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.

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.