"Metabolomics" (MO) refers to an approach in which many chemicals in the body ("metabolites") are measured together. This is an "expansion award" mechanism that seeks to follow up on our prior findings of mitochondrial (mt) alterations in Gulf War Veterans (GWV) with Gulf War illness (GWI). (Mitochondria make energy for cells, but also produce free radicals (oxidative stress), and control cell death [apoptosis].) It seeks to do so by comparing MO in GWI to controls. A preliminary study we conducted assessed MO in 20 male GWV with GWI and 20 matched controls. This identified a spectrum of critical, previously unidentified objective changes that match pathways reported to be altered in reports of MO alterations in mt dysfunction and reports of lipid alterations in mt dysfunction. Moreover, the MO approach completely separated GWV with GWI from controls matched in age, sex, and ethnicity. If these MO findings prove right, they not only radically expand our understanding of processes and mechanisms in GWI, but may provide for objective diagnosis and provide important targets for treatment. Promising findings like these are best trusted after they have been replicated and "validated." Ideally this should include new participants, and participants recruited in a different location. Thus, our plan is to assess MO in several groups including GWV with GWI, controls, and others, e.g., that fail some GWI qualification -- to replicate/validate, and extend and circumscribe the remarkable preliminary findings.

To validate the findings, we will reassess MO, on 20 "new" male GWV with GWI and 20 matched controls from San Diego, the original location. As a secondary aim, we will test MO from a geographically distant site: The Boston GWI Consortium's biorepository will provide samples from 20 male GWI cases and 20 controls. Tertiary aims explore boundaries of GWI -- important for a tool for diagnosis. To assess if GWI case definition matters, we will test MO in up to 20 GWI cases from Minneapolis, where GWI is defined by adherence to either, rather than both, the Kansas and Centers for Disease Control and Prevention (CDC) GWI criteria. We will assess MO in persons with GWI symptoms who were not Gulf-deployed and in GWV with GWI symptoms but who have conditions (like multiple sclerosis or lupus) that typically disqualify from a diagnosis of GWI, or participation in GWI studies. Since Gulf exposures may increase risk of other conditions, both conditions may be due to Gulf service -- and these GWV feel justifiably disenfranchised from GWI research. If diagnostic markers of GWI (or a subset) extend to these Veterans, MO might aid diagnosis and possibly enable involvement of these GWV in some research efforts. (MO findings can also be compared to MO signatures for these disqualifying conditions, in non-GWV). We will begin to assess females (GWI and controls) and those who were GW-deployed but are not ill (preliminary).

This study is important for many reasons. For instance, (1) MO profiling provides key insights into pathways and mechanisms in GWI. (2) It promises to provide an objective means to diagnose GWI -- and to distinguish GWI from other conditions, including other war-related conditions, and other chronic multisymptom illness. Co-Investigator Naviaux has MO data on both post-traumatic stress disorder (PTSD) and non-GWV chronic fatigue syndrome: the GWI signatures are clearly distinct from each of these. (3) The context provided by alterations in other metabolites, gained with MO, can radically revise the interpretation of an already reported finding. E.g., cortisol is altered in GWI, which might be interpreted in terms of mental "stress" involvement. But cortisol is made in mitochondria from cholesterol, and our MO data show that multiple other cholesterol products, also made in mitochondria, are also disrupted in GWI. These range from bile acids to vitamin (vit) D to gonadal (sex) hormones. Moreover, these same changes are reported in mt dysfunction. Thus, mt dysfunction better fits the cortisol finding than does stress-related dysregulation, once the MO context is known. (4) MO findings suggest why some already-posited treatments might help, and (5) they define new candidate treatments. Low levels of active 1,25 vit D relative to the levels of 25 vit D, in MO, suggest a block at a step called 1 alpha hydroxylation, which takes place in kidney mitochondria. This could account for informally observed benefits of vit D -- based on which we'd proposed testing of vit D and vit D containing cod liver oil in GWI. (Active vit D is important for muscle and other domains affected in GWI.) Phosphatidylcholine was altered in MO data -- providing a basis for the benefit reported from lecithin (rich in phosphatidylcholine) by a Veteran member of the Research Advisory Committee. Niacin, in Hubbard detoxification for other hypothesized reasons, is altered in GWI. Finally, MO data suggest new treatments, like membrane lipid therapy, or others, e.g., that target apoptosis signaling.

Since we have already secured preliminary data, the present study can validate the findings -- to "seal and deliver" definitive MO signatures. These may, we hope, instantly revise understanding of mechanisms, revolutionize diagnosis of GWI, and have already suggested promising treatments, available for immediate testing.