Author’s Accepted Manuscript

A preliminary study of bipolar disorder type I by

mass spectrometry-based serum lipidomics

Henrique Caracho Ribeiro, Aline Klassen, Mariana

Pedrini, Michelle S. Carvalho, Lucas B. Rizzo,
Mariane N. Noto, Maiara Zeni-Graiff, Sumit Sethi,
Ljubica Tasic, Mirian A.F. Hayashi, Quirino
Cordeiro, Elisa Brietzke, Alessandra Sussulini www.elsevier.com/locate/psychres

PII: S0165-1781(16)32015-7
DOI: http://dx.doi.org/10.1016/j.psychres.2017.08.039
Reference: PSY10755
To appear in: Psychiatry Research
Received date: 7 December 2016
Revised date: 20 July 2017
Accepted date: 16 August 2017
Cite this article as: Henrique Caracho Ribeiro, Aline Klassen, Mariana Pedrini,
Michelle S. Carvalho, Lucas B. Rizzo, Mariane N. Noto, Maiara Zeni-Graiff,
Sumit Sethi, Ljubica Tasic, Mirian A.F. Hayashi, Quirino Cordeiro, Elisa
Brietzke and Alessandra Sussulini, A preliminary study of bipolar disorder type I
by mass spectrometry-based serum lipidomics, Psychiatry Research,
http://dx.doi.org/10.1016/j.psychres.2017.08.039
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 A preliminary study of bipolar disorder type I by mass
spectrometry-based serum lipidomics

Henrique Caracho Ribeiroa, Aline Klassenb, Mariana Pedrinic,

Michelle S. Carvalhod, Lucas B. Rizzoc, Mariane N. Notoc, Maiara Zeni-
Graiffc, Sumit Sethic, Ljubica Tasice, Mirian A. F. Hayashid, Quirino
Cordeirof, Elisa Brietzkec*, Alessandra Sussulinia*

a
Laboratory of Bioanalytics and Integrated Omics (LaBIOmics), Department of Analytical
Chemistry, Institute of Chemistry, University of Campinas (UNICAMP), P.O. Box 6154, 13083-
970, Campinas, SP, Brazil
b
Department of Chemistry, Federal University of São Paulo (UNIFESP), Diadema, SP, Brazil

c
Research Group in Behavioral and Molecular Neuroscience of Bipolar Disorder, Department
of Psychiatry, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil

d
Department of Pharmacology, Federal University of São Paulo (UNIFESP), São Paulo, SP,
Brazil

e
Chemical Biology Laboratory, Department of Organic Chemistry, Institute of Chemistry,
University of Campinas (UNICAMP), P.O. Box 6154, 13083-970, Campinas, SP, Brazil

f
Department of Psychiatry, Santa Casa de São Paulo, School of Medical Sciences, São Paulo,
SP, Brazil

*Corresponding authors

E-mails: sussulini@iqm.unicamp.br (AS) and elisabrietzke@hotmail.com (EB)

Abstract

The present study aimed at investigating possible alterations in the

serum lipid profile of euthymic patients with bipolar disorder type I (BD)
compared to healthy controls (HC). Thirty-five individuals from both
genders were recruited, with 14 diagnosed and treated as BD patients (BD
group) and 21 healthy subjects (HC group). Clinical assessment was based
on the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-
I), Young Mania Rating Scale (YMRS), and 17-items of Hamilton
Depression Rating Scale (HDRS-17) data, which were used to confirm
diagnosis, to verify psychiatric comorbidities, and to estimate the severity
of manic and depressive symptoms. Ultra-high performance liquid
chromatography (UHPLC) coupled to high resolution mass spectrometry
(HRMS) was applied to analyze the lipids extracted from all serum samples
from both studied groups. In this pioneer and exploratory study, we
observed different serum lipid profiles for BD and HC groups, especially
regarding glycerophospholipid, glycerolipid, and sphingolipid distribution.
Multivariate statistical analyses indicated that 121 lipids were significantly
different between BD and HC. Phosphatidylinositols were identified as the
most altered lipids in BD patient sera. The results of this preliminary study
reinforce the role of lipid abnormalities in BD and offer additional
methodological possibilities for investigation in the field.

Keywords: Metabolomics; Lipids; Biomarkers; Mood disorders; Ultra-

high performance liquid chromatography; ESI-QTOF MS; Untargeted lipid
analysis; Phosphatidylinositol; Glycerophospholipid; Blood serum samples.
1. Introduction

Although significant progress in understanding the pathophysiology

of bipolar disorder (BD) has been reached in the last two decades, these
achievements are still not translated into tools that could support diagnosis
in clinical practice (Phillips and Kupfer, 2013). It has been well established
that BD misdiagnosis increases the risk for patients experience a myriad of
social, personal and occupational impairments, including alcohol and other
substances abuse, suicidal behavior, exposure to isolated antidepressants,
and evolution across clinical stages (Berk et al., 2014; Hirschfeld and
Vornik, 2004; Nasrallah, 2015; Passos et al., 2016). Therefore, the search
for possible biomarkers that are able to increase diagnosis accuracy is
required (Kapczinski et al., 2009). Characteristics such as the association
with functional outcomes, low cost, efficiency and preferably non-invasive
measurements are highly desirable for good biomarkers candidates
(Paulsen, 2013; Scarr et al., 2015).

At the moment, Psychiatry is witnessing the introduction of several

new platforms for measuring potential biomarkers, and we can expect that,
in the near future, a considerable amount of data could be combined in
predictive models to assist BD diagnosis (Brietzke et al., 2012; McIntyre et
al., 2014; Scarr et al., 2015). Amongst the most employed platforms are
proteomics, genomics and metabolomics (Hagenbeek et al., 2016; Martins-
de-Souza, 2014; Tasic et al., 2017; West et al., 2014). Due to a large
number of lipid subclasses and their presence in the metabolome,
lipidomics represent a promising and still underexplored omics
methodology in the study of mood disorders pathophysiology (Fahy et al.,
2009; Harkewicz and Dennis, 2011; Hyötyläinen and Orešič, 2014).

Alterations in the lipid metabolism related to BD have been

described in the literature. Different authors reported changes in the
arachidonic acid metabolism in the brain and periphery (Hamazaki et al.,
2013; McNamara et al., 2014; Sethi et al., 2015). Other lipid abnormalities
have also been described as being related to BD, such as changes in free
fatty acid (FFA), phosphatidylcholine (PC), and ceramide levels (Schwarz
et al., 2008). These changes might be correlated with central and peripheral
nervous system alterations that could be an intrinsic feature of BD.

Nevertheless, data on differences regarding lipid structural feature

distributions and/or their amounts in BD have not been sufficiently
explored or linked with abnormalities in lipid metabolic pathways, and,
therefore, not recognized enough to be used in BD diagnosis and/or
treatment. In the present exploratory study, we compared the lipid profiles
of blood serum samples from physically and mentally healthy individuals
(healthy controls, HC) and euthymic BD patients using an untargeted
lipidomics approach. Our results point to differential serum lipid profiles in
the HC and BD groups. These data may bring forth insights on metabolic
pathways alterations in BD and guide further studies in this field.
2. Methods

The protocol for this study was approved by the local Ethics
Committee (Hospital das Clínicas, University of Campinas, Brazil,
protocol number 053814/2015), and all the subjects provided written
informed consent before inclusion in the study. Clinical and laboratory
investigations were strictly conducted according to the principles expressed
in the Declaration of Helsinki.

2.1. Subjects and serum sampling

Thirty-five individuals from both genders were included in the study:

14 patients composed the euthymic BD group and 21 subjects were healthy
controls (HC group). The BD group was composed of individuals ranging
from 18 to 65 years old, which fulfilled the DSM-IV criteria for BD type I,
according to the Structured Clinical Interview for DSM-IV Axis I
Disorders (SCID-I). Euthymia was defined as not satisfying the criteria for
any current mood episode and presenting, at the same time, Young Mania
Rating Scale (YMRS) and 17-items Hamilton Depression Rating Scale
(HDRS-17) scores below 8. Functioning was estimated by General
Assessment of Functioning (GAF). Exclusion criteria were suicide risk,
presence of substance use disorders (except nicotine dependence), acute or
unstable chronic general medical comorbidity, pregnancy or postpartum
period, and the inability to read and understand the study procedures. All
subjects were inquired with regard to medical history, including lifetime
use of any medication. Body mass index (BMI) was also determined using
the formula: BMI = weight (kg) / height2 (m2).

The control group was composed of 21 healthy volunteers ranging

from 18 to 65 years old, with negative history of any psychiatric disorder
(current or lifetime) according to the SCID-I, and which had never made
use of any psychiatric medication. In addition, only volunteers with a
negative family history of major mental disorders (schizophrenia-spectrum
disorders, mood disorders and suicide) were included. The same exclusion
criteria used for the BD group were also applied to the HC group.

Blood samples were collected between 8 and 10 a.m. with all

individuals under fasting conditions. Blood was collected in vacutainer
tubes, allowed to clot for at least 30 min, and then centrifuged at 1,500 g
for 10 min. The obtained serum was aliquoted into polypropylene tubes,
and stored at -80 °C until analysis. The maximum storage time was two
weeks.

2.2. Lipid extraction and ultra-high performance liquid chromatography

(UHPLC) coupled to mass spectrometry (MS) analyses

2.2.1. Bligh and Dyer extraction

The lipids were extracted based on the Bligh and Dyer method
(Bligh and Dyer, 1959), where 30 µL of serum, 180 µL of methanol,
MeOH (J.T. Baker, Center Valley, USA) were mixed with 360 µL of
chloroform, CHCl3 (J.T. Baker, Center Valley, USA) in a microtube. After
vortexing, 120 µL of MilliQ water were added to the mixture, and then, the
mixture was vortexed again and then centrifuged at 8,000 g (10 min, 10
°C). Subsequently, the organic phase was separated and dried using N2.
Before analysis, the samples were dissolved in 240 µL of the mobile phase
A and 160 µL of the mobile phase B (described in Section 2.2.2).
2.2.2. UHPLC conditions

The samples were analyzed on an Agilent 1290 ultra-high

performance liquid chromatograph performing a 20 min chromatographic
run at a 0.3 mL min-1 flow rate and a 10 µL injection volume, using a
Titan™ C18 column (100 x 2.1 mm, 1.9 µm, Supelco, Bellefonte, USA)
for the separation. The mobile phases consisted of acetonitrile (ACN):H2O
(40:60, v/v) with 10 mmol L-1 ammonium acetate (Sigma-Aldrich, St
Louis, USA) (A) and ACN:isopropanol (10:90, v/v) with 10 mmol L-1
ammonium acetate (Fluka, St. Louis, USA) (B). The following gradient
elution condition was used: 40% B for 2 min, 50% B for 10 min, 70% B for
2 min, 100% B for 5 min, and 40% B for 2 min.

2.2.3. Quadrupole time-of-flight mass spectrometry (QTOF MS) analyses

Mass spectrometry analyses were performed on an Agilent 6500

series quadrupole time-of-flight (QTOF) mass spectrometer (Agilent,
USA), operating in the negative ionization mode. The VCap voltage was
maintained at 4 kV, the gas temperature was set to 290 °C and the drying
gas flow rate was of 11 L min-1, while the nebulizer pressure was 45 psi.
The fragmentor was maintained at 175 V and the skimmer voltage was of
320 V. A mixture of purine (m/z 121.0508; 5 mmol L-1) and hexakis (1H,
1
H, 3H-tetrafluoropropoxy)phosphazine (m/z 922.0097; 2.5 mmol L-1) was
used as internal standard. Data were recorded in a mass range of m/z 50-
1700, with a scan rate of 1 spectra s-1 and a MS/MS scan rate of 2 spectra s-
1
with an isolation width of 1.3 and collision energy of 35 eV. MS/MS
precursor selection was set to 4 precursors per cycle, where these were
chosen by abundance only mode.
2.2.4. Quality control (QC) and serum samples analyses

In order to detect possible sources of instrumental variation in the

batch analysis of the samples, a third group of samples was analyzed,
named quality control (QC), consisting in a pool of all samples (BD and
HC), prepared by mixing 5 µL of each sample. QC sample preparation was
performed as for the other samples, following the extraction method
described in Section 2.2.1. The QC samples were injected five times at the
beginning of the batch analyses, at every 5 injections, and one time at the
end of the batch. Analyses of the HC and BD groups serum samples were
randomized.

2.3. Data processing and statistical analyses

Raw data derived from the UHPLC-MS analyses were saved in mz

data format by the Agilent MassHunter Qualitative Analysis software
(version B.04.00) and preprocessed using the publically available XCMS
software (version 1.24.1) (Smith et al., 2006). For the dataset, the samples
were grouped in BD (n = 14) and HC (n = 21) groups, and QC samples
were treated as a separate group. Default settings were employed in the
XCMS, with the exception of width of overlapping m/z slices (mzwid =
0.25) and bandwidth for the grouping performed after retention time
correction (bw = 10 s). A median normalization was performed by using an
R script (Veselkov et al., 2011). The generated peak table containing the
retention time, m/z ratio and intensities of each variable in each sample was
adjusted by logarithmic transformation, 10 log (peak height), and
transferred to SIMCA 14 (Umetrics, Umea, Sweden) for a multivariate
analysis by Principal Components Analysis (PCA) after UV-scaling and by
Projections to Latent Structures Discriminant Analysis (PLS-DA).
Multivariate analysis results were evaluated by CV-ANOVA. The
differential features obtained from the PLS-DA loadings plot were further
evaluated by Variable Importance in Projection (VIP), using the software
SIMCA 14, and Student’s t-test (95% of confidence) followed by signal-to-
noise ratio (S/N) verification. When the VIP score  1 and p-value  0.005
for the t-test, the variable could be considered as a contributor for the
classification into BD or HC groups.

In metabolomics, supervised analysis, such as PLS-DA, is commonly

used to obtain discriminating features, mainly due to the correlation
between matrix X (with all information - variables) and matrix Y (with the
classes - control and test samples). In the PCA, the latent vector only
maximizes the total variance in the X matrix. Thus, in metabolomics, a
PCA is commonly used for the visualization of outliers, data evaluation,
tendencies in grouping and choosing the right set of variables for
subsequent model construction (Fonville et al., 2010).

2.4. Classification and putative identification of differential lipids

After the determination of the significant features by statistical

analyses, their experimental m/z values obtained by high resolution MS
were analyzed using LIPID MAPS (www.lipidmaps.org). All differential
lipids were firstly classified using the LIPID MAPS Lipid Classification
System, which considers eight lipid subclasses: fatty acyls (FA),
glycerolipids (GL), glycerophospholipids (GP), sphingolipids (SP),
saccharolipids (SL), polyketides (PK), prenol lipids (PR) or sterol lipids
(ST).

For putative identification, we selected the top five lipids whose

levels were the most altered in each group, i.e. had their levels enhanced in
BD or HC, and presented a VIP score ≥ 1.5. The putative identities for such
differential lipids were obtained by searches in the LIPID MAPS, METLIN
(metlin.scripps.edu) and HMDB (www.hmdb.ca) databases. The mass error
limit used for the acceptance of a putative identification for a feature was of
10 ppm. We selected the matches with the lowest mass errors compared to
the theoretical m/z input from the databases.
3. Results

Clinical and demographic characteristics of the studied samples are

displayed in Table 1. As expected, no statistically significant differences
for age, gender, BMI and education levels were observed between the
groups. In addition, the BD group presented very low severity of mood
symptoms as evaluated by analysis of the HDRS-17 and YMRS scores,
which is expected in the case of euthymic individuals.

3.1. QC evaluation

Prior to the UHPLC-MS analyses of the samples and identification of

the statistically significant lipids as possible biomarkers for BD, the
reproducibility of our data was evaluated by the coefficient of variation
(CV, %) from all features encountered in QC samples and clustering of the
QC samples in a PCA scores plot (FDA, 2001; Gika et al., 2008). For all
QC samples, 94% of the features were acceptable using the CV < 15%
criterion. Figure 1A displays the PCA scores plot including the QC
samples, with explained amount of variation of 0.654 (0.356 for PC1, 0.182
for PC2 and 0.116 for PC3). The QC samples were well clustered in the
PCA scores plot, although they were not centered due to the different
number of samples in each studied group.

3.2. Multivariate analysis of BD versus HC samples

Figures 1B and 1C present the PCA and PLS-DA score plots,

respectively, obtained for BD versus HC samples. In order to build the
PCA and PLS-DA models, QC samples were excluded from the data
treatment. The amount of explained variation was 0.302 and 0.188 for PC1
and PC2, respectively, in the PCA scores plot. A validation of the PLS-DA
model was performed by a permutation test (Lindgren et al., 1996;
Szymańska et al., 2012). The intercepts for R2 (0.0, 0.625) and Q2 (0.0, -
0.233) indicate that the model was validated. In addition, the PLS-DA
model with three components was statistically significant according to
cross-validation, indicating that the model could describe 52.7% of the
variation in X (R2X = 0.527) and 97.6% of the variation in response Y (R2Y =
0.976), with a predictive ability of 91% (Q2Y = 0.910), F value of 56.8591
and p-value of 2.16128 e-14.

The differential features were determined by a corresponding PLS-

DA loadings plot, which is based on the contribution of different variables
for the separation of the BD and HC groups. According to the VIP scores,
Student’s t-test and S/N verification, 107 features displayed enhanced
levels in the BD group, while 115 features presented augmented levels in
the HC group, contributing to the clustering of these groups in the PLS-DA
scores plot. Removing the lipid isotopologues, we obtained 63 lipids with
increased levels in the BD group and 58 lipids with enhanced levels in the
HC group. The selected features are ranked by VIP scores and presented in
Figure S1 (Supplementary Material).

3.3. Differential lipids between BD and HC groups

The 121 statistically differential lipids between the BD and HC

groups, determined by a multivariate analysis, were classified using the
LIPID MAPS platform, where matches with low or insignificant
differences between experimental and theoretical masses were processed.
As shown in Table 2, we obtained the distribution of lipid subclasses in BD
patients sera in comparison to HC individuals. The adducts considered for
this search were [M-H]-, [M+OAc]-, and [M+Cl]-. All experimental m/z
values for the differential lipids and their respective classification are
displayed in Table S1 (Supplementary Material).

The putative identities for the top five most differential lipids in each
studied group and their respective experimental m/z values used for the
identification are presented in Table 3. Even considering the acquired
MS/MS spectra, some lipids could not be identified, and appear as NI (Not
Identified) in this table.

3.4. Receiver operating characteristic (ROC) tests

A ROC test analysis was performed for the ten most significant
lipids, selected as described in Section 3.3, using the MetaboAnalyst
platform (Xia and Wishart, 2011). ROC curves were employed to verify the
specificity and predictive accuracy of the differential features to be
considered as biomarkers, as they allow for the visualization of true
positive and false positive rates. Thus, we verified the possibility of
defining the lipids listed in Table 3 as potential BD biomarkers. The area
under curve (AUC) values for those selected lipids are displayed in Table
3, and the ROC curves are shown in Figures S2 to S11 (Supplementary
Material).
4. Discussion

In this pioneer, but rather exploratory study, UHPLC coupled to MS

was successfully used in the untargeted analysis of lipids extracted from
blood serum samples from BD patients and HC individuals. Our main goal
was to detect statistically significant differences in the lipid profiles
between these two studied groups, in order to identify possible candidates
to be further explored as biomarkers for BD.

Our results pointed to different lipid subclass distribution in the

serum lipid profiles of individuals from the BD and HC groups, such as an
increased proportion of sphingolipids and glycerolipids in the serum of BD
patients and a decreased proportion of glycerophospholipids compared to
the HC group, as presented in Table 2.

The five most important lipids that were enhanced in the HC group
(in white) and in the BD group (highlighted in gray) are observed in Table
3. A heatmap presenting the distribution of the differential lipids among the
samples is displayed in Figure 2.

The ions with m/z 951.6 and 915.6 were putatively identified as
phosphatidylinositol (PI) (40:3), which are different adducts for the same
lipid. Several studies have reported abnormalities in the PI levels in BD
patients (Chang-Gyu and Eitan, 1999; Lachman and Papolos, 1989; Soares
and Mallinger, 1995). In addition, other recent research articles also
corroborate with the results obtained herein, especially with regard to the
increased level of the glycerophospholipids in controls compared to BD
patients (Knowles et al., 2017).

The ion with m/z 789.5 was putatively identified as

phosphatidylglycerol (PG) (32:4 (OH)). As PI also regulates the synthesis
of PG (Ju and Greenberg, 2003), we can correlate this finding with the
decreased levels of PI in BD patients reported above.

The ions with m/z 807.6 and 871.6, putatively identified as PA (44:4)
and PA (48:8(OH)), respectively, are phosphatidic acids that also play a
role in the glycerophospholipid metabolic pathway, as well as the ion with
m/z 856.6, putatively identified as phosphatidylethanolamine (PE) (42:5).
These findings indicate that the phospholipid biosynthesis is the most
altered pathway in BD.

The ion with m/z 775.6 was putatively identified as triacylglycerol

(TG) (42:3). Several studies have reported the increase of triglyceride
levels in BD patients (Chung et al., 2007; Glueck et al., 1994; Wysokiński
et al., 2015), indicating that glycerolipids can be potential candidates as
biomarkers for BD. Nevertheless, the serum lipids of the BD patients may
be altered due to the mood disorder category (Chung et al., 2007). Bülbül et
al. (2014) reported that patients in an euthymic state present increased
levels of triglycerides compared to BD patients in a manic state. As the
patients evaluated herein were in an euthymic state, we believe our data
corroborates with the hypothesis reported in these previous studies.

The reported results in this exploratory study might encourage the

application of lipidomics approaches, such as UHPLC coupled to MS, in
larger sample cohorts and in different populations for biological validation.
It is necessary to point out the limitations of the presented research study,
such as the small sample size, that does not allow for exploration of cause-
effect relations. Further studies including drug-naïve patients should be
performed to determine specific BD biomarkers, excluding the treatment
effects.

In summary, the application of MS-based untargeted lipidomics

strategy to study BD allowed for the identification of statistically
significant differences in the serum lipid profiles between BD and HC
groups. Glycerophospholipids, glycerolipids, and sphingolipids were the
most affected lipid classes in BD. Thus, BD lipid metabolism was
definitively altered, since decreases in the glycerophospholipid levels were
detected, as well as an increase in sphingolipid and glycerolipid distribution
in the BD patients. Alterations in glycerophospholipids can be supported by
the fact that PI controls the glycerophospholipid metabolic pathway leading
to the production of phosphatidylglycerol, phosphatidylcholine (PC),
phosphatidylserine (PS) and cardiolipins (CL). Increases in PI levels lead to
a decrease in PC and CL syntheses, which can lead to other metabolic
dysfunctions (Ju and Greenberg, 2003), possibly causing diverse
biochemical alterations in BD patients. In conclusion, our initial hypothesis
of alterations in the serum lipidome of BD patients was supported by the
results, which corroborate with previously published data on BD.
Hopefully, our findings might open new paths in BD molecular research
field, by possibly enabling the elucidation of the biochemical alterations
involved in BD.
Acknowledgements

The authors greatly acknowledge Prof. Dr. Marcos N. Eberlin -

ThoMSon Mass Spectrometry Laboratory (UNICAMP, Brazil) - and
Agilent Technologies Brazil for the possibility to perform the UHPLC-ESI-
QTOF MS experiments. The authors also thank FAPESP [grant numbers
2014/18938-8 and 2015/13229-1] and FAEPEX [grant number 1244/14]
for financial supports. H.C.R. and M.S.C. thank CAPES for their
scholarships. Conflicts of interest: none.
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Tables

Table 1. Clinical and demographic data of the samples (BD: Bipolar

Disorder patients; HC: Healthy Controls; SD: Standard Deviation; BMI:
Body Mass Index; YMRS: Young Mania Rating Scale; HDRS-17:
Hamilton Depression Rating Scale - 17 items; GAF: Global Assessment of
Functioning Scale; CGI: Clinical Global Impression Scale)

Variable BD (n = 14) HC (n = 21) Test-value p-value

Age in years; mean (SD) 46 (9) 34 (13) 2.693a 0.012a

Gender F/M; n (%) 10/4 (71/29) 14/7 (67/33) 3.836b 0.147b

Years of education; n (%)

≤ 10 years 5 (36) 8 (38) 2.213c 0.149c

more than 10 years 9 (64) 13 (62)

BMI in kg m-2; mean (SD) 25.4 (4.6) 25.2 (3.6) 0.122a 0.903a

YMRS score; mean (SD) 0.71 (1.68)

HDRS-17; mean (SD) 4.08 (3.68)

GAF score; mean (SD) 80.7 (10.2)

CGI score; mean (SD) 2.0 (0.9)

Medication use; n (%)

Lithium 1 (7.1)

Valproate 1 (7.1)

Carbamazepine 6 (43)

Chlorpromazine 1 (7.1)

Quetiapine 1 (7.1)

Fluoxetine 1 (7.1)

Clonazepan 1 (7.1)

a
Student’s t-test; b Pearson Chi-Square; c Levene’s Test
Table 2. Distribution of lipid classes for BD and HC groups, identified
using the LIPID MAPS database

Lipid Class BD group (%) HC group (%)

Fatty Acyls (FA) 27.86 26.53

Glycerolipids (GL) 8.20 6.12

Glycerophospholipids (GP) 52.46 63.27

Sphingolipids (SP) 11.48 4.08

Table 3. Putative identification for the top ten differential lipids for BD and
HC groups, selected according to the VIP scores. The features highlighted
in grey were enhanced in the BD group while the others were augmented in
the HC group (AUC: Area Under Curve; NI: Not Identified)

m/z Experimental Mass error

Putative IDs Ion AUC
(retention time, s) mass (ppm)

951.6 (503) PI (40:3) 951.5720 [M+Cl]- 1.58 1

1175.8 (812) NI 1175.8750 1
762.4 (365) NI 762.4404 1
789.5 (365) PG (32:4(OH)) 789.4547 [M+OAc]- 1.65 1
915.6 (503) PI (40:3) 915.5953 [M-H]- 1.64 1
775.6 (907) TG (42:3) 775.6054 [M+OAc]- 5.15 0.963
-
856.6 (893) PE (42:5) 856.5632 [M+Cl] 0.35 0.827
871.6 (904) PA (48:8(OH)) 871.5859 [M-H]- 0.11 0.952
753.4 (383) NI 753.3655 0.855
807.6 (900) PA (44:4) 807.5949 [M-H]- 4.95 0.844
Figure 1. PCA scores plot (PC1 vs. PC2) for QC and grouping evaluation.
QC samples are displayed in green, whereas BD samples are displayed in
blue and HC in red (A); PCA scores plot (PC1 vs. PC2) of BD (gray) and
HC (light gray) samples (B); PLS-DA scores plot of BD (gray) and HC
(light gray) samples (C).
Figure 2. Heatmap clustering of the selected differential lipids (m/z /
retention time in seconds) for BD and HC samples.

Highlights

1. Evaluation of the serum lipid profile of bipolar disorder (BD)

patients.

2. Serum analyzed by liquid chromatography and high resolution mass

spectrometry (MS).

3. Significant differences in lipid profile for BD patients in comparison

to controls.

4. Glycerophospho-, glycero-, and sphingolipids were the most altered

classes in BD.

5. First report of a MS-based untargeted lipidomics strategy applied to

study BD.