Sex-specific differences in plasma lipid profiles are associated with Gulf War Illness

Approximately 30% of the 700,000 veterans of the 1991 Gulf War (GW) suffer from Gulf War Illness (GWI), a chronic multi-symptom condition characterized by cognitive impairment, fatigue, and pain [1]. Veterans’ exposure to chemical warfare agents, pesticides, and pyridostigmine bromide are among the major contributors to GWI etiology [1‐4]. Currently, there are no biomarkers for indicating the underlying pathology of this condition. Advances in GWI research suggest lipid dysfunction, bioenergetics deficits, and inflammation as key contributors to its ongoing pathology [5, 6]. Blood biomarkers of these biological processes would be a cost-effective and minimally invasive avenue to assist clinicians with accurate detection of GWI.

Evidence suggests that GW pesticides affect cellular lipid metabolism and lipid homeostasis [2, 7‐11]. Altered lipid homeostasis can result in an abnormal accumulation of lipids that promote cellular inflammation [12, 13]. Abnormal plasma and brain lipid profiles after GW pesticide exposure in rodents and in veterans with GWI have been shown to accompany inflammation, neurobehavioral and bioenergetic deficits [14‐18]. As such, altered plasma lipid profiles may provide a biomarker signature for the bioenergetic deficits and inflammation associated with GWI.

It is expected that since the pesticides to which GW veterans were exposed tend to disturb lipid homeostasis, owing to their high affinity for lipids, neutral lipids classes, such as triglycerides (TG), diglycerides (DG), and cholesterol esters (CE), and phospholipid (PL) classes would be affected in GWI [2, 9‐11, 19, 20]. The degree of unsaturation of the fatty acids within the PL classes is known to alter metabolic and inflammatory systems, with many saturated fatty acid (SFA)-containing PL positively associated with chronic metabolic conditions whereas polyunsaturated fatty acid (PUFA)-containing PL are associated with inflammation [21‐24]. Among these, PUFA containing the omega-3 fatty acid, docosahexaenoic acid (DHA), generate anti-inflammatory or inflammation-resolving lipid metabolites and PUFA containing the omega-6 fatty acid, arachidonic acid (AA), can produce metabolites that promote pro-inflammatory responses [23‐25].

Changes in lipid profiles were also expected in relation to the sex of GW veterans, as many classes of plasma lipids are significantly associated with sex [26]. Women are up to four times more likely than men to be diagnosed with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a chronic multi-symptom disease clinically similar to GWI that has been shown to have sex-linked differences in blood lipid profiles [27, 28]. Female GW veterans have a higher prevalence of GWI diagnosis and severe GWI-related symptoms than male GW veterans [29]. These findings suggest potential sex-linked differences in pathophysiological responses to GW-era chemical exposures, necessitating sex-stratified analyses.

We hypothesized that blood lipid levels would be affected both by GWI status and the sex of GW veterans. We evaluated the levels of several major blood lipid classes and their molecular species in three cross-sectional cohorts of participants classified as GWI or control. This study will further our understanding of plasma lipids as potential biological markers to track the ongoing inflammation and metabolic dysfunction of GWI, which may help to provide better care and management of this illness.

Previous animal studies have shown that exposure to GW chemicals, such as those to which GW veterans were exposed, disturbs lipid homeostasis and contributes to inflammatory and metabolic dysfunction that are seen in veterans with GWI [7‐9, 14, 15, 41]. To evaluate the translational significance of prior mechanistic studies, the current study 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 TG are major dietary fats, but they are also synthesized in the liver [42]. 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 [43]. In plasma, an excess of TG, such as seen in the male veterans with GWI, is thought to reflect an overproduction of very low-density lipoprotein (VLDL) corresponding with the production of CE-depleted low-density lipoprotein (LDL) particles [42]. 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 [44]. 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 [42].

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 [45, 46]. As early as 1995, it was observed that veterans with severe GWI had slightly higher BMI than non-cases [33]. However, BMI did not differ between GWI and controls (in a subsample) and therefore unlikely to explain the observed differences in lipid profiles. 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 [47]. However, differences between GWI and controls were not evaluated in this prior study. In our current study, a lack of difference in cardiovascular risk factors between controls and GWI suggested that observed differences in lipid profiles between the two groups were not simply a reflection of the presence of cardiovascular risk factors in this study cohort. As many of the neutral lipid changes may be indicative of subsequent risk of cardiovascular disease with age, further assessement of blood lipids by clinicians may be helpful for monitoring cardiovascular health as veterans with GWI age.

In the current study, SFA-containing TG species were reduced 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, 48]. 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 [48]. However, their significance in the clinical presentation of female GWI is unclear. Since these same TG-SFA species were elevated among those with cardiovascular risk factors, 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, particularly cardiovascular health, of veterans with GWI.

Our previous studies using subsets of samples from these cohorts showed increases in proinflammatory cytokines, interleukin-1 beta (IL-1β), interferon gamma (IFN-γ) and IL-6, and increases of peripheral immune cells among veterans with GWI compared to controls [18]. Others have shown that veterans with GWI have increases in other blood inflammatory markers consisting of C-reactive proteins and matrix-metalloproteinase-2 [49]. Several proinflammatory cytokines, including IL-1β and IL-15, are associated with an increase in GWI symptom severity [50]. As such, an examination of lipids related to inflammation may provide additional information about the underlying biology of GWI. Since omega-6 AA or omega-3 DHA modulate inflammation and 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 the 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 species among male and female 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 [51]. 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, 52]. 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 [53]. These differences 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 [49, 54]. Hence, future studies are needed that interrogate systems-level omic data along with biological parameters of inflammation to identify specific inflammatory pathways that are associated with GWI pathogenesis.

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 [55, 56]. Production of long-chain ceramides is known to induce apoptosis [57]. The increase in the long chain Cer measured in this study (d32:0–44:2) may therefore indicate increased cell death in female GWI veterans. Ceramide levels are shown to be increased with obesity and metabolic disorders, particularly diabetes. For instance, Boon and colleagues also found that Cer elevation is another marker of the dyslipidemia of type 2 diabetes mellitus and promotes insulin resistance [55]. Others have shown that plasma Cer increases are associated with obesity and may be indicative of lipotoxicity, which may ultimately contribute to the development of diabetes and insulin resisistance [58, 59]. These studies suggest a different process in males and females in relation to Cer-mediated metabolic dysfunction that warrants further investigation.

This study provides key information about blood biomarkers associated with adverse metabolic and inflammation-related lipid profiles. The BMI in females was non-significantly lower than in males and therefore did not present as a confounding factors for the differences in lipids seen between the two sex groups. These findings are largely consistent with previous studies showing sex-specific differences in lipid profiles. 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 supplementation 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.

This study identifies distinct sex-specific plasma lipid profiles associated with GWI diagnosis, with significantly elevated TG and DG and lowered CE as a distinctive pattern in male GW veterans with GWI. Triglyceride AA-to-DHA ratios were also increased in male GW veterans with GWI. Since these lipid changes are associated with metabolic syndrome and inflammation, our findings suggest veterans with GWI have maladaptive biological processes that involve lipid transport and metabolism. While additional mechanistic work is needed to better elucidate lipid dysfunction in GWI, these blood lipid signatures suggest that focused studies are needed into the role of dyslipidemia in the ongoing pathophysiology of GWI. The current findings suggest that blood lipid monitoring may be beneficial for veterans with GWI. As such, further research is needed to characterize how these lipids and their associated pathways relate to GWI pathology to identify potential biomarkers for tracking general health related outcomes in GWI.