Role of arachidonyl triglycerides within lipid bodies in eicosanoid formation by human polymorphonuclear cells.

Increasing evidence suggests that the subcellular and glycerolipid localization of esterified arachidonic acid (AA) is a key factor in regulating its availability to lipases. The goal of the current study was to determine the potential of AA stored in triglycerides (TG) to serve as a substrate for lipases and 5-lipoxygenase during neutrophil (polymorphonuclear leukocytes, PMN) activation. PMN containing high concentrations of AA in TG were generated by culturing PMN in vitro with high concentrations of exogenous AA (eAA) for 12 h. Cellular AA increased 2- and 4-fold in PMNs incubated with 5 and 20 microM AA, respectively, and this increase was almost exclusively observed in neutral lipids (NL). Further analysis revealed that 88% of the AA in the NL fraction was associated with TG. Subsequent experiments were designed to determine whether this AA in TG could be mobilized and metabolized to eicosanoids during cell activation. TG pools of AA were increased as previously described and then PMN were stimulated with ionophore, A23187. In contrast to the 43-fold increase in TG AA after eAA loading (20 microM), free AA increased by only 1.9-fold after cell stimulation. Similarly, leukotriene (LT)B(4) production increased only 2-fold after loading TG with large quantities of AA. The magnitude of increase in free AA released and in LTB(4) formation was similar to the magnitude of increase in AA mass in phospholipase (PL), suggesting that PL, and not TG, served as the source of released AA and subsequent product generation. To confirm that AA in TG did not serve as a source for eicosanoid production, cellular pools of AA were differentially labeled with [(14)C]AA and [(3)H]AA, and the [(3)H]AA-to-[(14)C]AA ratio of LTB(4) and 20-hydroxyl LTB(4) produced during cell stimulation was measured. The [(3)H]AA/[(14)C]AA ratios of LTs were markedly different from the ratios in TG, thus providing further evidence that AA pools in TG are not a major source of AA for LT generation.

Dietary supplementation with gamma-linolenic acid alters fatty acid content and eicosanoid production in healthy humans.

To understand the in vivo metabolism of dietary gamma-linolenic acid (GLA), we supplemented the diets of 29 volunteers with GLA in doses of 1.5-6.0 g/d. Twenty-four subjects ate controlled eucaloric diets consisting of 25% fat; the remaining subjects maintained their typical Western diets. GLA and dihomo-gamma-linolenic acid (DGLA) increased in serum lipids of subjects supplemented with 3.0 and 6.0 g/d; serum arachidonic acid increased in all subjects. GLA supplementation with 3.0 and 6.0 g/d also resulted in an enrichment of DGLA in neutrophil phospholipids but no change in GLA or AA levels. Before supplementation, DGLA was associated primarily with phosphatidylethanolamine (PE) of neutrophil glycerolipids, and DGLA increased significantly in PE and neutral lipids after GLA supplementation. Extending the supplementation to 12 wk did not consistently change the magnitude of increase in either serum or neutrophil lipids in subjects receiving 3.0 g/d. After GLA supplementation, A23187-stimulated neutrophils released significantly more DGLA, but AA release did not change. Neutrophils obtained from subjects after 3 wk of supplementation with 3.0 g/d GLA synthesized less leukotriene B4 (P < 0.05) and platelet-activating factor. Together, these data reveal that DGLA, the elongase product of GLA, but not AA accumulates in neutrophil glycerolipids after GLA supplementation. The increase in DGLA relative to AA within inflammatory cells such as the neutrophil may attenuate the biosynthesis of AA metabolites and may represent a mechanism by which dietary GLA exerts an anti-inflammatory effect.

Metabolism of gammalinolenic acid in human neutrophils.

Gammalinolenic acid (GLA), when provided as a dietary supplement, has been reported to improve clinical symptoms of several inflammatory disorders. The goal of the current study was to examine the metabolism of GLA and its relationship to arachidonic acid (AA) in the human neutrophil. Initial studies indicated that neutrophils provided GLA in vitro rapidly elongate it (by two carbons) to dihomogammalinolenic acid (DGLA). The bulk of this newly formed DGLA is incorporated into neutral lipids and specifically triacylglycerides. Neutrophils from volunteers supplemented with GLA as borage oil also had elevated quantities of DGLA but not GLA, when compared with neutrophils from volunteers not consuming the GLA supplement. To determine whether DGLA could be mobilized from cellular glycerolipids, neutrophils were stimulated with ionophore A23187 and fatty acid levels were determined. DGLA and AA were both released during stimulation, and the quantities of DGLA mobilized increased threefold after in vitro GLA supplementation. Exogenously provided DGLA was converted to one major metabolite during cell stimulation; this product migrated on reverse-phase HPLC with the 15-lipoxygenase product, 15-hydroxy-eicosa-trienoic acid (15-HETre). Both 15-HETre and DGLA (provided exogenously) inhibited the formation of leukotriene B4, (LTB4) and 20-hydroxy-leukotriene B4 (20-OH-LTB4). The IC50 for 15-HETre inhibition of both LTR, and 20-OH-LTB4 in A23187-stimulated neutrophils was 5 microM. This inhibition could be reversed by removing the compounds from the cells. Taken together, these data reveal that there are enzymes within the human neutrophil that metabolize GLA or its elongation product DGLA, and that the metabolism of GLA and AA may interact at a number of critical junctures.