minimal fluorescence was assessed by adding 20 mM EDTA to chelate Ca2+. The difference in the signal ratio obtained with Triton X-100 and Triton X-100 plus 20 mM EDTA was set to 100% for each experiment.PMNL (1 × 102). Cells were washed and pre-incubated with 3 µM α-T-13′-COOH (4a), δ-TE-13′-COOH (4h) or vehicle control (0.1% DMSO) in PGC buffer  for 15 min at 37 °C prior to stimulation with A23187 . To analyze in situ protein interaction of 5-LO with FLAP by proximity ligation assay17, the incubation was stopped by fixation with paraformaldehyde . Cells were permeabilized using 100% ice-cold acetone  and incubated with primary antibodies against 5-LO 61 and FLAP 17 for 16 h at 4 °C and then treated for 1 h at 37 °C with oligonucleotide-labeled specific secondary antibodies . Two other circle-forming DNA oligonucleotides and a ligase were added to create a DNA circle from the antibody-bound oligonucleotides, which is possible when 5-LO and FLAP are <40 nm distant from each other. After rolling-circle-amplification , the newly generated DNA was visualized by hybridization with fluorescently labeled oligonucleotides . Nuclear DNA was stained with DAPI-containing ProLong diamond antifade mountant . Cells were visualized by a Zeiss Axiovert 200M microscope and a Plan Neofluar ×100/1.30 Oil (DIC III) objective , and image acquisition was performed using an AxioCam MR camera (Carl Zeiss). The interaction between 5-LO and FLAP appears as fluorescent spot (magenta). Overview images were obtained using a Plan Neofluar 40/1.30 Oil (DIC III) objective (Carl Zeiss).PBMC were seeded onto glass coverslips in growth medium for 1.5 h  were suspended in PBS pH 7.4 containing 1 mg/ml glucose. After incubation with vehicle , α-T-13′-COOH (4a) or δ-TE-13′-COOH  for 20 min at 37 °C, cells were centrifuged  and washed three times with PBS pH 7.4 plus 0.1% fatty acid-free BSA (Sigma-Aldrich). LCMs were extracted and analyzed by UPLC-MS/MS using γ-TE-13′-COOH  as internal standard. Intracellular vitamin E metabolite concentrations were calculated assuming spherical cell shape, an equal intracellular distribution, and an average diameter of 13 µm .Freshly isolated PMNL  were treated with LCMs for 10 min at 4 °C in potassium phosphate buffer  containing 5 mM glutathione. The conversion of LTA4 to LTC4 methyl esters was initiated by addition of LTA4 methyl ester . After 10 min at 4 °C, the reaction was terminated by ice-cold methanol (one volume), and LTC-d5 methyl ester  was added as internal standard. LTC4 was extracted and analyzed by UPLC-MS/MS as described for lipid mediators. Vehicle (DMSO)-treated microsomal membranes produced 7.1 ± 0.5 nmol LTC4. The LTC4 reference inhibitor MK-886  suppressed LTC4 formation by 95.6 ± 0.9%.To investigate the effect of LCMs on LTCS)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) was initiated by addition of AA . 12-HHT was extracted using Sep-Pak C18 35 cc Vac Cartridges (Waters) and separated by RP-HPLC on a Nova-Pak C18 Radial-Pak Column  as described for 5-LO products. The COX-1/2 reference inhibitor indomethacin  suppressed COX-1 product formation by 79.3 ± 2.3% and COX-2 product formation by 77.4 ± 3.1%.Purified bovine COX-1  or human recombinant COX-2  in 100 mM Tris buffer pH 8, 5 mM glutathione, 5 µM hemoglobin, and 100 µM EDTA were pre-incubated with LCMs for 5 min at 4 °C followed by 1 min at 37 °C. Formation of COX-derived 12 at 37 °C and 5% CO2 for 48 h, harvested and frozen in liquid nitrogen. Ice-cold homogenization buffer  was added, and cells were incubated for 15 min on ice prior to sonication . The microsomal fraction was obtained by differential centrifugation at 10,000 × g for 10 min and 174,000 × g for 1 h at 4 °C. Microsomes were suspended in homogenization buffer and diluted in potassium phosphate buffer  containing 2.5 mM glutathione. Membranes  were pre-incubated with LCMs for 15 min at 4 °C, and PGE2 formation was initiated by addition of PGH2 . The reaction was terminated after 1 min by addition of one volume stop solution . PGE2 was extracted using Sep-Pak C18 35 cc Vac Cartridges (Waters), separated by RP-HPLC on a Nova-Pak C18 Radial-Pak Column  under isocratic conditions (30% acetonitrile/70% water/ 0.007% trifluoroacetic acid) at a flow rate of 1 ml/min and detected at 195 nm67. The mPGES-1 reference inhibitor MK-886 (10 µM) suppressed PGE2 formation by 78.0 ± 2.5%.Microsomal membranes of interleukin-1β-treated A549 cells were prepared as source of mPGES-18) were pre-incubated with LCMs for 5 min at room temperature. Formation of COX-1-derived 12-HHT was initiated by addition of exogenous AA (5 µM). After 5 min at 37 °C, 12-HHT was extracted using Sep-Pak C18 35 cc Vac Cartridges (Waters) and separated by RP-HPLC on a Nova-Pak C18 Radial-Pak Column  as described for 5-LO products.Freshly isolated human platelets (1 × 1070. Sf9 cells were infected with a recombinant baculovirus (72 h) and sonicated  in lysis buffer . Supernatants were centrifuged  and subjected to benzylthio-sepharose affinity chromatography. sEH was eluted with 4-fluorochalcone oxide in PBS pH 7.4, 1 mM dithiothreitol and 1 mM EDTA and then dialyzed and concentrated. The purified enzyme (60 ng) was diluted in 25 mM Tris HCl, pH 7 plus 0.1 mg/ml BSA and pre-incubated with LCMs for 10 min at room temperature. The enzymatic reaction was started by addition of PHOME , which was converted to the fluorescent 6-methoxy-naphtaldehyde. After 60 min in darkness, the reaction was terminated by ZnSO4 (200 mM), and the fluorescence was measured using a NOVOstar fluorescence microplate reader . The reference inhibitor AUDA  suppressed sEH activity by 42.2 ± 1.9%.To investigate the effect of LCMs on sEH activity, we purified the human recombinant enzyme from sEH expressing Sf9 insect cells by benzylthio-sepharose affinity chromatographyn observations, where n represents the number of experiments or the number of animals, as indicated. Sample size was not pre-determined by statistical methods. Data from animal studies were confirmed as normally distributed with similar variance between groups using a Kolmogorov–Smirnov test. Samples of the vitamin E library were blinded for screening purposes but not for further biological evaluation, and samples from mouse peritoneum were blinded for counting infiltrated cells. Outliers were determined using a Grubb’s test. Comparison of different groups was performed by one-way or two-way ANOVA for independent or correlated samples followed by Tukey or Bonferroni HSD post hoc test or by two-tailed Student t test for paired or unpaired samples. Tests were conducted using a two-sided α level of 0.05. P values < 0.05 were considered statistically significant. Statistical calculations were performed using GraphPad Prism 4.0 . IC50 values were determined by graphical analysis using SigmaPlot 13.0 .Data are presented as mean ± s.e.m. of Supplementary InformationPeer Review File