We compared metabolites from the tryptophan, tryrosine, purine and methionine pathways between rMDD and dMDD subjects and healthy controls using the LCECA platform. This approach detects a subset of the metabolome, namely compounds amenable to oxidation-reduction, with greater sensitivity for studying monoamine neurotransmitter pathways than can be achieved using other technologies. Since cellular metabolism reflects the integrated interconversion of metabolic substrates through enzyme-catalyzed biochemical reactions, an alteration in the enzyme function at one reaction can affect the fluxes of one or several subsequent reactions28
. In this CSF metabolite pathway analysis we observed that the levels of several metabolites within the investigated pathways were altered in unmedicated-remitted individuals with MDD. In contrast, unmedicated-depressed individuals did not exhibit statistically significant perturbations within the same metabolic branches, consistent with the results of most previous studies on 5-HIAA, HVA, and purine metabolites in depressed individuals selected according to similar diagnostic criteria.
The counterintuitive findings that the CSF 5-HIAA levels and the 5-HIAA/TRYP ratio were significantly lower in the rMDD versus the dMDD subjects conceivably may reflect greater biochemical heterogeneity within the dMDD group. One source of this heterogeneity may arise from variation in symptom severity, as suggested by the associations observed between the tryptophan, tyrosine, and purine pathway metabolites and depression or anxiety ratings. For example, Nordin29
found that the relationship between 5-HIAA concentrations and depressive symptoms in MDD was curvilinear, such that individuals with the lowest and highest 5-HIAA levels had the lowest depression ratings. Our data appear compatible with this finding, since most of the currently depressed subjects we studied had depression ratings in the moderate severity range. Moreover, the observation that anxiety ratings correlated positively
with 5-HIAA levels but negatively
with TRYP levels suggests that the greater anxiety symptoms associated with active depressive illness may contribute to the higher 5-HIAA levels and the higher 5-HIAA/ TRYP ratio in the currently ill versus the currently remitted MDD samples. In preclinical studies of anxiety, rats exposed to stressors or threats show adaptive increases in serotonin turnover in several brain structures30
, which putatively would increase the CSF 5-HIAA levels. Thus the anxiety symptoms associated with depressive episodes may result in an elevation of 5-HT turnover, potentially obscuring a basal deficit in 5-HIAA levels that is evident during remission.
Another potential explanation for the more prominent difference in the remitted sample is that the entrance criteria used to select the rMDD subjects yielded a more homogenous subphenotype of MDD. The rMDD subjects were characterized by manifesting an episodic course (i.e., episodes of illness separated by epochs of remission) and the ability to remain in remission while unmedicated. In contrast, while the depressed sample included some subjects with an episodic course, most of the dMDD subjects manifested chronic illness. Thus, one interpretation of the marked reduction in 5-HIAA and HVA levels in the rMDD subjects is that the entrance criteria for this sample identifies a subphenotype that is more likely to maintain euthymia because of having a lower dopamine and serotonin turnover rate, potentially due to genetic variation that results in slower metabolism of these neurotransmitters (so that their availability within the synapse is prolonged). Compatible with such a hypothesis, drugs that achieve such a condition through monoamine oxidase inhibition exert potent antidepressant effects. Thus the persistently remitted group may manifest biological differences that confer resilience and prevent illness chronicity in the course of MDD. Nevertheless, the cross-sectional design of our study did not allow us to distinguish whether these CSF abnormalities reflect trait-like abnormalities or compensatory changes arising in response to past illness.
In either case the findings in remitted MDD subjects appear compatible with other evidence that, despite symptom remission, such individuals show behavioral and biochemical deficits relative to never-depressed controls. Clinically, unmedicated-remitted MDD subjects show a greater risk of depressive relapse, higher rates of suicide attempts and ideation, lower pleasure ratings and impaired socio-occupational functioning relative to controls31
. Such individuals also show neurobiological abnormalities that in some cases resemble32
, but in other cases contrast with, those shown by their currently-depressed counterparts. For example, under acute tryptophan depletion or catecholamine depletion, unmedicated rMDD subjects redevelop depressive symptoms, neuropsychological deficits, and neurophysiological abnormalities characteristically seen in the depressed phase33,34,35
, while unmedicated-depressed
MDD subjects show no exacerbation of depressive symptoms under these challenges36,37
. These findings suggest that alterations in the tryptophan or tyrosine pathways limit the capacity to remain in remission in rMDD subjects, consistent with their abnormal reductions in CSF 5-HIAA and HVA levels shown herein.
The cross-sectional design also did not allow us to exclude the possibility that persistent effects of previous treatment influenced our findings, since only a minority of the subjects was treatment-naïve. Nevertheless, while several studies reported decreased CSF concentrations of 5-HIAA following acute or subacute administration of selective serotonin reuptake inhibitors (SSRIs)38,39,40,41,42
, after chronic SSRI treatment the CSF 5-HIAA levels return to the pre-treatment baseline43
. Similarly, HVA levels are not changed significantly by chronic SSRI treatment43
. Thus our findings more likely relate to the pathophysiology of MDD, and as such may underlie predisposing factors or adaptive responses to illness, as opposed to homeostatic mechanisms arising in response to past treatment44
. For example, the abnormal reduction in CSF 5-HIAA levels in the rMDD sample is consistent with evidence that MDD is associated with neuropathological involvement of brainstem serotonergic nuclei45
, and that maintenance treatment with drugs that increase 5-HT transmission reduce the risk of illness recurrence in rMDD patients46,47
Our findings in the rMDD sample appear compatible with those of previous studies that measured CSF 5-HIAA in isolation from the remainder of the tryptophan pathway. van Praag and de Hann48
reported that 33 of 54 depressed individuals with low post
probenecid accumulation of CSF 5-HIAA also showed this low accumulation after recovery. Furthermore, Vestergaard et al.27
reported that CSF 5-HIAA and HVA levels were significantly lower following recovery than during depression in 16 mood disordered subjects, although the differences in the 5-HIAA levels with respect to healthy controls did not reach significance in either the depressed or the recovered phases. Our data thus were the first to document that remitted MDD subjects show significantly reduced mean 5-HIAA levels relative to controls in the absence of probenicid challenge.
The significantly smaller 5HIAA/TRP and 5HIAA/KYN ratios in the rMDD group versus the dMDD and HC groups are noteworthy, in light of evidence that the pathophysiology of MDD involves activation of pro-inflammatory pathways49
. Activation of the KYN pathway is initiated by pro-inflammatory cytokines via induction of the enzyme, indoleamine 2,3-dioxygenase (IDO), which metabolizes TRP into KYN. Activation of the KYN pathway is hypothesized to contribute to reductions in 5-HT synthesis in MDD and in individuals who develop depressive symptoms during cytokine administration50,51
. Potentially compatible with this hypothesis, the reduced 5HIAA/KYN ratio in the rMDD sample suggests that more TRP is being shunted toward the KYN pathway than toward 5-HT synthesis. These findings thus appear compatible with evidence that a pro-inflammatory state exists in remitted MDD subjects, despite their recovery from depressive symptoms52
Within the tyrosine pathway, the rMDD group showed significantly higher TYR/4HPLA ratios, lower TYRA levels, and smaller HVA/MHPG and HVA/TYR ratios versus the HC group. This combination of findings suggests that of the TYR routed toward catecholamine synthesis, relatively more is being routed toward norepinephrine utilization than toward dopamine utilization (). Given the role of dopamine in reward processing, these data together with the abnormal reduction in HVA levels in the rMDD group appear compatible with evidence that hypohedonia (diminished ability to enjoy pleasurable activities) is a trait-like marker of MDD53
. In addition, the quartile data () suggested that a subset of the currently depressed subjects also manifested reduced HVA levels, consistent with previous reports that subjects with the melancholic subtype of MDD show reductions in the CSF HVA concentration (see Introduction).
Our data further suggest that a balance between dopaminergic and serotonergic function may be relevant to the switch between depression and remission. Previously Roy et al.54
reported that the CSF HVA-to-5-HIAA ratio was lower in depressed MDD subjects than in controls. In our study the significant difference in the HVA/5HIAA ratio across groups () was attributable to a lower ratio in the dMDD subjects versus the HC and rMDD subjects (consistent with Roy et al.54
). These data imply that the ratio of dopamine turnover to serotonin turnover is lower in dMDD than in rMDD cases, raising the possibility that imbalances between 5-HT and DA neurotransmission contribute to the development of depressive symptoms
. Notably the literature characterizing interactions between dopaminergic and serotonergic neurotransmission has emphasized functionally antagonistic relationships between these systems55
The MET concentration was higher and the GSH/MET ratio was lower in the rMDD group than in the HC and dMDD groups. These data conceivably reflect a reduction in the conversion rate of MET to GSH, possibly implicating a defect in 1-carbon metabolism in MDD. Methionine plays a critical role in protein synthesis, methylation, and polyamine biosynthesis, and its derivative S-adenosyl methionine (SAM) serves as a methyl donor for several processes including DNA methylation. A role for DNA methylation in mood regulation is suggested by the antidepressant effect of S-adenosyl methionine56,57,58
, the post mortem
evidence that DNA methylation of genes involved in catecholamine metabolism are altered in mood disorders59,60,61
, and preclinical evidence that epigenetic processes underlie both the resilience against and the vulnerability toward the development of depressive behaviors under stress62
. Our data raise the possibility that higher MET concentrations support the maintenance of symptom remission in MDD58
. Conversely, the reduction in GSH levels observed in the remitted sample conceivably may confer the vulnerability of the rMDD population to illness recurrence. Glutathione is the major antioxidant in the brain, and thus plays a key role in defending against oxidative damage. Glutathione is thought to play an important role in the neuroprotective effects of mood stabilizing drugs63
, given the accumulating evidence for oxidative stress mechanisms as common pathophysiological pathways in mood disorders64,65,66,67
. We previously reported10
that glycine levels differed significantly between remitters and non-remitters to SSRI treatment, and proposed that glycine's link to folate and methionine cycles and methylation processes might play a role in recovery from the depressed state.
Although none of the purine metabolites showed significant effects on their own, some ratios involving the purine metabolites and the metabolites within other pathways differed across groups (i.e., XAN/HVA and 5-HIAA/XAN). Previous studies reported strong positive correlations between CSF levels of xanthine and hypoxanthine with corresponding levels of HVA and 5HIAA, despite finding no significant difference in the mean concentrations of these purine metabolites between depressives and controls68,69
. The strong correlation in the lumbar CSF pool between HVA and XAN across the purine and tyrosine pathways has been observed in previous studies70
. This cross pathway correlation appears to extend the relationship between XAN and tyrosine/tryptophan evident in the brain and the ventricular CSF to the lumbar CSF, as the end product metabolites of tyrosine and tryptophan resulting from spinal cord metabolism. The purine and tyrosine pathways also share the pterin compounds as common cofactors, potentially contributing both to the similarities in the direction of correlation and to the differences in the ratio obtained across the rMDD group and the other two groups.
This study provides new insights about the metabolic state associated with remission from MDD, and distinguishes this state from the normative metabolic state. The relatively small sample size in our study limited the statistical sensitivity to detect other potentially meaningful differences between groups. The lack of difference between the dMDD and HC groups may reflect both the limited statistical power and the greater biochemical heterogeneity seen among chronically depressed subjects. Moreover, greater coverage of the metabolome using complementary platforms may reveal perturbations in dMDD that were not detected in our study. Finally, correlations between biochemical changes in the CSF with those observed in plasma might enable the connection of central and peripheral changes.
In conclusion, the correlation of metabolites within and across the tryptophan, tyrosine and methionine pathways revealed differences in remitted MDD subjects which suggest alterations in the regulatory feedback within these pathways. Our data thus demonstrate that metabolomics approaches hold the potential to elucidate failures in the regulation of specific biochemical pathways and networks in mood disorders. The differentiation of whether these abnormalities reflect consequences of prior depressive episodes or vulnerabilities toward the development of future mood episodes awaits longitudinal studies in at-risk samples that assess the function of these pathways before and following illness-onset. Furthermore, the biochemical heterogeneity extant within the depressed MDD population suggests that future studies should characterize sufficiently large samples that subphenotypes can be identified which manifest more homogenous metabolomic signatures (potentially analogous to those found in the rMDD sample).