ARTICLES
Neurometabolic Disorders: Potentially Treatable
Abnormalities in Patients With Treatment-Refractory
Depression and Suicidal Behavior
Lisa A. Pan, M.D., Petra Martin, B.S., Thomas Zimmer, B.S., Anna Maria Segreti, B.S., Sivan Kassiff, B.S.,
Brian W. McKain, R.N., M.S.N., Cynthia A. Baca, R.N., M.S.N., Manivel Rengasamy, M.D., Keith Hyland, Ph.D.,
Nicolette Walano, M.S., Robert Steinfeld, M.D., Marion Hughes, M.D., Steven K. Dobrowolski, Ph.D., Michele Pasquino, B.S.,
Rasim Diler, M.D., James Perel, Ph.D., David N. Finegold, M.D., David G. Peters, Ph.D., Robert K. Naviaux, M.D., Ph.D.,
David A. Brent, M.D., Jerry Vockley, M.D., Ph.D.
Objective: Treatment-refractory depression is a devastating
condition with signicant morbidity, mortality, and societal
cost. At least 15% of cases of major depressive disorder re-
main refractory to treatment. The authors previously iden-
tied a young adult with treatment-refractory depression
and multiple suicide attempts with an associated severe
deciency of CSF tetrahydrobiopterin, a critical cofactor
for monoamine neurotransmitter synthesis. Treatment with
sapropterin, a tetrahydrobiopterin analogue, led to dramatic
and long-lasting remission of depression. This sentinel case
led the authors to hypothesize that the incidence of met-
abolic abnormalities contributing to treatment-refractory
depression is underrecognized.
Method: The authors conducted a case-control, targeted,
metabolomic evaluation of 33 adolescent and young adult
patients with well-characterized histories of treatment-
refractory depression (at least three maximum-dose,
adequate-duration medication treatments), and 16 healthy
comparison subjects. Plasma, urine, and CSF metabolic
proling were performed by coupled gas chromatography/
Major depressive disorder is the second leading cause of
disability worldwide and affects an estimated 350 million
people (1). Although strides have been made in the treatment
of depression, an estimated 15% of depressed individuals do
not respond despite adequate pharmacotherapy, psycho-
therapy, and even ECT (2). Current predictors of treatment
response for depression, such as severity, chronicity, and
exposure to adverse life events, are nonspecic and without
clear therapeutic implications in treatment-refractory de-
pression. The clinical management of treatment-refractory
depression has not advanced signicantly in the past two
decades, and the suicide rate in the United States has been
increasing (3). Recent emphasis on treatment-refractory
depression has focused on neuromodulatory treatments,
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mass spectrometry and high-performance liquid chroma-
tography electrospray ionization tandem mass spectrometry.
Results: CSF metabolite abnormalities were identied in
21 of the 33 participants with treatment-refractory de-
pression. Cerebral folate deciency (N=12) was most
common, with normal serum folate levels and low CSF
5-methyltetrahydrofolate (5-MTHF) levels. All patients with
cerebral folate deciency, including one with low CSF levels
of 5-MTHF and tetrahydrobiopterin intermediates, showed
improvement in depression symptom inventories after
treatment with folinic acid; the patient with low tetrahy-
drobiopterin also received sapropterin. None of the healthy
comparison subjects had a metabolite abnormality.
Conclusions: Examination of metabolic disorders in treatment-
refractory depression identied an unexpectedly large pro-
portion of patients with potentially treatable abnormalities. The
etiology of these abnormalities remains to be determined.
AJP in Advance (doi: 10.1176/appi.ajp.2016.15111500)
such as deep brain stimulation, which, while promising,
requires neurosurgical intervention. We propose a novel
diagnostic and therapeutic approach to treatment-refractory
depression based on a targeted analysis of metabolites in
blood, urine, and CSF.
Metabolomics is the study of patterns of metabolic in-
termediates to characterize dysfunctional metabolic path-
ways potentially related to symptomatic presentation. This
approach has previously contributed to our understanding of
the pathophysiology of depression. For example, over three
decades ago, decreased CSF levels of 5-hydroxyindoleacetic
acid (5-HIAA) in depressed patients were shown to be as-
sociated with a markedly elevated risk of suicide (4). This
work helped spur the development of selective serotonin
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NEUROMETABOLIC DISORDERS IN TREATMENT-REFRACTORY DEPRESSION
reuptake inhibitors, which are now widely used for the
treatment of depression (5, 6). In addition, neuropsychiat-
ric symptoms are a common manifestation of many inborn
errors of metabolism. Adolescent and adult neuropsychiatric
symptoms, possibly caused by metabolic abnormalities, are
also a common comorbidity in both primary (7) and sec-
ondary forms of mitochondrial dysfunction (8).
We previously reported on the case of a 19-year-old male
patient with unremitting treatment-refractory depression
and repeated suicide attempts who had decient CSF levels
of tetrahydrobiopterin intermediates and responded to re-
placement with sapropterin, a tetrahydrobiopterin analogue
(9). CSF testing of ve additional adolescent patients with
treatment-refractory depression revealed that three had low
CSF levels of 5-methyltetrahydrofolate (5-MTHF) and nor-
mal blood levels of folate, indicating cerebral folate deciency
(10). None of the conventional clinical or research diagnostic
or therapeutic approaches would have identied the source
of these patients difculties, nor would they have suggested
the subsequent successful treatment strategies.
We hypothesized that the incidence of metabolic abnor-
malities contributing to treatment-refractory depression
would be signicantly greater than in healthy comparison
subjects. Broader identication of such metabolic disorders
could transform psychiatric practice, particularly in severe
depression, when standard treatments are ineffective. We
describe the systematic evaluation of 33 patients with
treatment-refractory depression for primary and secondary
disorders of CNS metabolism. Targeted studies of CSF and
peripheral samples (blood and urine) were utilized to identify
patients with abnormalities in the monoamine neurotrans-
mitter synthesis pathway, as well other metabolomic ab-
normalities leading to apparent isolated CNS dysfunction
with depression.
METHOD
Sample Characteristics
Participants 1440 years of age with depression that has not
responded to at least three maximum-dose medication trials
of at least 6 weeks each were recruited by advertisement
through the Clinical and Translational Science Institutes
Research Participant Registry at the University of Pittsburgh
or by clinical referral. All adult participants provided in-
formed consent; adolescent participants provided informed
assent for involvement in the study and parents provided
informed consent. We compared this group with young adult
healthy comparison subjects who had no personal or rst-
degree-relative history of psychiatric disorder or suicidal
behavior. Adolescent comparison subjects were not included
because lumbar puncture confers greater than minimal risk.
The study was approved by the Institutional Review Board of
the University of Pittsburgh.
A total of 38 patients with treatment-refractory depres-
sion and 22 healthy comparison subjects were enrolled.
One comparison subject was subsequently excluded after
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discovery of substance use disorder and suicidal behavior in
one of the participants parents and another after discovery of
self-administration of large quantities of B vitamins. One
participant in the depression group was excluded because of a
diagnosis of Aspergers syndrome. Four participants in the
depression group and four in the comparison group were
excluded because they declined lumbar puncture.
Participants were assessed with a structured psychiatric
interview, including the Family Interview for Genetics
Studies (11), at the time of referral to characterize depression
course, comorbidity, family history, and history of trauma,
psychosis, and anxiety. Treatment-refractory depression
was conrmed with the Antidepressant Treatment History
Questionnaire (12). All participants completed the Beck
Depression Inventory (BDI) (13) and the Suicidal Ideation
Questionnaire (14), and for adolescents, the Child Depression
Rating ScaleRevised (15) was completed on the basis of
clinical assessment. Patients remained on their current
medications and other treatments during the course of the
study.
Study Procedures
The research staff and principal investigator (L.P.) completed
structured psychiatric interviews, administered self-report
symptom questionnaires, and arranged for study laboratory
testing. Assessment consisted of a psychiatric interview,
review of records, and self-report instruments at intake to
characterize depression course (BDI), suicidal ideation and
behavior (Suicidal Ideation Questionnaire, Columbia Suicide
History Form [16], and Beck Suicide Ideation Scale [17]),
comorbidity (anxiety, psychosis, substance use, attention
disorders, assessed with the DSM-5 Level 1 Cross-Cutting
Symptom Measure), and family history (Family Interview for
Genetic Studies and three-generation pedigree) (11). A
neurologic examination was conducted by the principal
investigator.
Testing for CNS-specic metabolic abnormalities in-
cluded 5-methyltetrahydrofolate, tetrahydrobiopterin, neo-
pterin, pyridoxal-5-phosphate, 5-hydroxyindoleacetic acid,
homovanillic acid, and amino-adipic semialdehyde. Analysis
of blood and CSF began immediately after collection. Plasma
and urine testing were performed by the clinical biochemical
genetics and clinical chemistry laboratories of University
of Pittsburgh Medical Center (UPMC), per their standard
protocols, including gas chromatography-mass spectrometry
of urine and liquid chromatography tandem mass spec-
trometry and high-performance liquid chromatography
tandem mass spectrometry proling of blood to examine
groups of metabolites known to contribute to depression.
Lumbar puncture for CSF collection was performed under
uoroscopic guidance by the Neuroradiology Department
at UPMC, and samples were analyzed as for blood and urine
by the clinical biochemical genetics and clinical chemis-
try laboratories of UPMC and Medical Neurogenetics, Inc.
(Atlanta). If a specic abnormality was suspected on the basis
of the initial testing, the patient was referred to a biochemical
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 PAN ET AL.

geneticist for additional conrmatory testing. Once results of TABLE 1. Demographic Characteristics of Patients With
testing became available, a follow-up appointment was Treatment-Refractory Depression and Healthy Comparison
Subjects Assessed for Neurometabolic Disorders
scheduled for all participants in the depression group to
review results and provide additional referrals if needed. At Depression Group Comparison Group
Characteristic (N=33) (N=16)
this appointment, the BDI, the Suicidal Ideation Question-
naire, and the DSM-5 Level 1 Cross-Cutting Symptom Mean SD Mean SD
Measure were repeated. Participants for whom a novel Age (years) 26.21 7.63 26.12 6.11
treatment was available were recontacted at least 6 weeks
N % N %
after start of treatment to determine outcome with repeated
Female 25 76 9 56
administration of the BDI, the Suicidal Ideation Question-
Race
naire, and the DSM-5 Level 1 Cross-Cutting Symptom African American 1 3 2 13
Measure. Asian 0 0 4 25
Analyses were conducted in IBM SPSS Statistics, version Caucasian 30 91 9 56
21 (IBM, Armonk, N.Y.). We employed independent t tests to Multiple races 2 6 1 6
examine the differences between the depression and healthy Hispanic 2 6 0 0
comparison groups in depression severity (BDI), suicidal
ideation (Suicidal Ideation Questionnaire), and age. A chi-
square goodness-of-t test was used to compare the sex either of the two groups, with the exception of one adolescent
distribution of each group. Because of the small number of in the depression group who had a benign ne hand tremor.
participants who were assessed before and after intervention
(N=10), Wilcoxon signed-ranks tests were performed be- Metabolic Clinical Studies
tween pre- and posttreatment BDI and Suicidal Ideation We identied evidence of metabolite abnormalities in 21 of the
Questionnaire scores among the participants who were de- 33 participants in the depression group (Tables 3 and 4).
cient in cerebral folate. Cerebral folate deciency (CFD), a metabolic abnormality in
which the serum folate level is normal but the CSF 5-MTHF
level is low (,40 nmol/L), was the most common metabolic
RESULTS
abnormality identied, present in 12 participants (36%). One
The treatment-refractory depression group (N=33) and the participant with CFD also had low CSF levels of tetrahy-
healthy comparison group (N=16) were similar with respect drobiopterin metabolites, as seen in our previously published
to age, sex, and ethnicity (Table 1). case of a patient who responded to treatment with sapropterin
(9). Abnormalities were identied in nine additional partici-
Characterization of Depression pants: ve with an acylcarnitine prole abnormality, one with a
The characteristics of each of the 33 patients in the low guanidinoacetate level and a creatine/creatinine ratio
treatment-refractory depression group are summarized consistent with a creatine synthesis decit, one with Fabrys
in Table 2. Participants reported long-standing unremit- disease, one with a 20p12.1 microdeletion, and one with a
ting or recurrent depression, with a mean of 5.1 episodes 15q13.3 microduplication on microarray analysis.
(SD=15.98) and a mean longest duration of episode with-
out symptom remission of 453.9 weeks (SD=386.51). The Treatment Outcomes
mean age at onset of depressive symptoms in the de- Of the 12 patients with CFD, all were treated with folinic acid
pression group was 12.4 years (SD=5.05). The mean BDI (12 mg/kg per day) for at least 6 weeks (range 679 weeks),
score was much higher in the depression group than in while continuing their pre-evaluation treatment regimen.
the healthy comparison group (mean=29.2 [SD=8.4] com- Ten of the 12 patients showed reductions in symptom in-
pared with mean=0.6 [SD=1.2]; t=13.60, df=47, p,0.001). ventory scores at follow-up. One patient was lost to follow-up,
The depression group also had a much higher mean Sui- and one was nonadherent with the folinic acid treatment.
cidal Ideation Questionnaire score than the comparison Eight participants with CFD who completed follow-up did
group (mean=36.39 [SD=21.40] compared with mean=1.18 not have any comorbid biochemical ndings. Of these, four
[SD=1.08]; t=6.57, df=47, p,0.001). Half of the participants had reductions in BDI score to below the threshold for de-
in the depression group (N=17) reported a history of at least pression. Five of the eight patients met threshold scores for
one suicide attempt. suicidal ideation on the Suicidal Ideation Questionnaire at
On the Family Interview for Genetic Studies, 88% of par- intake, and three of these ve had reductions in suicidal
ticipants in the depression group had a positive family history ideation scores to below threshold after treatment. One pa-
in at least one rst-degree relative for any disorder, 82% for tient with CFD was also treated with sapropterin (20 mg/kg
depression, and 24% for suicide attempts. By denition, the per day) for low CSF tetrahydrobiopterin levels. FOLR1 gene
healthy comparison subjects had no personal or rst-degree- sequencing was conducted for eight of the 12 patients, and no
relative history of psychiatric disorders or suicidal behavior. sequence abnormalities were identied. In patients with
On neurologic examination, no abnormalities were noted in CFD, the mean BDI score decreased from 30.6 (SD=7.29) to

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NEUROMETABOLIC DISORDERS IN TREATMENT-REFRACTORY DEPRESSION

TABLE 2. Characterization of Depression Among Patients With Treatment-Refractory Depression Assessed for Neurometabolic
Disordersa
Age at Duration of
Onset of Longest Suicide Family History Family History
b
Depressed Depression Episodeb Attemptsb of Major of Bipolar Medication
b
Patient Episodes (N) (years) (weeks) (N) Comorbid Disorders Depression Disorderc Trialsd (N)
Cerebral folate deciency
1 2 15 600 1 PTSD, anxiety Yes Yes 6
2 1 9 468 1 Anxiety Yes No .10
3 2 12 468 5 PTSD, anxiety, ADHD Yes No 8
4 1 26 208 4 PTSD No Yes 4
5 .100 9 50 0 PTSD, ADHD, substance Yes Yes 9
use disorder
6 2 16 312 0 Substance use disorder Yes No 4
7 2 12 72 0 Anxiety No No 4
8 2 13 156 5 PTSD Yes No .10
9 1 20 28 1 None No No 3
10 1 4 1,300 3 PTSD, anxiety Yes No 9
11 1 11 362 0 PTSD Yes Yes 3
12 90 14 12 0 Substance use disorder No Yes 3
Other metabolic disorders
13 1 5 1,092 0 OCD, PTSD Yes No 4
14 2 5 468 0 Anxiety Yes No 5
15 1 12 1,352 10 PTSD, psychosis not Yes Yes .10
otherwise specied
16 2 13 364 0 Anxiety Yes No 6
17 .100 21 104 1 Anxiety No No .10
18 1 17 1,092 0 PTSD Yes Yes .10
19 1 13 364 1 Anxiety, ADHD Yes No 9
20 2 11 208 0 Anxiety, OCD No No 4
21 15 8 12 2 Anxiety Yes Yes 10
No metabolic disorder
22 6 9 468 1 PTSD, anxiety Yes No 6
23 1 12 884 2 None Yes Yes 8
24 5 9 728 0 None Yes No 7
25 1 23 572 0 Anxiety No No 10
26 2 6 364 0 None Yes No 8
27 1 10 416 2 Anxiety Yes No 6
28 1 13 364 4 PTSD, psychosis Yes No .10
29 1 14 1,042 0 PTSD, anxiety, substance Yes Yes 7
use disorder
30 1 7 780 0 None Yes No 4
31 4 13 36 1 Anxiety Yes No 4
32 1 12 208 1 Anxiety No No 5
33 5 16 24 0 None Yes No .10
a
For all patients, level of impairment on the Family Interview for Genetic Studies was rated 2 (incapacitated), except patient 29, for whom it was rated 1 (impaired).
ADHD=attention decit hyperactivity disorder; OCD=obsessive-compulsive disorder; PTSD=posttraumatic stress disorder.
b
Assessed from Family Interview for Genetic Studies reports.
c
Only four patients had a personal history of bipolar disorder: patients 5, 15, 17, and 18.
d
Five patients had also received ECT: patients 2, 4, 11, 16, and 28.

11.0 (SD=11.03) (Wilcoxon signed ranks test, Z=2.0, p,0.022).
The mean Suicidal Ideation Questionnaire score decreased
from 38.0 (SD=23.13) to 23.1 (SD=17.09), although the change
was not statistically signicant (Table 3, Figure 1).
DISCUSSION
To our knowledge, this CNS-specic metabolomic survey
is the rst to systematically evaluate abnormalities of neu-
rotransmitter, vitamin, pterin, and energy metabolism in
peripheral and CSF samples from patients with isolated
psychiatric symptoms in the absence of primary neuro-
logic symptoms. It was spurred by our initial identication
of a young man with a severe deciency of CSF tetrahy-
drobiopterin who responded to treatment with sapropterin,
followed by our discovery of CFD in three of ve additional
patients (9). In this case-control study of 33 individuals
with treatment-refractory depression, 21 had metabolite
abnormalities. Results were determined based on established
clinical norms for all tests reported.

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TABLE 3. Findings and Treatment Results for Patients With Treatment-Refractory Depression and Cerebral Folate Deciencya
Interval From
CSF Serum Initial Visit Posttreatment Visit Initial to
5-MTHF Folate Posttreatment
Level Level Psychiatric SIQ BDI Psychiatric SIQ BDI Self-Report
Patient Metabolic Findings (nmol/L) (ng/mL) Medications Score Score Medications Score Score (weeks)
1 Low CSF 5-MTHF 40 6.1 None 13 22 Folinic acid 8 15 6
2 Low CSF 5-MTHF 39 .20.0 Citalopram, 36 25 Folinic acid, 17 14 36
risperidone, amitriptyline,
amitriptyline, trazodone, 5-HTP,
trazodone carbidopa
3 Low CSF 5-MTHF 37 .20.0 Trazodone 61 29 Folinic acid, trazodone 45 29 8
4 Low CSF 5-MTHF 39 12.2 Zolpidem, 70 37 Folinic acid, zolpidem, 58 36 11
vilazodone vilazodone
5 Low CSF 5-MTHF 36 .20.0 Baclofen, 23 33 Folinic acid, baclofen, 18 17 20
gabapentin, gabapentin,
tramadol tramadol, lithium
carbonate,
dextroamphetamine
6 Low CSF 5-MTHF 15 .20.0 Fluoxetine 14 24 Folinic acid, 13 13 15
uoxetine
7 Low CSF 5-MTHF 14 18.3 Sertraline, 41 28 Folinic acid, sertraline, 20 16 7
carbidopa, carbidopa,
lamotrigine lamotrigine
8 Low CSF 5-MTHF 35 14.4 Sertraline 67 43 Folinic acid, sertraline 15 0 79
9 Low CSF 5-MTHF 37 .20.0 Bupropion 20 20 Lost to follow-up
10 Low CSF 5-MTHF 40 17.6 Lamotrigine, 53 31 Nonadherent with
escitalopram, folinic acid
clonazepam,
trazodone
11 Low CSF 5-MTHF, low 31 .20.0 Sertraline, 47 40 Folinic acid, 5-HTP, 33 35 52
tetrahydrobiopterin escitalopram, carbidopa,
lisdexamfetamine, sapropterin,
hydroxyzine lisdexamfetamine,
hydroxyzine
12 Low CSF 5-MTHF, 36 .20.0 None 8 25 Folinic acid 4 21 10
abnormal
acylcarnitine
prole
a
5-HTP=5-hydroxytryptophan; 5-MTHF=5-methyltetrahydrofolate; BDI=Beck Depression Inventory; SIQ=Suicide Ideation Questionnaire.

Twelve patients had CFD, and in 10 of these patients BDI
and Suicidal Ideation Questionnaire scores were reduced
after treatment with folinic acid; of the remaining two pa-
tients, one was nonadherent with folinic acid treatment and
the other was lost to follow-up. Half of the eight patients
who completed follow-up and did not have a comorbid
identied metabolic abnormality had reductions in BDI
score to below the threshold for depression. In all cases,
patients had normal serum metabolites associated with
folate pathways and therefore would have eluded conven-
tional diagnostic approaches without a more comprehen-
sive evaluation.
It should be noted that the abnormalities identied may
be due either to a primary genetic disorder in a metabolic
pathway or a secondary abnormality of indirect etiology
(e.g., injury to the CNS from infection, autoimmune dis-
ease, asphyxia, or epilepsy), typically with other obvious
systemic manifestations (18). The abnormalities identied
could also be sequelae of unremitting depression. CFD
is a CNS-specic syndrome characterized by low CSF
5-MTHF levels and normal plasma folate levels (19, 20). It
can be caused primarily by mutations in the cerebral fo-
late receptor gene (FOLR1) but can be secondarily re-
duced in any metabolic defect that increases use of methyl
groups, or by changes in transport across the blood-brain
barrier. FOLR1 gene sequencing was normal in the rst
eight patients with identied CFD. Secondary CFD is also
seen in disorders of the mitochondrial respiratory chain,
such as Alpers syndrome (21), Kearns-Sayre syndrome
(22), and others (23). Folate is involved in nearly 100
metabolic reactions (24), including the purine synthetic
pathway, contributing to impaired tetrahydrobiopterin,
serotonin, norepinephrine, and dopamine synthesis, and
thus polygenic effects due to the accumulation of muta-
tions in multiple genes may play a role in this population
(25, 26).
The ndings in this patient population are different from
those in reports of empirical treatment with L-methylfolate
(2730). Adjunctive treatment with L-methylfolate may im-
prove depressive symptoms because of its role as a cofactor

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NEUROMETABOLIC DISORDERS IN TREATMENT-REFRACTORY DEPRESSION

TABLE 4. Findings for Patients With Treatment-Refractory Depression Who Had Metabolic Disorders Other Than Cerebral Folate
Deciency or Who Had No Metabolic Disordera
Initial Visit
CSF 5-MTHF
Level Serum Folate SIQ BDI
Patient Metabolic Findings (nmol/L) Level (ng/mL) Psychiatric Medications Score Score

Other metabolic disorders

13 Abnormal acylcarnitine prole 68 17.6 Citalopram, melatonin 53 37
14 Abnormal creatine/guanidinoacetate 55 13.4 Aripiprazole, diazepam, methylphenidate, 15 38
ratio lamotrigine, bupropion
15 Abnormal acylcarnitine prole 60 .20.0 Lamotrigine, clonazepam, uoxetine 33 35
16 Abnormal acylcarnitine prole 109 19.3 Levothyroxine sodium, lorazepam, 36 29
sertraline, lurasidone, hydroxyzine
17 Fabrys disease 77 14.5 Lithium carbonate, trazodone 15 19
18 Abnormal acylcarnitine prole 92 10.2 Gabapentin, lithium carbonate, 32 32
lamotrigine, clonazepam, olanzapine
19 Abnormal acylcarnitine prole, 59 .20.0 Nortriptyline, sertraline 72 32
abnormal creatine/
guanidinoacetate ratio
20 20p12.1 microdeletion on 67 .20.0 Venlafaxine, uvoxamine 22 24
chromosome analysis
21 15q13.3 microduplication on 45 16.6 Desvenlafaxine, divalproex sodium, 17 26
chromosome analysis aripiprazole

No metabolic disorder
22 60 17.5 Imipramine, venlafaxine, quetiapine 37 41
fumarate
23 46 .20.0 Sertraline, quetiapine fumarate 59 33
24 54 .20.0 Amitriptyline, citalopram, levothyroxine 12 12
sodium
25 58 9.8 Bupropion, vilazodone, temazepam, 11 19
clonazepam
26 49 10.3 Lamotrigine, melatonin 22 25
27 52 17.6 None 41 22
28 59 .20.0 Clozapine 88 49
29 53 .20.0 Venlafaxine 11 17
30 59 18.6 Clonazepam 55 39
31 51 .20.0 None 20 22
32 78 .20.0 Fluoxetine, lamotrigine, mirtazapine 48 24
33 48 .20.0 Lisdexamfetamine, trazodone, 49 30
levothyroxine sodium
a
5-MTHF=5-methyltetrahydrofolate; BDI=Beck Depression Inventory; SIQ=Suicide Ideation Questionnaire.

in monoamine metabolism (31). This is different from our
ndings in CFD (18). Also, L-methylfolate is most helpful
in those with the MTHFR polymorphism, which was neg-
ative in the eight patients with CFD in whom we conducted
gene testing. We continue to work to understand the bi-
ologic mechanism of our biochemical ndings. It is clear
that treatment with L-methylfolate is not sufcient in these
individuals, because of a decit earlier in the folate meta-
bolic pathway that produces bioactive metabolites. Fur-
thermore, the administration of L-methylfolate will falsely
correct the CSF test to identify these individuals. While
empirical treatment with folinic acid may seem appealing,
this again is problematic with our current understanding.
After folinic acid administration, denitive testing cannot
be conducted, and the full effects of treatment with folinic
acid in an individual with CFD occur over years. This time
frame is due to neuronal turnover and growth in the set-
ting of availability of active folate metabolites needed for
appropriate white matter structure. We cannot determine
whether the metabolite abnormalities are primary or sec-
ondary. However, our patients had no other overt clini-
cal disease, and thus secondary deciencies related to
other systemic conditions are unlikely. Applications of next-
generation sequencing technologies (i.e., whole exome or
genome sequencing) on a focused, homogeneous patient
population with treatment-refractory depression will pro-
vide further insight into genetic components of the meta-
bolic ndings in our patients. After further evaluation and
characterization of a larger population of patients, animal
models of biochemical abnormalities will be an important
next step.
Although the majority of patients in this study were
recruited by advertisement through a research registry,
four participants were clinically referred, and the possi-
bility of an ascertainment bias cannot be excluded. This
was a relatively small sample of mostly Caucasian patients,

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FIGURE 1. Symptom Inventory Outcomes in Patients With Treatment-Refractory Depression and Cerebral Folate Deciency (CFD), Before
and After Treatment With Folinic Acid (N=10)a
Suicide Ideation Questionnaire Beck Depression Inventory
80 50

45
70

Patient 1 40
60 Patient 2
35
Patient 3
50 Patient 4
30
Patient 5

Score
Score

Patient 6 25
40
Patient 7
Patient 8 20
30
Patient 11 (CFD and low
tetrahydrobiopterin) 15
20 Patient 12 (CFD and
abnormal acylcarnitine) 10

10
5

0 0
Baseline Follow-up Baseline Follow-up
a
Follow-up interval ranged from 6 to 79 weeks. The mean score on the Suicide Ideation Questionnaire was 38.0 (SD=23.13) at baseline and
23.1 (SD=17.09) at follow-up (Z=1.24, p=0.107). The mean score on the Beck Depression Inventory was 30.6 (SD=7.3) at baseline and 19.6 (SD=11.04)
at follow-up (Z=2.0, p=0.022).

and the treatment component of the study was an open
trial. Continued treatment as usual resulted in medica-
tion changes during the course of the study, meaning that
in three cases of CFD, there were concomitant medica-
tion changes in addition to folinic acid supplementa-
tion. Preliminary experience with treatment has been
promising; however, long-term outcomes remain to be
determined.
CSF testing is required to determine the presence of
CFD and need for treatment. Review of the CSF meta-
bolic prole directs the diagnosis and avoids missing other
metabolic disorders that may be present. L -Methylfolate
(5-L-methyltetrahydrofolate) is the treatment specic
to methylenetetrahydrofolate deciency (32), but in the
majority of cases, folinic acid is preferable because of
its superior stability and lower costs when compared
with L-methylfolate. Dihydrofolate reductase converts
folic acid to dihydrofolate and then to tetrahydrofolate
(Figure 2). Folinic acid (5-formyl-tetrahydrofolate) is
converted to 5-,10-methenyltetrahydrofolate, then 5-,10-
methylenetetrahydrofolate, and eventually to 5-MTHF.
Folinic acid therefore allows the availability of active
metabolites in the folate pathway in individuals with CFD
that is not caused by methylenetetrahydrofolate de-
ciency. Furthermore, folinic acid has therapeutic effect
in the setting of folate receptor autoantibodies, although
there is controversy surrounding the assessment of these
autoantibodies (33).
Folinic acid should be given by prescription. Dos-
ages of 12 mg/kg per day may be administered with
prescription-strength folinic acid. Treatment with either
folinic acid or L-methylfolate will preclude testing after
administration because they correct decient CSF 5-MTHF
levels. A clinical response should be expected in patients
with treatment-refractory depression and low CSF 5-MTHF
levels. Experience with more severe neurologic disorder
associated with CFD (34) indicates that the response may
take several months, but the full effect of treatment may
take 13 years. This may be related to neuronal growth and
turnover in an environment in which folate metabolites are
newly available. Folic acid is not recommended because of the
potentially low activity of dihydrofolate reductase in CFD and
because of its tight binding to folate receptor alpha, which
results in reduced cerebral transport of 5-MTHF. Folinic
acid treatment in our patients did have some side effects
(facial ushing and initial anxiety). It is important to note
that vitamin B12 acts as a cofactor in this pathway. Blood
folate and B12 levels should be evaluated in all patients with
treatment-refractory depression. The best option for patients
with treatment-refractory depression for whom metabolic
disorder is suspected is consultation with a biochemical
geneticist.
Our ndings indicate a need for further evaluation of
the role of neurometabolic abnormalities in treatment-
refractory depression. The remarkably high incidence
of actionable abnormalities and some evidence of symp-
tom improvement with treatment strongly support the
need for larger studies. Historically, a diagnosis of a
metabolic disorder has been considered in the context of
a family history of a known disorder or symptoms that

ajp in Advance ajp.psychiatryonline.org 7

NEUROMETABOLIC DISORDERS IN TREATMENT-REFRACTORY DEPRESSION

FIGURE 2. Summary of the Folate Pathwaya

Folate

Folinic Acid DHFR

(5-Formyl-THF)
Formate + THF
Dihydrofolate NADH
NAD+
DHFR

10-Formyl-THF
5,10-Methenyl-THF

NADP+

NADP+ NADPH Purine nucleotides

NADPH

5,10-Methylene-THF dUMP
B2

MTHFR Glycine
(Methylene-THF
reductase)
dTMP
B6
5-Methyl-THF DHF

Serine Pyrimidine nucleotides

THF NADPH
Transport B12
across intestine
and choroid
plexus Homocysteine Methionine + THF

a
Folinic acid (5-formyl-THF) enters the pathway without the action of DHFR and contributes active metabolites. 5-Methyl-THF is a distal metabolite.
DHFR=dihydrofolate reductase; DHF=dihydrofolate; dTMP=deoxy-thymidine phosphate; dUMP=deoxy-uridine phosphate; NAD=nicotinamide
adenine dinucleotide; NADP=nicotinamide adenine dinucleotide phosphate; NADPH=nicotinamide adenine dinucleotide phosphate, reduced;
THF=tetrahydrofolate.

are exacerbated by signicant physiologic stresses (e.g.,
fever, fasting, surgeries), especially with multisystem in-
volvement (35), neither of which was present in our pa-
tient population. Our ndings, if replicated, suggest that
neurometabolic disorders may contribute to treatment-
refractory psychiatric disorders even without other sys-
temic illness. An important question is whether early
identication and treatment of an underlying metabolic
abnormality early in the course of psychiatric illness could
prevent long-term emotional and cognitive complications.
If these ndings are replicated, they suggest that the iden-
tication of new inborn errors of metabolism or second-
ary disorders of metabolism contributing to psychiatric
illness may allow repurposing of currently approved
orphan drugs.
AUTHOR AND ARTICLE INFORMATION
From the Departments of Psychiatry, Pediatrics, Human Genetics,
Pathology, and Obstetrics and Gynecology, University of Pittsburgh,
School of Medicine, Pittsburgh; Medical Neurogenetics Laboratory,
Atlanta; the Department of Pediatrics, University Medical Center
Gttingen, Gttingen, Germany; and the Departments of Medicine,
Pediatrics, and Pathology, School of Medicine, University of California,
San Diego.
Address correspondence to Dr. Pan (thomasla@upmc.edu).
Supported by the Brain and Behavior Research Foundation NARSAD
Young Investigator Award; and by the Clinical and Translational Science
Institute, University of Pittsburgh, through NIH grants UL1 RR024153 and
UL1 TR000005. Ongoing research is supported by the American
Foundation for Suicide Prevention Standard Research Award, the
Childrens Hospital of Pittsburgh Foundation, and departmental funding
supported by David A. Brent, M.D., Jerry Vockley, M.D., Ph.D., and David
A. Lewis, M.D.
The authors thank the participants who made this research possible, and
they thank Katherine Wisner, M.D., and Vishwajit Nimgaonkar, M.D., Ph.D.,
for their review of the manuscript.
Dr. Hyland is executive vice president of Medical Neurogenetics Labo-
ratories. Dr. Finegold is founder of Diavacs and Personalized Genomics
Laboratories. Dr. Brent receives research support from NIMH, royalties
from Guilford Press, royalties from the electronic self-rated version of the
Columbia Suicide Severity Rating Scale from ERT, Inc., honoraria for
performing duties as an UpToDate Psychiatry section editor, and con-
sulting fees from Healthwise. The other authors report no nancial re-
lationships with commercial interests.
Received Nov. 29, 2015; revisions received March 11 and May 8, 2016;
accepted May 20, 2016.

8 ajp.psychiatryonline.org ajp in Advance


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