ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation

Abstract

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20-59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats ("55" genotype) than in subjects with one deletion (d) (three or four repeats) and one common ("d5" genotype) allele or with two deletion ("dd") alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk.

Fig. 1.

A consort diagram describes subject recruitment and retention. Of the 667 subjects excluded, 338 subjects were excluded based on criteria evaluated during initial screening; 28 subjects were excluded due to ineligible genotype; 100 subjects with eligible genotypes were excluded because these genotype groups were full; 38 subjects were excluded with abnormal blood test results at a second screening step; and 113 subjects declined to participate. Subjects were randomized within six ALOX5 genotypes, as shown in Table 1.

Fig. 2.

Western blot analyses of arachidonate 5-LO protein and β-actin are shown. These representative blots show K562 control cells and monocyte samples (0.5 × 10⁶ cells per lane) from three subjects at baseline (week 0) and follow-up (week 6). The 5-LO bands are approximately 80 and 78 kDa.

Fig. 3.

Relative mean concentration values of oxylipid metabolites are shown 60 min after A23187 stimulation of monocyte cultures from subjects with different ALOX5 promoter genotypes at baseline. Bars show percentages relative to the mean levels for the 55 control group and dd and d5 subjects (top panel); 33 and 35 subjects (middle panel); and 44 and 45 subjects (bottom panel). *, Significant differences from the 55 group mean by one-factor ANOVA. Concentrations, standard errors and numbers of subjects are given in Table 4 (also see supplementary Tables IV and V).

Fig. 4.

Ratios of EPA-derived (5-HEPE and 15-HEPE) to AA-derived (5-HETE and 15-HETE) 5-LO and 15-LO metabolites are shown at 60 min after A23187 stimulation of monocyte cultures from subjects with different ALOX5 promoter genotypes at baseline. Box plots show median, 10th, 25th, 50th, 75th, and 90th percentiles for subjects with the three ALOX5 genotypes of interest. *, Significant difference from the corresponding 55 genotype group. Numbers of subjects in the dd, d5, and 55 genotype groups were 32, 53, and 30, respectively.

Fig. 5.

Mean (±SEM) changes in oxylipid concentrations are shown from baseline to follow-up among the three ALOX5 genotype groups of interest in response to placebo and fish oil interventions. Significant increases were seen in all of these EPA-derived (5-HEPE, 15-HEPE, and LTB5) and DHA-derived (17-HDoHE) metabolites in the fish oil treatment compared with the placebo group (P < 0.05). Genotype X treatment interactions were seen for 5-HEPE and 15-HEPE. For these metabolites, either a single (*) or double (**) asterisk indicates a within-genotype difference between the treatment and placebo groups, while the double asterisk additionally indicates that the 55 genotype, within the fish oil treatment group, had a significantly greater increase than the other genotype groups. The numbers of fish oil/placebo subjects in the dd, d5, and 55 genotype groups were 12/13 subjects, 24/25 subjects, and 13/12 subjects, respectively.