Quantitative lipidomic analysis of mouse lung during postnatal development by electrospray ionization tandem mass spectrometry
Quantitative lipidomic analysis of mouse lung during postnatal development by electrospray ionization tandem mass spectrometry
Srikanth Karnati - 0 1
Vannuruswamy Garikapati 0 1
Gerhard Liebisch 1
Paul P. Van Veldhoven 1
Bernhard Spengler 1
Gerd Schmitz 1
Eveline Baumgart-Vogt 0 1
0 Institute for Anatomy and Cell Biology II, Division of Medical Cell Biology, Justus Liebig University Giessen , Giessen, Germany , 2 Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen , Giessen, Germany , 3 Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg , Regensburg, Germany , 4 Laboratory for Lipid Biochemistry and Protein Interactions, Campus Gasthuisberg, KU Leuven , Leuven , Belgium
1 Editor: Harald Ehrhardt, Center of Pediatrics , GERMANY
Lipids play very important roles in lung biology, mainly reducing the alveolar surface tension at the air-liquid interface thereby preventing end-expiratory collapse of the alveoli. In the present study we performed an extensive quantitative lipidomic analysis of mouse lung to provide the i) total lipid quantity, ii) distribution pattern of the major lipid classes, iii) composition of individual lipid species and iv) glycerophospholipid distribution pattern according to carbon chain length (total number of carbon atoms) and degree of unsaturation (total number of double bonds). We analysed and quantified 160 glycerophospholipid species, 24 sphingolipid species, 18 cholesteryl esters and cholesterol from lungs of a) newborn (P1), b) 15-day-old (P15) and c) 12-week-old adult mice (P84) to understand the changes occurring during postnatal pulmonary development. Our results revealed an increase in total lipid quantity, correlation of lipid class distribution in lung tissue and significant changes in the individual lipid species composition during postnatal lung development. Interestingly, we observed significant stage-specific alterations during this process. Especially, P1 lungs showed high content of monounsaturated lipid species; P15 lungs exhibited myristic and palmitic acid containing lipid species, whereas adult lungs were enriched with polyunsaturated lipid species. Taken together, our study provides an extensive quantitative lipidome of the postnatal mouse lung development, which may serve as a reference for a better understanding of lipid alterations and their functions in lung development and respiratory diseases
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: We thank German Academic Exchange
Service (DAAD) for granting the Graduate School
Scholarship Programme ªLipids in Nutrition and
Metabolismº (DAAD-GSSP-2015). V.G. is a holder
of a doctoral fellowship (DAAD-ID91566181)
within this programme. Our study was supported
also by LOM (Leistungsorientierte Mittel)
associated with lipids.
performance related resource allocation-funds of
the Medical Faculty of the Justus Liebig University
Giessen, Germany. Financial support by the
Deutsche Forschungsgemeinschaft (DFG, Sp 314/
13-1) is gratefully acknowledged. The research
leading to these results also received funding from
the European Community's Seventh Framework
Programme (FP7/2007-2013) under grant
agreement nÊ 202272, IP-Project LipidomicNet.
Competing interests: The authors declare that they
have no conflict of interests in relation to the work
Abbreviations: PC, phosphatidylcholine; LPC,
lysophosphatidylcholine; PC O,
etherphosphatidylcholine; PG, phosphatidylglycerol; PE,
phosphatidylethanolamine; PE P, PE based
plasmalogen; PI, phosphatidylinositol; PS,
phosphatidylserine; SM, sphingomyelin; Cer,
ceramide; HexCer, hexosylceramide; GP,
glycerophospholipids; SP, sphingolipids; SL, sterol
lipids; CE, cholesteryl esters; ESI, electrospray
ionization; MS/MS, tandem mass spectrometry;
P1, newborn mice; P15, 15-day-old mice; P84,
12week-old adult mice.
The lung is composed of more than 40 different pulmonary cell types, whose cellular
membranes are enriched with lipids that perform a variety of functions including maintenance of
the lung architecture [1±3]. In addition, alveolar epithelial type II cells of the pulmonary
epithelium that are lining the alveolar surface synthesize and secrete surfactant into the alveolar
space. Pulmonary surfactant is a complex mixture of lipids (phospholipids, triglycerides, fatty
acids and cholesterol, etc.), surfactant proteins (A-D) and a small amount of carbohydrates.
The majority of the pulmonary lipids comprise glycerophospholipids (GP) in which
phosphatidylcholine (PC) is a predominant lipid class making up to 50% of the phospholipids.
Phosphatidylethanolamine (PE) makes up to 20% of the lipids and phosphatidylserine (PS),
phosphatidylinositol (PI) and phosphatidylglycerol (PG) constitute 12%-15% of the total
phospholipid pool [
Pulmonary lipids are important and diverse biomolecules that are involved in many
biological processes. The thus far known functions of the major lung lipids include 1) prevention of
alveolar collapse and preservation of bronchiolar patency [
], 2) improvement of mucociliary
], 3) involvement in innate immunity and viral protection [
], 4) action as potent
intracellular signalling molecules in lung inflammation , 5) involvement of lipid mediators
like leukotrienes, lipoxins and prostaglandins in specific reactions of inflammation and
], 6) suppression of the proliferation, immunoglobulin production and cytotoxicity
of lymphocytes . In fact, alterations in whole lung lipid composition and/or deficiency of
pulmonary surfactant lipids are closely associated with a) respiratory distress syndrome (RDS)
], b) bronchopulmonary dysplasia (BPD) [
], c) asthma , d) chronic obstructive
pulmonary disease (COPD) [
], e) cystic fibrosis , f) pneumonia [
], g) lung
injury , h) cancer [
] and in other lung diseases [
High heterogeneity of lung tissue (e.g., bronchial versus alveolar regions) and differences in
the lipid composition of individual pulmonary cell types create a complex mixture of lipid
classes and molecular species associated with a set of potential complications. Despite these
difficulties, several studies analyzed the lipid composition of lung tissue in different mammalian
species such as in pig [
], rat [
], rabbit [
], monkey [
], dog [
], bovine [
], mice [
and human [
] and also compared the lung lipid composition among different mammalian
]. Actually, the major lipid classes were similar among different mammalian
species and minor differences were observed only in the class of phospholipids. Interestingly,
surfactant has proven to be highly diverse across species in its molecular design, especially in the
concentration of individual surfactant proteins and its GP profile [
]. Further, most of
these studies focused on the analysis of PC, which are the prominent class of lipids in whole
lung tissue and pulmonary surfactant. Furthermore, among the analyzed PC,
dipalmitoylphosphatidylcholine (DPPC; PC 32:0), a species with two saturated acyl chains, is believed to be a
major compound of the pulmonary surfactant [
]. However, recent studies on homeothermic/
heterothermic mammalian species surfactant showed that DPPC is not the only major
surfactant phospholipid component. In addition to major PC lipid molecular species, there are minor
lipid classes such as PG and PI, which also play an important role in lung biology [
Lung development in the majority of mammalian species (e.g., rat, mice and humans)
continues postnatally. One important aspect of postnatal lung development is alveolarization, a
process, in which the total number of terminal gas exchange units increase total size and
surface area of the lung [35±37]. To address the lipidomic changes in the fetal and postnatal lung
development, various studies were conducted in several mammalian species (e.g., rat, rabbit,
lamb, pig and human) and in birds (e.g., duck and chicken) by using morphometric and
biochemical approaches [35±41]. However, the existing lipidomic studies of postnatal lung
2 / 25
development primarily focused on the composition of surfactant PC and very few PG lipid
31, 40, 41
]. Dautel et al., recently reported postnatal developmental changes of mouse lung
using a multi-omics approach [
]. These data are consistent with our results, but in
comparison to their study (day7, day14 and 6±8 week old animals), we have used a wider time frame
between birth (new born) and the 12th week of life. Furthermore, the highly abundant
cholesterol, as well as cholesteryl ester species were not analysed in the Dautel et al., study during
postnatal development. Moreover, we performed direct infusion lipidomics using triple-quadrupole
MS analytical setup and provided the quantitative information (nmol/mg wet weight) of
individual lipid species of possible major lipid classes during postnatal development.
In our study, we employed electrospray ionization tandem mass spectrometry (ESI-MS/
MS) to 1) investigate total lipid quantity and 2) to perform a detailed analysis of lipid classes
(PC, LPC, PG, PS, PE, PE P, PI, SM, Cer, HexCer, CE, and cholesterol), and 3) the composition
of individual lipid species (e.g. PC 32:0, PC 32:1 etc.), and 4) to analyse their distribution
pattern based on the carbon chain length (total number of carbon atoms). Additionally, we
analysed the degree of unsaturation (total number of double bonds) of whole lung homogenates
in newborn, 15-day-old and 12-week-old adult mice in order to provide a detailed lipidomic
information during the postnatal development. Our current study provides an extensive
quantitative lipidome of mouse whole lung, which may serve as a reference for a better
understanding of the development of lung and molecular mechanisms underlying various pulmonary
diseases associated with the lipid alterations.
Materials and methods
Unless otherwise mentioned, all chemicals were procured from Sigma-Aldrich (Deisenhofen,
Germany). Phospholipid standards were obtained from Avanti Polar Lipids (Alabaster, AL,
USA). Cholesterol and cholesteryl ester standards of purity greater than 95% were obtained
from Sigma (Taufkirchen, Germany). High purity cholesterol-(25, 26, 26, 26, 27, 27, 27-d7)
was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). HPLC grade
solvents methanol and chloroform were obtained from Merck (Darmstadt, Germany). Analytical
grade ammonium acetate and acetyl chloride were obtained from Sigma-Aldrich (Buchs,
Switzerland). All other reagents used were of high purity and analytical grade.
Twelve-week-old adult male mice, 15-day-old males and pregnant females of C57BL/6J genetic
background were obtained from Charles River, Sulzfeld, Germany. Mice were kept on a
normal laboratory diet and water ad libitum and housed in cages under standardized
environmental conditions (12 hours light/dark cycle, 23ÊC ± 1ÊC and 55% ± 1% relative humidity) at the
central animal facility of the Justus Liebig University Giessen, Germany. After delivery of the
newborn pups in the morning, they were taken directly out of the animal facility. 15-day-old
and 12-week-old adult mice were killed by cervical dislocation and the newborn pups were
killed by decapitation. All experiments with laboratory mice were approved by the
governmental ethics committee for animal welfare (RegierungspraÈsidium Giessen, Germany, permit
number: V 54±19 C 20/15 c GI 20/23).
Lipid extraction and sample preparation
The newborn (P1), 15-day-old (P15) and 12-week-old adult (P84) male mice fur was vertically
incised from the pelvis to the mandibles and removed to both sides. The abdomen was opened
3 / 25
and a bilateral pneumothorax was produced by puncturing the abdominal surface of the
diaphragm. The sternum was cut in the middle and the thorax was opened with a thorax spanner.
The lungs were isolated carefully and snap-frozen immediately.
Fresh snap-frozen lungs from P1, P15 and P84 male mice were homogenized with a
Precellys homogenator (peQlab Biotech GmbH, Erlangen, Germany) at a concentration of 50 μg
wet weight per μL. Homogenate corresponding to 2 mg wet weight was used for extraction,
and lipids were extracted according to the procedure described by Bligh and Dyer [
phase separation, the chloroform phase was transferred to a fresh tube and dried under a
stream of nitrogen gas. For each lipid class (except for SM and PE P), two naturally not
occurring lipid species were added as internal standards, to compensate for variations in sample
preparation and ionization efficiency. PC 14:0/14:0 (28:0), PC 22:0/22:0 (44:0) for PC and SM,
PE 14:0/14:0 (28:0), PE 20:0/20:0 (40:0) for PE and PE based plasmalogens (PE P), PS 14:0/14:0
(28:0), PS 20:0/20:0 (40:0), PG 14:0/14:0 (28:0), PG 20:0/20:0 (40:0), PI 16:0/16:0 (32:0), LPC
13:0, LPC 19:0, Cer 14:0, Cer 17:0 (d18:1/17:0), cholesterol-d7, CE 17:0 and CE 22:0 internal
standards were added for the analysed lipid classes.
Mass spectrometric analysis of lipids
Lung homogenates were subjected to lipidome analysis by electrospray ionization-tandem
mass spectrometry (ESI-MS/MS) in positive-ion mode as described [43±46]. Briefly, the
samples were analyzed on a triple-quadrupole mass spectrometer (Quattro Ultima, Micromass,
Manchester, UK) by direct flow injection analysis using an autosampler (HTS PAL, Zwingen,
Switzerland) and a binary pump (Model 1100, Agilent, Waldbronn, Germany) with a solvent
mixture of methanol containing 10 mM ammonium acetate and chloroform (3:1, v/v). A flow
gradient was performed starting with a flow of 55 μL/min for 6 seconds followed by 30 μL/min
for 1 minute and an increase to 250 μL/min for another 12 seconds. The mass spectrometer
was equipped with electrospray ionization and operated in positive-ion mode using following
tune parameters as capillary voltage, 3.5 kV; cone voltage, 110 V; collision energy, 30 V;
collision gas, argon at a pressure of 0.13 Pa.
A precursor ion of m/z 184, which is specific for phosphocholine-containing lipids, was
used for the analysis of phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and
sphingomyelin (SM) lipid species [
]. Neutral loss scans of 141 and 185 were used for the
phosphatidylethanolamine (PE) and phosphatidylserine (PS) respectively . Fragment ions
of m/z 364, 390, and 392 were used for the quantification of PE P-16:0, PE P-18:1 and PE
P18:0 plasmalogens according to Zemski et al. [
]. Neutral loss scans of 189 and 277 were used
for the ammonium adducts of phosphatidylglycerol (PG) and phosphatidylinositol (PI)
]. Sphingosine (d18:1) based ceramides (Cer) were analysed using a fragment ion of
m/z 264 [
]. Cholesterol and cholesteryl esters (CE) were quantified using a fragment ion of
m/z 369 after selective derivatization of cholesterol using acetyl chloride [
After identification of relevant lipid species, selective ion monitoring analysis was
performed to increase precision of the analysis of lipids. Quantification of different classes of lipid
species was achieved by plotting the standard calibration curves of naturally occurring lipid
species of PC 34:1, 36:2, 38:4, 40:0 and PC O-16:0/20:4; SM d18:1/16:0, 18:1, 18:0; LPC 16:0,
18:1, 18:0; PE 34:1, 36:2, 38:4, 40:6 and PE P-16:0/20:4; PS 34:1, 36:2, 38:4, 40:6; Cer d18:1/16:0,
18:0, 20:0, 24:1, 24:0; cholesterol, CE 16:0, 18:2, 18:1, 18:0. Correction of isotopic overlap of
lipid species as well as data analysis were performed using self-programmed Excel Macros for
all lipid classes according to principles described previously [
]. In brief, data analysis was
performed with MassLynx software, which included the NeoLynx tool (Micromass) for
averaging the scans at half peak height of the total ion count. NeoLynx generates centroid peak
4 / 25
data from the continuum spectra and allows selection of the intensities of certain peaks. Neolynx
includes background subtraction and smoothing according to Savitzky Golay of the combined
spectra. These NeoLynx results were exported to Excel spreadsheets and further processed by
selfprogrammed Excel macros, which sort the results, correct for isotopic overlap, calculate the ratios
to the internal standards, generate calibration curves, and calculate quantitative values [
Lipid species were annotated according to the proposal for shorthand notation of lipid
structures derived from mass spectrometry [
]. Glycerophospholipid species annotation was
based on the assumption of even numbered carbon chains only and presented as the sum of
carbon chain length and degree of unsaturation, without specifying fatty acid location at the
sn-1 or sn-2 position. SM species annotation was based on the assumption that sphingosine
d18:1 is present [
]. Final quantities of lipid species and total lipid (sum of analysed lipid
species) were calculated and expressed in nanomoles per milligram wet weight of tissue.
All data are expressed as mean ± standard deviation (SD) with at least three mice (n = 3) for
each group. Two-way analysis of variance (ANOVA) was calculated using GraphPad Prism
Version 5.4 (GraphPad Software, San Diego, CA). Statistical comparisons among the groups
were performed by Bonferroni post-test using the same software. A p-value of 0.05 or lower
was considered as significant. Significance is indicated as P < 0.0001, P < 0.001,
P < 0.01, P < 0.05.
Overview and analysis of the pulmonary lipidome
The current study presents an extensive quantitative lipidome analysis of the total mouse lung
homogenates during the postnatal development performed with the help of ESI-MS/MS. In
total, we performed quantitative analysis of 160 GP, 24 SP, 18 CE species and cholesterol in
different stages of the postnatal lung development (lungs of newborn, 15-day-old and
12-weekold adult mice). The glycerophospholipids consist of 35 PC, 15 LPC, 16 PG, 23 PE, 33 PE P, 15
PI and 23 PS lipid species. Sphingolipids consist of 15 SM, 7 Cer and 2 cerebroside (HexCer)
lipid species. The overview of total lipid analyses from mouse lung homogenates during
development is depicted in Fig 1A and 1B.
Total lipid quantitation during postnatal lung development
To evaluate the total lipid content in mouse lungs during the postnatal development, lipid
quantity of phospholipids (sum of all analyzed GP classes), cholesteryl esters and cholesterol
was calculated and expressed as nmol/mg wet weight (Fig 2). Total phospholipid content
significantly increased during the development from P1 (28.26 ± 3.08 nmol/mg) to P84 (35.20 ±
1.42 nmol/mg) and from P15 (30.68 ± 0.85 nmol/mg) to P84 (35.20 ± 1.42 nmol/mg).
However, we did not observe a statistically significant increase between P1 (28.26 ± 3.08 nmol/mg)
and P15 (30.68 ± 0.85 nmol/mg). In contrast, cholesterol was significantly increased during
progressing from P1 (7.9 ± 0.83 nmol/mg) to P15 (13.16 ± 0.70 nmol/mg) and a significant
increase of the values was observed during the development from P1 (7.9 ± 0.83 nmol/mg) to
adult lung (12.81 ± 0.55 nmol/mg). However, while developing from P15 to P84, cholesterol
remained constant in P84 (12.81 ± 0.55 nmol/mg). The esterified cholesterol species remained
at the level of <0.5 nmol/mg wet weight.
Individual lipid class (sum of the analysed lipid species) quantities are presented in S1
Table. For instance, the PC species gradually increased from the stage of P1 (16.47 ± 2.23
5 / 25
Fig 1. Overview of the quantitative lipidomic analyses of mouse lung homogenates during postnatal development
by mass spectrometry. The numbers represent number of lipid species quantified for particular lipid class.
nmol/mg) to P84 (18.03 ± 0.70 nmol/mg), but in P15 lung the PC species were lower
(14.80 ± 0.57 nmol/mg) as compared to P1 and adult. In contrast, we observed a gradual
increase in the total amount of PS, PI, PE, PE P, LPC and SM lipid classes during the postnatal
development of the mouse lung. Among these, only PS (3.05 ± 0.19 to 5.35 ± 0.22 nmol/mg)
and PE P (2.55 ± 0.23 to 3.82 ± 0.12 nmol/mg) showed statistical significance during the
change from P1 to P84.
Individual lipid species analysis during postnatal lung development
To gain deeper insights into the postnatal developmental alterations, we evaluated the
individual lipid species profile of respective lipid classes from mouse lung. The
diacylglycerophospholipids were denoted with total number of carbon atoms and double bonds (C:D). LPC and CE
species containing single fatty acids were denoted according to lipid species nomenclature.
Phosphatidylcholine species. In total, 35 different PC (24 PC, 11 PC O) species with
different chain length and degree of unsaturation were documented in the lung extracts and their
composition is depicted in Fig 3A and 3B. As expected, PC 32:0, PC 32:1, PC 30:0, PC 34:1 and
PC 34:2 were the most abundant phosphatidylcholine species in the tested stages in the mouse
lung. The predominant PC 32:0 increased significantly while progressing from P1 (4.11 ± 0.6
nmol/mg) to P84 (6.27 ± 0.2 nmol/mg). Similarly, PC 34:2, PC 36:4, PC 38:6, PC 38:4 and PC
40:6 lipid species were significantly elevated in P84 as compared to P1 (Fig 3A & 3B).
In contrast, monounsaturated lipid species (total number of double bonds = 1) PC 34:1
showed a significant reduction during the development from P1 (2.23 ± 0.23 nmol/mg) to P15
(1.73 ± 0.06 nmol/mg) and remained constant in P84 (1.79 ± 0.09 nmol/mg). Similarly, the
values of PC 32:1 also significantly dropped during the maturation from P1 (4.08 ± 0.6 nmol/mg)
to P15 (2.21 ± 0.19 nmol/mg) and thereafter slightly increased from the stage P15 to P84
(2.74 ± 0.11 nmol/mg). The monounsaturated lipid species (PC 30:1, 32:1, 34:1) were elevated
in P1 lungs (Fig 3A).
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Fig 2. Total lipid quantity of phospholipids, cholesteryl esters and cholesterol in mouse lung during postnatal development. Displayed are nmol/mg wet
weight of the lipid class of all analyzed lipid species. Values are represented as mean ± SD, p-value summary: P < 0.0001, P < 0.001, P < 0.01.
Where significance is not mentioned, values are considered as being not significant. Phospholipids represents the sum of PC, LPC, PE, PE P, PG, PI, PS, and
The PC 30:0 gradually increased from the stage of P1 (1.3 ± 0.1 nmol/mg) to P15 (1.8 ± 0.07
nmol/mg) and then significantly decreased in P84 (0.91 ± 0.01 nmol/mg). Interestingly, higher
levels of PC 30:0 were detected in P15 lungs in comparison to P1 and P84.
The analyzed ether-phosphatidylcholine (PC O) lipid species were present at low
concentrations and their quantitative information during postnatal pulmonary development is
showed in Fig 3B.
Lysophosphatidylcholine species. In total, 15 different lysophosphatidylcholine lipid
species were analyzed and their composition pattern is depicted in Fig 4. The major LPC
species detected were LPC 16:0 followed by 18:0, 18:1, 16:1, 18:2 and 20:4. The values of LPC
16:0 significantly increased during the maturation from P1 (0.103 ± 0.02 nmol/mg) to P15
(0.210 ± 0.01 nmol/mg) and P1 to P84 (0.212 ± 0.03 nmol/mg). Similarly, during development
process from newborn to adult the amounts of all other LPC species including LPC 18:0,
polyunsaturated LPC species 18:2 and 20:4 increased. Remaining LPC species occurred at lower
concentrations and did not show any significant differences.
Phosphatidylglycerol species. We analyzed 16 individual lipid species of
phosphatidylglycerol and their composition during the postnatal lung development is represented in Fig 5.
As expected, PG 34:1, PG 34:2, PG 32:0 and PG 32:1 constituted highly abundant lipid species
that were detected in all three groups. Interestingly, in contrast to PC 32:0, disaturated PG 32:0
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Fig 3. Composition of individual phosphatidylcholine (PC) and ether-phosphatidylcholine (PC O) lipid species during postnatal
development of mouse lung. Values are represented as nmol/mg wet weight. A) PC B) PC and PC O. Values are mean ± SD, p-value
summary: P < 0.0001, P < 0.001, P < 0.01, P < 0.05. Where significance is not mentioned, values are considered as being
decreased while progressing from P1 to P15. Notably, monounsaturated PG 32:1 significantly
dropped in P84 (0.075 ± 0.002 nmol/mg) as compared to P1 (0.205 ± 0.04 nmol/mg). Similarly,
the values of PG 34:1 dropped in P15 as compared to P1 and slightly increased while maturing
from P15 to P84.
In contrast, diunsaturated (total number of double bonds = 2) PG 34:2 shows a significant
increase during the development from P1 (0.117 ± 0.023 nmol/mg)) to P15 (0.173 ± 0.020
nmol/mg) and from P15 to P84 (0.367 ± 0.016 nmol/mg).
Similar to PC 30:0, the PG 30:0 was higher in P15 in comparison to P1 and P84.
Interestingly, we detected higher levels of PG 36:4 in P15 lungs. Remaining PG lipid species did not
show any statistically significant differences during development.
Fig 4. Composition of individual lysophosphatidylcholine (LPC) lipid species during postnatal development of mouse lung. Values are represented as nmol/mg
wet weight. Values are mean ± SD, p-value summary: P < 0.0001, P < 0.001, P < 0.01, P < 0.05. Where significance is not mentioned, values are
considered as being not significant.
9 / 25
Fig 5. Composition of individual phosphatidylglycerol (PG) lipid species during postnatal development of mouse lung. Values are represented as nmol/mg wet
weight. Values are mean ± SD, p-value summary: P < 0.0001, P < 0.001, P < 0.01, P < 0.05. Where significance is not mentioned, values are considered as
being not significant.
Phosphatidylethanolamine species. Total 23 individual phosphatidylethanolamine lipid
species were quantified and their composition is depicted in Fig 6. Interestingly, in comparison
to the other lipid classes, PE lipids exhibited higher abundance of long chain polyunsaturated
species. Strikingly, PE 38:4 showed higher abundance in all three groups in comparison to
other PE species. Further, PE 38.4 significantly increased during maturation from P1 (0.50 ±
0.038 nmol/mg) to P15 (0.712 ± 0.046 nmol/mg) and remained constant in P84 (0.726 ± 0.066
nmol/mg). Similarly, PE 40:4, PE 40:5, PE 40:6 and PE 38:6 were relatively abundant in P84
and significantly increased during the development of the lung. In contrast, less abundant PE
34:1, 36:2 and 38:5 exhibited higher concentrations in P1 but then significantly decreased in
P84. The concentration of other lipid species were low abundance and it did not show any
significant differences in all three groups.
PE based plasmalogens. PE P-16:0, PE P-18:0 and PE P-18:1 (sn-1) individual
plasmalogen compositions were calculated and their values have been displayed in Fig 7. Interestingly,
PE P-16:0 (sn-1 substituent) plasmalogens are present in higher amounts in all three groups.
Regardless of the alkenyl chain in sn-1, the plasmalogens containing PUFAs in sn-2 position
10 / 25
Fig 6. Composition of individual phosphatidylethanolamine (PE) lipid species during postnatal development of mouse lung. Values are represented as nmol/mg
wet weight. Values are mean ± SD, p-value summary: P < 0.0001, P < 0.001, P < 0.01, P < 0.05. Where significance is not mentioned, values are
considered as being not significant.
were the most abundant, represented mainly by 20:4 followed by 22:6, 22:4 and 22:5 in all
The PE P-16:0/22:4 and PE P-16:0/22:6 significantly rose during the postnatal lung
development (from P1 to P15 and P1 to P84). In contrast, the predominant PE P-16:0/20:4 and PE
P16:0/22:5 also rose during development from P1 to P15, however, a slight decrease from P15 to
P84 was noted. Similarly, PE P-18:0 based 20:4, 22:4, 22:6 and PE P-16:0/18:1 lipid species
increased significantly during the development process (P1 to P84). However, the values of
low abundant PE P-16:0/16:1 were significantly lower in P84 as compared to P1. Other
individual ethanolamine based plasmalogens were not significant during the postnatal lung
Phosphatidylinositol and phosphatidylserine species. The individual composition of 15
species of phosphatidylinositol and 23 species of phosphatidylserine is depicted in Fig 8A and
8B respectively. Similarly, to PE and PE based plasmalogens, both PI and PS were found to be
highly enriched with polyunsaturated species. PI 38:4 was the most abundant during all
developmental stages. PI 38:4 (0.806 ± 0.065 nmol/mg to 1.173 ± 0.027 nmol/mg), PI 36:4 were
11 / 25
Fig 7. Composition of individual PE based plasmalogen (PE P) lipid species during postnatal development of mouse lung. Values are represented as nmol/mg wet
weight. Values are mean ± SD, p-value summary: P < 0.0001, P < 0.001, P < 0.01, P < 0.05. Where significance is not mentioned, values are considered as
being not significant.
significantly increasing while progressing from P1 to P84, whereas less abundant PI 36:2 and
PI 38:5 species were higher in newborn and significantly gradually decreasing during the
postnatal lung development (Fig 8A).
Among PS, polyunsaturated species (total number of double bonds >2) 40:4, 40:5, 40:6,
38:4 and the PS 36:1 were highly abundant in all stages of lung development. PS 40:4 was
significantly gradually increasing from the phase of P1 (0.566 ± 0.04 nmol/mg) to P15 (1.218 ±
0.09) and from P15 to P84 (1.381 ± 0.06 nmol/mg). PS 38:4 and 36:1 species followed the same
pattern (rise from P1 to P15 and P15 to P84). In addition, PS 36:2, 38:3 and 40:6 was detected
in P1 and their increase was observed in P84. Among the PS species, PS 40:5 exhibited higher
levels in P15 (0.827 ± 0.039 nmol/mg) as compared to P1 (0.335 ± 0.021 nmol/mg) and P84
(0.662 ± 0.031nmol/mg). The other analyzed PS species did not reach significance during lung
development (Fig 8B).
Sphingomyelin and ceramide species. Within sphingolipids, individual 15
sphingomyelin, 7 ceramide and 2 cerebroside species compositions are depicted in Fig 9A and 9B
respectively. SM 34:1 was the most dominant SM species in all developmental stages and gradually,
12 / 25
Fig 8. Composition of individual phosphatidylinositol (PI) and phosphatidylserine (PS) lipid species during postnatal development
of mouse lung. Values are represented as nmol/mg wet weight. A) PI B) PS. Values are mean ± SD, p-value summary: P < 0.0001,
P < 0.001, P < 0.01, P < 0.05. Where significance is not mentioned, values are considered as being not significant.
but significantly increasing from the phase of P1 (0.46 ± 0.052 nmol/mg) to P15 (0.716 ± 0.05
nmol/mg), and thereafter slightly increasing from P15 to P84 (0.821 ± 0.057 nmol/mg).
Similarly, SM 42:2 followed the same pattern during development.
The very long chain SM lipid species 40:1, 42:1and 42:3 were significantly rising during the
postnatal lung development and their elevated levels were detected in P84 (Fig 9A).
Cer d18:1/16:0 was present in higher amounts in P15 lungs in comparison to P1 and P84.
The very long chain fatty acid containing ceramide lipid species Cer d18:1/24:0 exhibited
higher levels in P84. Interestingly, both of the analyzed HexCer d18:1/16:0 and HexCer d18:1/
24:1 was present at higher levels in P1 and a significant gradual decrease was observed during
the postnatal lung development up to the stage of P84 (Fig 9B).
Cholesteryl esters. Eighteen esterified forms of cholesterol were analysed and their
composition is depicted in Fig 10. Similar to LPC, the myristic acid (14:0) and palmitic acid (16:0)
containing CE species were significantly elevated in the phase of P15 in comparison to P1 and
P84. Oleic (18:1), palmitoleic (16:1) containing CE species were more elevated in P1 and
significantly lowered during development. In contrast, linoleic (18:2) and arachidonic (20:4) acid
containing CE species were significantly gradually increasing from the stage of P1 and reached
higher levels in P84.
Furthermore, the distribution pattern of GP according to their carbon chain length (total
number of carbon atoms), degree of unsaturation (total number of double bonds) was
elaborated and the results are shown in S2 Table, S1 Fig. Overall, the PC and PG comprised higher
amounts of lipid species with carbon chain length 36. Whereas PE, PS and PI contained
higher amount of lipid species with carbon chain length >36.
Lipids constitute a diverse group of biomolecules, playing many roles in lung biology,
especially in reducing the surface tension of alveoli to prevent the alveolar collapse and thereby
stabilizing the lung parenchyma. The lung major lipid classes originating either from the
pulmonary surfactant or from the bronchoalveolar lavage fluid (BALF) were already partly
characterized, but a detailed distribution of total lipid classes and of their individual lipid
molecular species composition in mouse lung during its postnatal development however, are
not fully understood. In our current study, we used direct flow injection electrospray
ionization tandem mass spectrometry to provide the total lipid quantity, and significant stage specific
alterations of individual lipid species during the process of mouse postnatal pulmonary
development. Furthermore, we showed the distribution pattern of lipid classes according to their
carbon chain length and degree of unsaturation during development process.
In the present study, we analyzed and quantified total 202 lipid species (GP, SP, and CE)
and cholesterol of the pulmonary lipidome from the mouse lung homogenates of the P1, P15
and adult lungs (Fig 1A & 1B and Fig 2 & S1 Table). In general, an increase in the total lipid
quantity (nmol/mg wet weight) of pulmonary tissue is a characteristic change during the lung
development process. Our results revealed increased levels of the total phospholipid and
cholesterol content during development. Indeed, these results are in consistence with the findings
of Williams and colleagues as well as of Hahn, et al. in which rat lung and other organ lipids
were studied during maturation [
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Fig 9. Composition of individual sphingomyelin (SM) and ceramide (Cer) lipid species during postnatal development of mouse
lung. Data are represented as nmol/mg wet weight. A) SM B) Cer. Values are mean ± SD, p-value summary: P < 0.0001,
P < 0.001, P < 0.01, P < 0.05. Where significance is not mentioned, values are considered as being not significant.
Furthermore, pulmonary lipid distribution patterns of the whole lung tissue in comparison
to the pulmonary surfactant lipid composition in different mammalian species were studied
(see book chapters in ref) [
]. In this context, it is important to mention that the
distribution pattern of the lipids in the lung tissue is heterogeneous between distinct species. In
general, PC's were the most predominant class of lipids of all tested species . The second most
abundant lipid class of the surfactant GP was PG, however, this lipid class was detected in
lower amounts in the whole lung tissue. In contrast, PE represented the minor class of lipids in
the surfactant but constituted second major lipid class following PC in the whole lung tissue.
Similarly, SM and PS lipid classes were detected in lower amounts in the pulmonary surfactant,
however, they also were described as being abundant in the whole lung tissue. In fact, our
results of the lipid class distribution of the mouse lung homogenates are in agreement with
these findings [
]. Our data clearly indicate that PC and cholesterol occupy major parts of
Fig 10. Composition of individual cholesteryl ester (CE) lipid species during postnatal development of mouse lung. Data are represented as nmol/mg wet weight.
Values are mean ± SD, p-value summary: P < 0.0001, P < 0.001, P < 0.01, P < 0.05. Where significance is not mentioned, values are considered as being
16 / 25
the whole lung lipidome (Panel A in S2 Fig). Among the phospholipids, PC is the most
dominant lipid class followed by PE (PE+PE P), PS, SM, PI and PG present during the postnatal
mouse lung development (Panel B in S2 Fig). Based on these composition values, we calculated
the molar ratios of lipid classes (S3 Table). For instance, the PC/LPC ratio is decreased from
P1 to P15, and from P15 to P84. In contrast, SM/Cer molar ratio significantly increased from
P1 to P15 and from P15 to P84.
Interestingly, we observed a significant increase in the concentration of cholesterol from
the phase of newborn to P15 (Fig 2, S1 Table & Panel A in S2 Fig), suggesting that cholesterol
may play an important role in the process of alveolarization. Cholesterol is an integral
component of various cell membranes, involved in the maintenance of membrane fluidity, membrane
functions and signal transduction. In fact, cholesterol is the major neutral lipid component of
the lung and up to 80% of the cholesterol present in the lung is in surfactant [
], and it is
considered as a protosurfactant in immature lungs, lungs with lack of septation and in saccular
]. Moreover, cholesterol enhances the adsorption of DPPC by increasing membrane
fluidity and control the surface viscosity of the surfactant [
In our study, we focused on the alterations of individual lipid species of mouse lungs during
the postnatal development. Our results showed a significant increase in the abundance of PC
30:0 during the alveolarization (P1 to P15) (Fig 3A) process. These results are supported by
previous observations on the postnatal development of the lung tissue and surfactant lipid
analyses from 8-day-old mice and adult animals [
]. Bernhard and co-workers reported
about significant alterations in the abundance of major PC lipid molecular species in the
pulmonary surfactant of different mammalian species during lung development [
31, 40, 41
In contrast to PC findings of surfactant lipidome, we showed a significant gradual increase
of PC 32:0 (most likely to be DPPC) during the postnatal lung development. DPPC is the
primary surface-active material found in majority of the mammalian species pulmonary
surfactant. Maintenance of adequate DPPC within air space is essential for normal lung function
]. RDS is the major cause of mortality and morbidity in premature infants diagnosed with
mainly DPPC deficiency in quantity and quality of pulmonary surfactant [
surfactant replacement therapy with added products of DPPC is an effective therapeutic strategy
available for RDS management [
]. Interestingly, high contents of monounsaturated lipids of
PC 32:1 and PC 34:1 was detected in P1 mice in comparison to P15 and adult lungs, suggesting
that PC 32:1 might be involved in the establishment of the air-liquid interface in newborn
animals. Furthermore, PC 34:1 is known to be crucial and plays an important role in the
adsorption of DPPC immediately after birth [
]. In contrast to mice, PC 30:0 is completely
absent and PC 32:1 is minimal in nonalveolar species (birds), in which the lung contains
capillaries instead of alveoli, suggesting that, PC 30:0 and PC 32:1 species are important and
play an active dynamic role in the alveolarization process . In this context, it is important
to mention the recent findings that PC 30:0 inhibited macrophage-triggered proliferation of
T-lymphocytes and decreased the production of reactive oxygen species (ROS) during
]. Moreover, PC 30:0 was significantly reduced in the emphysema patients
and infants with BPD as well as in the neonatal rat models of reduced alveolarization
suggesting that PC 30:0 may serve as a diagnostic marker for alveolar size during diseases .
Similarly to these observations, in the pig model, PC 30:0 was found high in abundance in
newborns and gradually decreased with age in adolescent pigs [
], whereas in humans and
guinea pigs, PC 30:0 was increased during the lung development [
]. The specific functions
of PC 30:0, PC 32:1 and PC 34:1 lipid species during postnatal lung development are not clear
yet. Also, the molecular mechanism and alterations of lipid species during the lung
development are not clear yet. These alterations may be specific for individual mammalian species, a
supposition that requires further investigation.
17 / 25
Phosphatidylglycerol lipid concentrations are highly concentrated in the lung compared to
other mammalian tissues [
]. It is well documented that PG lipids are involved in the
adsorption and, spreading of surfactant over the epithelial surface, as well as influence innate
immunity and protect against viral infections [
9, 67, 68
]. Interestingly, surfactant deficiency in
premature infants and also in mouse models of BPD showed the complete absence of PG lipids
]. Indeed, the presence of PG lipids in amniotic fluid is an indicator for the fetal lung
maturity and PG lipids are known to be vital in the management of neonatal RDS and other
obstetric conditions . It is known that PG lipid species are crucial for the lung function. There
are, however, no reports on the composition of PG 30:0 during the postnatal lung
development. In fact, Bernhard and colleagues were not able to measure PG 30:0 from rat surfactant
during the postnatal lung development [
]. Remarkably, in our study, we observed a
significant increase in PG 30:0 abundance during alveolarization (P1 to P15), similar to PC 30:0,
probably because of the high content of myristic acid during the postnatal lung development.
In contrast to DPPC, we observed a significant decrease of abundance of DPPG during
postnatal lung development. Recent findings suggest that DPPG interacts with vaccinia and variola
virus strains and reduces the infection of pneumocytes in respiratory poxvirus infection [
Moreover, several reports demonstrated that PG 34:1 (palmitoyl-oleoyl-phosphatidylglycerol,
POPG) acts as a potent antiviral lipid against influenza A and respiratory syncytial virus [
]. Interestingly, we observed a high content of this antiviral lipid PG 34:1 (POPG) in the
newborn mouse lungs in comparison to P15 and P84 (Fig 5), suggesting that they might
improve the innate immunity against viruses during the perinatal period.
In addition to PC and PG, we measured other lipid species in the lung tissue such as PE, PI
and PS (Figs 6, 8A & 8B). In contrast to PC and PG, we found that these lipid classes were
found to be mostly enriched with long chain polyunsaturated species. Monounsaturated lipid
species (PE 32:1) were found in high abundance in P1 in comparison to P15 and P84. In fact,
long chain polyunsaturated species serve as substrates for the pro-inflammatory (leukotrienes,
prostaglandins, etc.), as well as anti-inflammatory and pro-resolution (lipoxins, etc.) lipid
]. Furthermore, mass spectrometry imaging of adult mouse lungs showed that
these long chain polyunsaturated lipids are highly abundant at the epithelial lining of airways
]. We observed that 38:4 lipids were abundant in case of PE and PI lipid classes in all three
age groups, which may serve as a source for arachidonic acid (AA) for the generation of lipid
mediators. Proteomics data of a recent study revealed that proteins (cyclooxygenases,
lipoxygenases, etc.) responsible for the generation of bioactive lipid mediators, are significantly
upregulated in the adult mouse lungs, suggesting that these long chain polyunsaturated lipid
species serve as a source for AA [
Quantitative information of less abundant species of these lipid classes would help to
understand postnatal developmental alterations in detail. However, the mechanism of
alterations of individual lipid species of these lipid classes during lung development needs to be
Plasmalogens are glycerophospholipids characterized by a vinyl ether linkage in sn-1 and
an ester linkage in sn-2 position of the glycerol backbone. Plasmalogens are involved in the
membrane dynamics, serve as an endogenous antioxidants, protect against ROS and prevent
lipoprotein oxidation [
]. Plasmalogen biosynthesis starts in the peroxisomes and deficiency
of plasmalogens is associated with various peroxisomal disorders [
] and other respiratory
diseases like BPD [
], asthma and COPD . We observed that PE-based plasmalogens are
much higher abundant compared to ether-phosphatidylcholines (PC O) during the postnatal
mouse lung development. In ethanolamine plasmalogens, PE P-16:0 plasmalogens comprised
the highest amount, whereas, PE P-18:0 and PE P-18:1 made up a smaller amount (Fig 7).
Interestingly, we observed a high content of 20:4, 22:6, 22:5 and 22:4 (most likely to be
18 / 25
arachidonic acid AA, docosahexaenoic acid DHA, docosapentaenoic acid DPA and adrenic
acid)-rich plasmalogens (Fig 7). Further, the total quantities of plasmalogens (sum of all
analyzed PE P species) were gradually increased from P1 to P84 during the postnatal lung
development (S1 Table). These results are in consistent with the previous study, in which high
abundance of PE P species were noted in adult mouse lungs during postnatal pulmonary
developmental processes [
]. Arachidonic acid enriched plasmalogens seem to play an
important role in immune defence and normal lung physiology [
]. Plasmalogens are reported to
serve as a reservoir for the precursor molecules (e.g., AA, EPA, DHA, and DPA etc.) of
eicosanoids, which are biologically active secondary lipid signalling messengers or for maresin and
resolvins, lipid derivatives involved in the regulation of inflammation [77, 78]. RuÈdiger and
colleagues showed that addition of plasmalogens to surfactant-like phospholipid mixtures
reduces surface tension [
] and high content of plasmalogens in tracheal aspirate of preterm
infants reduces the risk of respiratory diseases [
]. Likewise, another study reported that high
contents of plasmalogens protects the endothelial cells from hypoxia and ROS mediated stress
Sphingolipids are primarily found in cell membranes and are involved in diverse biologic
processes such as migration, proliferation, differentiation, senescence, cell death, autophagy,
and efferocytosis [
]. In the lungs, sphingolipids are associated with cystic fibrosis, asthma,
pulmonary edema, BPD, inflammation, lung injury and various types of lung cancers [
Ceramides show both proliferative and apoptotic effects depending on their concentration and
chain length [
]. In analyzed SM lipid species, we detected that SM 34:1 lipid species as being
predominant in all stages (Fig 9A). Both sphingomyelin 34:1 and ceramide species (Cer d18:1/
16:0) showed high contents during alveolarization, especially in P15 (Fig 9A & 9B) mouse
lungs, suggesting that, these lipid species are involved in the remodelling of tissue, also
observed in the rat lung development [
]. In contrast to our findings (P1 to P15), a recent
study on mouse lungs using LC-MS/MS approach showed no significant alterations in the Cer
d18:1/16:0 levels from P7 to P14 [
]. The total content of sphingomyelin gradually increased
with the age and the necessary transfer of biochemical substances across the semipermeable
membranes (S1 Table).
So far, no reports are available about the developmental changes of less abundant
cholesteryl ester species in the mouse lung. Saturated fatty acids such as myristic acid (14:0)- and,
palmitic acid (16:0)-containing lysophosphatidylcholines (Fig 4) and cholesteryl esters (Fig 10)
were found highly abundant during P15 in comparison to P1 and P84. Monounsaturated fatty
acid (MUFA) containing CE species were elevated in the newborn, whereas polyunsaturated
fatty acid (PUFA) containing CE species were elevated in adult lungs. Physicochemical
properties of lipids depend on their chain length and their degree of unsaturation. In this aspect, we
calculated the distribution patterns of glycerophospholipids according to their carbon chain
length (number of carbon atoms). We observed that, PC and PG glycerophospholipids are
highly abundant of lipid species with carbon chain length C 36, whereas PE, PS and PI
glycerophospholipids are highly abundant with long chain lipid species (C>36). The majority of the
monounsaturated glycerophospholipids were found to be highly abundant in newborns,
whereas polyunsaturated lipid species were highly abundant in adult lungs (S1 Fig). In
contrary, P15 lungs exhibited high contents of myristic (14:0)- and palmitic (16:0)-acid containing
Limitations and weaknesses of the study
In the current study, we performed an extensive quantitative lipidomic analysis of P1, P15 and
P84 mouse whole lung tissue homogenates to understand the changes occurring during
19 / 25
postnatal development. The data provides lipidomic alterations in mouse lung during
developmental process. However, at this stage we are not able to discriminate the lipidomic changes
occurring specifically at cellular (membrane or intracellular) and extracellular (alveolar) level.
Comprehensive comparative (quantitative) lipidomic analysis of bronchoalveolar lavage fluid
(BALF) and whole lung tissue homogenates in mice and other mammalian species in which
alveolarization continues beyond extra-uterine life (e.g. rats) needs to be investigated in the
near future, which can provide deeper insights for a better understanding of pulmonary
developmental process at molecular and cellular level.
In our study, we have provided the total lipid quantity and given a detailed overview of lipid
classes as well as absolute quantitative information on the individual lipid species and their
distribution pattern according to carbon chain length and degree of unsaturation during
postnatal mouse lung development using high-throughput tandem mass spectrometry. Our study
provides an extensive quantitative lipidome of whole mouse lung tissue (including less
abundant lipid species, neutral lipid components such as cholesterol and their esters), which may
serve as reference for understanding the occurring lipid alterations, which in turn affect lung
function during development or in pulmonary diseases.
S1 Table. Quantitative analyses of individual lipid classes in mouse lung during postnatal
development. Values are expressed as nmol/mg wet weight and represented as mean ± SD.
S2 Table. Distribution of glycerophospholipids according to carbon chain length (total
number of carbon atoms) and alkenyl chain (PE P) during postnatal development of
mouse lung. Values are represented as nmol/mg wet weight.
S3 Table. Molar ratios of lipid classes. Displayed are the values of molar ratios of lipid classes.
Values are represented as mean ± SD.
S1 Fig. Distribution of glycerophospholipids according to degree of unsaturation (total
number of double bonds) during postnatal development of mouse lung. Saturated (total
number of double bonds = 0), Monounsaturated (total number of double bonds = 1),
Polyunsaturated (total number of double bonds 2) lipids.
S2 Fig. Total lipid composition of mouse lung during postnatal development. The
displayed values are mol% of the respective lipid class of all analyzed lipids. Panel A)
Glycerophospholipids (GP), sphingolipids (SP), cholesteryl esters (CE) and cholesterol. Panel B) only GP,
SP without CE and cholesterol. Values are represented as mean ± SD, p-value summary:
P < 0.0001, P < 0.001, P < 0.01, P < 0.05.
S1 File. Lipid profiling of mouse lung during postnatal development used in this study.
The list of identified and quantified lipid molecules, experimental protocol, abbreviations and
20 / 25
Formal analysis: Vannuruswamy Garikapati, Gerd Schmitz.
Investigation: Gerhard Liebisch.
Methodology: Gerhard Liebisch, Paul P. Van Veldhoven.
Supervision: Srikanth Karnati.
Validation: Bernhard Spengler, Eveline Baumgart-Vogt.
Visualization: Paul P. Van Veldhoven, Bernhard Spengler, Eveline Baumgart-Vogt.
Writing ± review & editing: Srikanth Karnati, Vannuruswamy Garikapati.
21 / 25
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Regulatory, integrative and comparative physiology. 2005; 289(5):R1426±39. https://doi.org/10.1152/
ajpregu.00496.2004 PMID: 16037124.
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