In this study, we analyzed plasma lipid profiles of veterans with GWI and control participants. Most of the differences pertained to the neutral lipids (CE, TG and DG), where the degree of unsaturation and ratios of AA-to-DHA were changed in GWI compared to controls. Our study shows that male veterans with GWI had significant increases in total neutral lipid content compared to male controls. Female veterans with GWI had elevated levels of Cer and lower levels of SFA TG species as compared to female controls. These sex-specific plasma lipid compositions in GWI veterans require further attention as they may indicate progressive metabolic and inflammatory processes, particularly in male veterans with GWI.
In the plasma, neutral lipids primarily appear in lipoprotein compartments due to their hydrophobic nature. The neutral lipid class of triglycerides are major dietary fats, but they are also synthesized in the liver (41). Because dietary TGs are digested to free fatty acids and monoglycerides in the intestines, plasma TG levels within lipoprotein particles are reflective of TG synthesis in liver via acylation with DG (42). In plasma, an excess of TG, such as seen in the male veterans with GWI, is thought to reflect overproduction of very low-density lipoprotein (VLDL) corresponding with the production of CE-depleted low-density lipoprotein (LDL) particles (41). This production of CE-depleted LDL may explain the significant decrease in CE in male veterans with GWI. In the current study, female participants, irrespective of their diagnosis, had lower levels of TG and DG compared to their male counterparts. This has also been reported by the Genetics of Lipid-Lowering Drugs and Diet Network (GOLDN) study of plasma lipidome, who attributed this to normal sex-linked lipid differences (26). In terms of the clinical implications of elevated TG in male veterans with GWI, elevated plasma TG is thought to be a risk factor for cardiovascular disease(43). This may require further monitoring by clinicians as veterans with GWI age. Elevated TG and the generation of CE-depleted LDL particles are also the defining features of dyslipidemia associated with insulin resistance and type 2 diabetes mellitus (41).
Veterans with GWI may be more sedentary and exercise less than healthy controls due to their condition, so this TG increase could reflect heightened body weight in veterans with GWI. A positive association between high TG and elevated body mass index (BMI) has been previously reported (44, 45). As early as 1995, it was observed that veterans with severe GWI had slightly higher BMI than non-cases (33). Unfortunately, not all the GWI cohorts in this study collected BMI data and so we cannot control for BMI as a covariate in this study. A recent health survey of GW veterans from the Fort Devens cohort suggested increases in the prevalence of high blood pressure, diabetes, and cardiovascular disease over the last 30 years (4). The Fort Devens Cohort was also assessed to have significantly higher rates of diabetes in both male and female GW veterans than the general population (46). Collectively, these studies suggest that a regular monitoring of blood lipids is required to better assess their contributions to the risk of developing cardiovascular disease and metabolic disorder among veterans with GWI.
In the current study, SFA-containing TG species were decreased in female veterans with GWI. The role of SFA in promoting cardiovascular disease remains controversial as the source of these SFA-containing lipids and the individual identity of each SFA influences their potential role in promoting cardiovascular disease (21, 47). As the SFA-TG species measured ranged from TG 46:0 to TG 54:0, and therefore consisted of 46 to 54 carbons split amongst 3 fatty acid chains, it is likely that the SFA-TG species measured contained long-chain fatty acids, which are over 12 carbons in length (47). However, their significance in the clinical presentation of female GWI is unclear. Future studies aimed at different aspects of dietary modification in male vs. female GW veterans may be required to better understand the role of SFA-containing TG species in the health of veterans with GWI.
Male GW veterans with GWI had elevated levels of PUFA within the DG and TG classes. Since omega-6 AA or omega-3 DHA comprise a large proportion of PUFA, we focused on lipids that contain AA and DHA as their fatty acid side chains. Our previous studies have shown that AA and DHA content within certain PL classes were associated with GWI (17). We now show that ratios of AA-to-DHA were increased in TG among male and female GW veterans with GWI. The ratio of AA-to-DHA was also increased in PE among male GWI veterans. As AA and DHA compose 50% and 40% of brain PUFAs respectively, sufficient AA is necessary for the growth, repair, and maintenance of neurons and DHA is used in neurotransmission (48). However, AA metabolites, such as prostaglandins, are known to activate pro-inflammatory pathways whereas DHA metabolites, such as resolvins, contribute to inflammation resolution pathways (24, 49). In addition, studies have shown that patients with a low DHA/AA ratio, (i.e. a higher AA-to-DHA ratio) had a higher risk of acute coronary syndrome than those with a high DHA/AA ratio, and this was significant for men in particular (50). These changes in AA and DHA composition could reflect a generalized pro-inflammatory state in veterans with GWI, which would be consistent with reports of markers of chronic-low grade inflammation being associated with GWI diagnosis (51, 52).
Another indication of pro-inflammatory states in GWI would be the increase in Cer in female GWI veterans, as ceramides are known to stimulate the production of pro-inflammatory cytokines, such as IL-6 and TNF-α, in macrophages and are correlated with IL-6 levels in serum (53, 54). Production of long-chain ceramides is known to induce apoptosis (55). The increase in the long chain ceramides measured in this study (d32:0-44:2) may therefore indicate increased cell death in female GWI veterans. Boon and colleagues also found that ceramide elevation is another marker of the dyslipidemia of type 2 diabetes mellitus and promotes insulin resistance (53).
This study provides key information about blood biomarkers associated with adverse metabolic and inflammation-related lipid profiles. However, there are certain limitations which restrict the generalizability of our findings to the current cohort only. First, due to a lack of availability of BMI data across cohorts, we were unable to adjust for BMI differences as a potential confounder between male and female veterans. However, as our findings are largely consistent with previous studies showing sex-specific differences in lipid profiles, it is therefore unlikely that BMI differences significantly confound this relationship and may be inherent to lipid dysfunction. Another limitation of our study includes unavailability of dietary data and medication data, particularly on the use of statins and omega-3/fish oil supplements. Since statins lower TG levels in blood and our findings suggest increases of TG in male veterans with GWI, we anticipate that it is unlikely that statin use affected our study results. Similarly, we have previously shown that AA-to-DHA ratios are decreased after DHA supplementation (39). Since we observed an increase in AA-to-DHA ratios in male GWI veterans, it is similarly unlikely that omega-3 or fish oil supplement was a confounder in our study. As CE levels were lower in male GWI veterans, measuring free cholesterol would be useful in future studies to determine if either the level of free cholesterol or the rate of cholesterol esterification had decreased. Unfortunately, we could not reliably detect the labelled free cholesterol in our assay but will correct this in future studies. While we have previously examined free fatty acids in a smaller cohort of GW veterans, which suggested that free forms of PUFA were indeed affected in GWI (18), a large study evaluating free fatty acids and their bioactive lipid metabolites will help better elucidate the role of these fatty acids in ongoing inflammatory processes associated with GWI.