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Elevated serum measures of lipid peroxidation and abnormal prefrontal white matter in euthymic bipolar adults: toward peripheral biomarkers of bipolar disorder

Diffusion tensor imaging (DTI) studies consistently reported abnormalities in fractional anisotropy (FA) and radial diffusivity (RD), measures of the integrity of white matter (WM), in bipolar disorder (BD), that may reflect underlying pathophysiologic processes. There is, however, a pressing need to identify peripheral measures that are related to these WM measures, to help identify easily-obtainable peripheral biomarkers of BD. Given the high lipid content of axonal membranes and myelin sheaths, and that elevated serum levels of lipid peroxidation are reported in BD, these serum measures may be promising peripheral biomarkers of underlying WM abnormalities in BD. We used DTI and probabilistic tractography to compare FA and RD in ten prefrontal-centered WM tracts, 8 of which are consistently shown to have abnormal FA (and/or RD) in BD, and also examined serum lipid peroxidation (lipid hydroperoxides, LPH and 4-hydroxy-2-nonenal, 4-HNE), in 24 currently euthymic BD adults (BDE)and 19 age- and gender- matched healthy adults (CONT). There was a significant effect of group upon FA in these a priori WM tracts (BDECONT:F[1,41]=10.3;p=0.003), and a significant between-group difference in LPH (BDE>CONT:t[40]=2.4;p=0.022), but not 4-HNE. Multivariate multiple regression analyses revealed that LPH variance explained, respectively, 59% and 51% of the variance of FA and RD across all study participants. This is the first study to examine relationships between measures of WM integrity and peripheral measures of lipid peroxidation. Our findings suggest that serum LPH may be useful in the development of a clinically-relevant, yet easily obtainable and inexpensive, peripheral biomarkers of BD.


Introduction
There is a pressing need to identify biomarkers reflecting underlying pathophysiologic processes for psychiatric disorders [1][2][3] to facilitate more accurate diagnosis of these disorders than reliance on behavioral measures alone, and ultimately provide biological targets to optimize treatment choice and new treatment developments. While neuroimaging may identify biomarkers reflecting central (neural circuitry) pathophysiologic processes, neuroimaging facilities are not available in all clinical settings, and remain relatively expensive to administer routinely in clinical practice. Identifying peripheral biomarkers related to underlying central pathophysiology is one way of obtaining clinically-relevant, yet easily-obtainable and inexpensive biological measures to help with psychiatric diagnosis, and ultimately treatment choice. This is especially important for bipolar disorder (BD), one of the top six most debilitating of all non-communicable illnesses [4][5] , yet misdiagnosed as recurrent major depressive disorder in 60% of BD individuals seeking treatment for depression 4,[6][7] .
Diffusion tensor imaging (DTI) is sensitive to the water diffusivity in brain tissue. Given the hydrophobic nature of the lipid components of WM tracts, specifically, axonal membranes and myelin sheaths, water molecules diffuse along the principal (longitudinal) direction of highly-packed fibers in collinear axons, but in two or more directions in non-collinear WM fibers (e.g., crossing tracts). DTI measures include: the diffusivity along the principal axis, λ 1 , longitudinal/axial diffusivity (L1); the diffusivity along transverse directions (λ 2 and λ 3 ) perpendicular to the principal axis of diffusion, radial diffusivity (RD), high in crossing tracts; and fractional anisotropy (FA), the ratio of longitudinal vs. transverse diffusivity in WM tracts. DTI studies consistently reported abnormalities in WM in BD 8,[9][10][11][12][13] (especially BD type-I), including reduced FA and elevated RD in 8 WM tracts connecting prefrontal with subcortical and other cortical regions [14][15][16][17][18][19][20][21][22][23] . These include: forceps minor (interhemispheric fibers connecting left and right frontal cortices); anterior thalamic radiation (connecting thalamus with prefrontal cortex); and six associative tracts interconnecting functional subdomains of the cerebral cortex: cingulum; inferior longitudinal fasciculus; superior longitudinal fasciculus; and uncinate fasciculus. The cingulum is further divided into the angular bundle and bundle of the cingulate gyrus 24 ; and the superior longitudinal fasciculus, into the arcuate and I-III bundles 24 .
Animal studies suggest that increased RD may reflect myelin abnormalities [25][26][27][28] , and human postmortem evidence 29 suggests that greater RD may reflect myelin abnormalities and axonal damage. Given that WM mostly comprises neuronal axons and glia cell-containing myelinated sheaths, and both axonal membranes (axolemma) and myelin comprise 80% lipids, potential peripheral measures of abnormal WM, suggested by elevated RD, are serum measures of lipid damage (e.g., lipid oxidative stress). Lipid peroxidation is the specific process by which reactive oxygen species (ROS) induces lipid oxidative stress damage, and involves three phases: initiation, propagation and termination of lipid peroxides 30 . During early stages, lipid hydroperoxides (LPH) are generated by reaction of fatty acids with ROS that can be used as an early stage biomarker of lipid peroxidation. If the lipid peroxidation cascade progresses, two carbon or hydroperoxide radicals react and form non-radical species, that can be detected in serum as measures of late-stage lipid damage. 4-hydroxy-2nonenal (4-HNE) is one example of a late-stage biomarker of lipid peroxidation 31 . Myelin is vulnerable to lipid oxidative stress because it contains a large amount of polyunsaturated fatty acid side-chains, specific targets for lipid peroxidation 30 . Increasing evidence suggests that oxidative stress may be implicated in the pathophysiology of mood disorders, particularly BD type-I 32-33 , and post mortem findings reported elevated 4HNE in the anterior cingulate cortex in BD 34 . Assessing early-and late-stage measures of lipid peroxidation in serum samples can provide insight into the level of lipid oxidative stress damage that in turn may relate to abnormal WM (elevated RD and reduced FA) in BD individuals.
As a first stage toward identifying peripheral biomarkers of pathophysiologic processes in BD, the goal of the present study was first to determine whether adults with BD showed abnormalities in both WM and peripheral measures of lipid peroxidation vs. CONT, and then to determine the nature of the relationships between WM and peripheral measures across all participants. We focused on BD type-I adults, given that the majority of DTI findings are reported in BD type-I (see above), and BD adults in the euthymic stage of illness, to avoid any potential confounds of depressed or manic mood episode upon peripheral and WM measures. For DTI analyses, we employed an automated global probabilistic approach in tractography to study 10 major WM tracts, including the above 8 tracts consistently shown to have abnormal FA and RD in BD (Figure1A), and two other "control" tracts that have not been consistently shown to be abnormal in BD: the forceps major, the posterior bundle of the corpus callosum passing through the splenium, and the cortico-spinal tract, a major ascending tract.
We had the following specific hypotheses: 1. BD (type-I) vs. healthy adults would show significantly reduced FA and elevated RD in the above a priori eight WM tracts.

3.
There would be a negative relationship across all adults between FA, and a positive relationship between RD, in these WM tracts and peripheral measures of lipid peroxidation.

Participants
Forty-three right-handed participants were recruited, including 24 euthymic BD individuals, type-I (BDE), diagnosed according to DSM-IV criteria and the Structured Clinical Interview for DSM-IV (SCID-I/P) 35

Statistical analysis
Demographic data, clinical data, peripheral measures, FA RD and L1 were all imported into well-established statistical software (Predictive Analytics SoftWare Statistics 17.0, for Windows) to test main hypotheses and exploratory analyses. Rather than considering the 10 WM tracts separately, our approach was to examine them simultaneously. This allowed us to balance type-I and type-II errors and avoid problems associated with multiple comparisons. Furthermore, given the absence of a group by laterality (left, right hemispheres) interaction for the overall mean FA (and RD/L1) for the 8 bilateral WM tracts in BDE and CONT (FA:F [1,41] =0.1;p=0.732; RD:F [1,41] =0.2;p=0.669), we computed mean FA (or RD/L1) across left and right hemispheres for each of the 8 bilateral tracts, and entered these mean values, together with the values of the 2 interhemispheric fibers, forceps major and minor, into the same model (total of 10 WM tracts). 2(group)×10(tract) repeated measures analyses of variance (ANOVAs; one each for FA, RD, and L1) were used. Main effects of group (BDE, CONT),tract (10 tracts) and between-effect interaction were examined for FA, RD, and L1. We then performed post-hoc analyses of individual measures for testing our a priori approach (8 tracts of interest; 2 control tracts), for hypothesis generating and future metaanalytic work. We examined the main effect of group upon peripheral measures (LPH, 4-HNE), using a 2×2 repeated measures ANOVA.
To examine possible effects of age and gender on these a priori dependent measures, Pearson or Spearman coefficients were used, as appropriate, for each group, and across groups. Given the no significant effects of age and gender upon WM and peripheral measures (for statistical values, see footnotes of Tables 2A,2C and 3A), these variables were not entered as covariates in the ANOVA models.
To estimate the variance in the WM measures explained by the peripheral measures of lipid peroxidation across all study participants, multivariate multiple regression analyses were used, with the two peripheral measures as independent variables, and FA(or RD/L1) of the 10 WM tracts as 10 dependent variables. For any significant relationships between WM and peripheral measures, post hoc Spearman rank order correlational analyses determined the direction (positive or negative correlation) between individual tract FA (and RD/L1) and peripheral measures across all participants, and in each group separately, using a statistical threshold of p<0.005 (p<0.05/10), to control for the 10 parallel tests for individual tracts.
In BDE, to assess potential effects of medication on peripheral and WM measures, independent-sample t-tests were performed in BDE not taking vs. BDE taking each medication class (antidepressants, antipsychotics, mood stabilizers and anxiolytics). To assess the potential contribution of lifetime comorbid anxiety disorders and substance use/ dependence on peripheral and WM measures, independent sample t-tests were performed with absence vs. presence of history of comorbidity as between-subjects factor.

Demographic and Clinical Characteristics
There were no significant between-group differences in age, gender ratio, years of education, premorbid IQ, handedness. As expected, BDE had significantly more anxious (STAI), depressive(HDRS) and manic(YMRD) symptoms than CONT(Table1).
Between group-differences in FA and RD in the ten a priori WM tracts FA-There was a significant main effect of group (BDE<CONT:F [1,41]  L1-There were no significant between-group differences in L1 in any tracts (Supplemental Table2).

Between group-differences in peripheral measures of lipid peroxidation
There was a significant main effect of group (BDE>CONT:F [1,39]

Discussion
The major goal of the present study was to identify whether peripheral biomarkers of lipid peroxidation reflected alterations in WM in individuals with BD, as a primary step toward identification of clinically-relevant peripheral biomarkers. We demonstrated that BDE vs. CONT showed significantly greater RD and reduced FA overall, and specifically in the forceps minor, cingulum, superior longitudinal fasciculus and uncinate fasciculus, tracts connecting prefrontal cortex with other cortical and subcortical limbic regions that have previously been shown to be abnormal in BD. We also showed significantly greater LPH, but not 4-HNE, in BDE vs. CONT. Furthermore, across BDE and CONT, LPH explained 59% of the variance in FA, and 51% of the variance in RD in a priori WM tracts.
Myelin is a specific target for lipid peroxidation 30 . Lipid peroxidation of myelin has been shown in brain injury 42 , and spinal cord dysfunction 43 , and myelin isolated in vitro studies is subject to oxidative stress in a time and dose-dependent manner 44 . The forceps minor, an inter-hemispheric tract connecting left and right prefrontal cortices, showed the most consistent pattern of significantly reduced FA and elevated RD in BDE vs. CONT, and was the WM tract for which there were significant relationships among peripheral (LPH) and WM (FA, RD) measures, across all study participants. To a lesser extent, BDE showed significantly altered FA and RD in the cingulum and uncinate fasciculus, and there were trend-level relationships among LPH and FA and/or RD in these tracts across all study participants. Our significant findings in the forceps minor may reflect the proximity of this WM tract to the dopamine-rich ventral prefrontal cortical-striatal reward system, known to be dysfunctional in BD 45 , given that dopamine can be auto-oxidized in the brain to form quinones, that increase oxidative stress by inhibiting mitochondrial functioning 46 . There were no consistent patterns of abnormal WM integrity in our two control tracts in BDE, namely, the forceps major and the cortico-spinal tract, and no significant relationships among LPH and either FA or RD in these tracts across all participants. Together, these findings support our three main hypotheses, and suggest that LPH may be a peripheral measure of WM integrity, particularly of key tracts connecting prefrontal cortical with subcortical and other prefrontal cortical regions supporting reward and mood regulation 47 .
Our findings of significantly greater RD (and reduced FA) in the forceps minor, cingulum and uncinate fasciculus in BDE vs. CONT support previous findings of abnormalities in WM integrity in the genu of corpus callosum and in the (anterior) cingulum in region-ofinterest 14 , voxel-based 15 , tract-based spatial statistics 16 , and tractography-based [17][18] studies, and in studies of the uncinate fasciculus [18][19][20][21][22][23] in BD. Our findings of reduced FA in these tracts in BDE vs. CONT likely resulted from elevated RD, given that there were no between-group differences in L1 in these tracts. Given that elevated RD may be associated with myelin and axolemma abnormalities 25-28 , our findings may reflect myelin and axonal membrane abnormalities in these WM tracts in BDE.
BDE vs. CONT also showed significantly greater RD and reduced FA in the arcuate fasciculus of the superior longitudinal fasciculus, supporting previous findings in BD [18][19] . Unlike the forceps minor, cingulum, and uncinate fasciculus, there were no significant relationships between FA or RD in this tract and peripheral measures of lipid peroxidation in study participants. This may suggest that WM abnormalities in this fasciculus may reflect a more complex architecture, rather than abnormalities in myelin or axolemma components, in BD vs. CONT. The specific role of this tract in the pathophysiology of BD remains unclear.
In a recent meta-analysis, we 48 found that thiobarbituric acidic reactive substances (TBARS), a measure of late-stage lipid peroxidation, was significantly elevated in all phases of BD type-I. In the present study, we measured both LPH and 4-HNE, and found betweengroup differences only in LPH. Elevated early-stage measures of lipid peroxidation (i.e., LPH) can be removed from cells by antioxidant enzymes 31,49 , and increases in these enzymes, e.g., glutathione-S-transferase, are reported in individuals with BD 50 . Such increases in antioxidant systems are thought to be activated in BD in response to oxidative stress to brain lipids, given that reduced levels of total glutathione are observed in postmortem prefrontal cortex of BD subjects 51 . The activation of this antioxidant system may help prevent a rise in measures of late-stage lipid peroxidation, and may explain in part the absence of elevated 4-HNE in BDE in the present study. Future studies assessing other late-stage products of lipid peroxidation (e.g., 8-isoprostane and acrolein) and antioxidant enzyme levels can clarify this. Further focus on the mechanism through which oxidative damage to lipids may affect specific WM tracts in BD is clearly needed.
There were limitations to the present study. BDE were medicated, as is frequently the case to allow BDE to remain in remission 52 . Exploratory analyses revealed that BDE taking antidepressants had greater RD in a priori WM tracts than BDE not-taking antidepressants, although BDE with higher subsyndromal depression severity also had greater RD in the forceps minor and uncinate fasciculus. Furthermore, BDE taking higher dosage antidepressants had higher subsyndromal depression severity, suggesting that greater RD may be associated with greater depression severity, rather than with antidepressants, and parallel previous findings of greater mean diffusivity (an indirect index of RD) in depressed BD individuals vs. BDE 53 .
While significant relationships between LPH and FA/RD in the forceps minor, and trendlevel relationships between LPH and FA/RD in the cingulum(angular bundle), were observed in all participants, only CONT showed linear relationships between these measures in these tracts (and also in uncinate and inferior longitudinal fasciculi). Interindividual variability (e.g., in subsyndromal symptoms, treatments and response to treatment) in BDE may in part explain the loss of linear relationships between LPH and FA/RD in these tracts. Additional studies should examine relationships between WM and peripheral measures in young BDE in early stages of illness, and also between symptom severity, medication, and WM and peripheral measures in BDE, and whether other factors (e.g., diet 54 ) impact these measures. Future studies should also examine relationships between WM and peripheral measures in BD individuals across different mood states, and in individuals with other mood disorders, to determine whether these relationships represent persistent or mood statedependent features of BD, or BD-specific pathology versus dimensions of pathology across the mood disorders spectrum.
This is the first study to use a combination of DTI, to examine measures of WM integrity, and peripheral (serum) measures of lipid peroxidation in BDE and CONT. Our findings suggest that peripheral measures of early-stage lipid peroxidation are associated with underlying pathophysiologic processes involving WM abnormalities in key prefrontalsubcortical and prefrontal-prefrontal cortical tracts in BD that, in turn, may underlie the mood dysregulation that is characteristic of the illness. These findings offer insight into one of the first steps for biomarker development, the identification of a robust biological candidate. There are critical next steps to understand whether serum LPH is a potential biomarker, including replication and determination of the sensitivity, specificity and predictive value for the biomarker. Our findings suggest that examination of these peripheral measures is a promising way forward to help identify pathophysiologically-relevant, yet easily obtainable and inexpensive, peripheral biomarkers of BD.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

18
Significance level was set at p<0.005 (p<0.05/10), to control for the ten parallel tests for individual tracts. Trend-level significance was set between 0.05 and 0.005.
Given the overall non-normality of the data (Levene's test < 200), Spearman's coefficients were reported between LPH and FA (and RD) in BDE, CONT and across the whole sample.