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An Improved High-Throughput Lipid Extraction Method For The Analysis of Human Brain Lipids
An Improved High-Throughput Lipid Extraction Method For The Analysis of Human Brain Lipids
DOI 10.1007/s11745-013-3760-z
METHODS
Abstract We have developed a protocol suitable for phosphatidylserines) were analyzed via electrospray ioni-
high-throughput lipidomic analysis of human brain sam- zation mass spectrometry and sterol species were analyzed
ples. The traditional Folch extraction (using chloroform using gas chromatography mass spectrometry. No differ-
and glass–glass homogenization) was compared to a high- ences in lipid species composition were evident when the
throughput method combining methyl-tert-butyl ether lipid extraction protocols were compared, indicating that
(MTBE) extraction with mechanical homogenization MTBE extraction with mechanical bead homogenization
utilizing ceramic beads. This high-throughput method provides an improved method for the lipidomic profiling of
significantly reduced sample handling time and increased human brain tissue.
efficiency compared to glass–glass homogenizing. Fur-
thermore, replacing chloroform with MTBE is safer (less
Keywords Lipidomics Human-brain Ceramide
carcinogenic/toxic), with lipids dissolving in the upper
Sphingomyelin Phosphatidylcholine
phase, allowing for easier pipetting and the potential for
Phosphatidylethanolamine Phosphatidylserine Sterol
automation (i.e., robotics). Both methods were applied to
the analysis of human occipital cortex. Lipid species
(including ceramides, sphingomyelins, choline glycero- Abbreviations
phospholipids, ethanolamine glycerophospholipids and BHT Butylated hydroxytoluene
BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide
CE Collision energy
Cer Ceramide
CerPCho Sphingomyelin
ChoGpl Choline glycerophospholipid
S. K. Abbott A. M. Jenner T. W. Mitchell CV Coefficient of variation
S. H. J. Brown B. Garner CXP Collision cell exit potential
Illawarra Health and Medical Research Institute, University DP Declustering potential
of Wollongong, Wollongong, NSW 2522, Australia EP Entrance potential
S. K. Abbott (&) A. M. Jenner B. Garner ESI–MS Electrospray ionization mass spectrometry
School of Biological Sciences, University of Wollongong, EtnGpl Ethanolamine glycerophospholipid
Wollongong, NSW 2522, Australia GC-MS Gas chromatography mass spectrometry
e-mail: [email protected] MTBE Methyl-tert-butyl ether
T. W. Mitchell S. H. J. Brown MRM Multiple reaction monitoring
School of Health Sciences, University of Wollongong, NL Neutral loss
Wollongong, NSW 2522, Australia PI Precursor ion
PtdSer Phosphatidylserine
G. M. Halliday
Neuroscience Research Australia and the University SGalCer Sulfatide
of New South Wales, Sydney, NSW 2031, Australia TMCS Trimethylchlorosilane
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Lipids
Materials and Methods 60, 78 and 88 years with post-mortem delay of 25, 6.5 and
9 h respectively.
Materials and Internal Standards
Lipid Extraction
MTBE, chloroform, methanol, acetone, hexane and aceto-
nitrile were HPLC grade and purchased from Thermo A comparison of the two lipid extraction methods is shown
Scientific, Scoresby, VIC, Australia; analytical grade in Fig. 1 and each is described in detail below.
butylated hydroxytoluene (BHT) and sodium hydroxide
(98 % minimum) from Sigma Aldrich, Sydney, NSW, Traditional Glass-Chloroform
Australia; analytical grade ammonium acetate from Crown
Scientific, Moorebank, NSW, Australia; 15 9 45 mm This protocol was based on the traditional Folch extraction
(4 mL) screw thread vials and 12 9 32 mm (1.8 mL) wide [13] with modifications previously described (see [3, 27,
mouth vials (both with PTFE/silicone septa caps) from 28]). Each 10-mg pulverized brain aliquot was homoge-
Grace Davison, Rowville, VIC, Australia. All solvents nized on ice with a glass–glass homogenizer (1 mL Ten-
contained 0.01 % w/v BHT. broeck Tissue Grinder; Edwards Instrument Company,
Two internal standard mixtures were added to each Narellan, NSW, Australia) in 500 lL methanol containing
sample. Firstly, 4 lL/mg of tissue of the phospholipid/ internal standards (plus 166 lL methanol to rinse homog-
sphingolipid standard mixture containing: 75 lM CerP- enizer). The homogenate was transferred to a 4-mL vial
Cho(d18:0/12:0), 75 lM Cer(d18:1/10:0), 500 lM Ptd- and 1,332 lL chloroform added (chloroform/methanol 1:2,
Cho(19:0/19:0), 175 lM PtdSer(17:0/17:0), 225 lM by vol). The homogenate was split, with 1/3 transferred to a
PtdEtn(17:0/17:0), 25 lM Lyso PtdCho (17:0) and 25 lM
Lyso PtdEtn (14:0) in chloroform/methanol (2:1, by vol);
Avanti Polar Lipids purchased from Auspep, Tullamarine,
VIC, Australia. Secondly, 10 lL/sample of sterols standard
mixture in ethanol was added, including: desmosterol-d6,
14-demethyl-lanosterol-d6, (Avanti Polar Lipids), b-sitos-
terol-d7, campesterol-d3 and 7-ketocholesterol-d7 (CDN
Isotopes, Quebec, Canada) and 27-hydroxycholesterol-d5
and 24-hydroxycholesterol-d7 (Medical Isotopes, Inc, Pel-
ham, AL, USA). Lanosterol, squalene, stigmasterol and
24,25-dihydrolanosterol were quantitated using other
heavy sterol internal standards.
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Lipids
separate 4-mL vial (for phospholipid analysis), while the centrifugation and combining the upper phase with the first
remaining 2/3 was used for sphingolipid and sterol analy- extract. This lipid extract was then dried under nitrogen at
sis. Both fractions were treated equally except for the 37 °C and resuspended in chloroform/methanol (1:2, by
addition of sodium hydroxide (final concentration 0.7 M) vol) as described above.
to the sphingolipid and sterol fraction (to promote hydro-
lysis of phospholipid ester linkages). Following overnight Mass Spectrometry and Lipid Analysis Settings
rotation at 4 °C, 0.15 M ammonium acetate was added
(chloroform/methanol/0.15 M ammonium acetate 8:4:3, by Phospholipid and sphingolipid extracts were analyzed via
vol; 333 lL for sphingolipid/sterols and 167 lL for phos- ESI–MS using a hybrid triple quadrupole ion trap mass
pholipids), the sample centrifuged (10 min at 2,0009g at spectrometer (QTRAPÒ 5500, AB SCIEX, Framingham,
4 °C) and the chloroform phase (lower phase) containing Massachusetts, USA) and an attached chip-based, auto-
lipids transferred to a new 4-mL vial. Chloroform/methanol mated nanospray source (Triversa NanomateÒ, Advion,
(1:2, by vol) was added to the original homogenate Ithaca, NY, USA). See Table 1 for ESI–MS settings. Prior
(equivalent to original volume), centrifuged as above and to introduction on the mass spectrometer lipid extracts were
the lower phase combined with the first chloroform extract. diluted 1/50 with chloroform/methanol (1:2, by vol) and
A second phase separation was performed on this chloro- ammonium acetate was added (final concentration 5 lM).
form extract by adding 0.15 M ammonium acetate (333 lL Samples (50 lL) were loaded onto a 96-well plate, sealed
for sphingolipid/sterols and 167 lL for phospholipids), with foil and centrifuged for 10 min at 2,2049g (Eppendorf
centrifuging as above and transferring the lower phase to a Centrifuge 5430). The spray parameters were set with a gas
new 4-mL vial (1.8-mL vial for phospholipid fraction), pressure of 0.4 psi and voltage applied at 1.2 and 1.1 kV for
which was then dried under nitrogen at 37 °C. The positive and negative mode respectively. Multi-channel
phospholipid fraction was resuspended in 300 lL chloro- acquisition (MCA) was employed for sphingomyelin and
form/methanol (1:2, by vol). The volume for the sphingo- ceramide precursor ion scans only.
lipid and sterol fraction was 700 lL, with half (350 lL) Phospholipid and sphingolipid species were quantified
transferred to a 1.8-mL vial for sphingolipid analysis and using AB SCIEX LipidviewTM Software (Framingham,
the remainder (350 lL) used for sterol analysis (see below Massachusetts, USA; see [29]). The processing settings for
for derivatization procedures). All phospholipid and LipidviewTM were: mass tolerance 0.5 and minimum %
sphingolipid extracts were stored at -80 °C prior to anal- intensity 0 (all species) and minimum S/N 50 (Cer, Cer-
ysis via ESI–MS. PCo, ChoGpl, EtnGpl) or 0 (PtdSer). Head group scans
were used to quantify each lipid species (see Table 1) and
Bead-MTBE the fatty acyl chain scans were used to determine each
molecular species (for phospholipids only). Masses that
The MTBE extraction was modified from [20] with could be assigned to ether or odd-chain fatty acids were
PrecellysÒ24 settings based on previous work [16]. Each assumed to be ether-linked phospholipids. Where two or
10-mg pulverized brain aliquot was weighed directly into a more isomeric species from the same class were detected
0.5-mL Precellys tube (Sapphire Bioscience, Waterloo, the relative proportion of the peak areas from each fatty
NSW, Australia) and homogenized in 300 lL ice-cold acyl scan was used to calculate the final corrected areas for
methanol containing internal standards, using a Precel- each molecular species. For example, ChoPCo (16:0/18:1)
lysÒ24 (Bertin Technologies; 2 9 30 s at 6,000 rpm). and ChoPCo (18:0/16:1) made up 95 and 5 % of total
After transferring the homogenate to a 4 mL glass vial the ChoPCo 34:1 peak areas respectively. Any species \0.5 %
Precellys tube was rinsed with 162 lL methanol and of lipid class were removed from the analysis. Ether-linked
1,540 lL MTBE added to the glass vial (MTBE/methanol EtnGpl do not produce the neutral loss of 141 Da to the
10:3, by vol). The homogenate was split into phospholipids same extent as diacyl EtnGpl [30] and previous work in our
and sphingolipid/sterol fractions as described above. Fol- group has demonstrated that plasmalogens produce the 141
lowing overnight extraction/hydrolysis, 0.15 M ammonium NL at about 29 % the efficiency of diacyl EtnGpl [31]. As
acetate was added (MTBE/methanol/0.15 M ammonium a diacyl internal standard was used to quantify all EtnGpl
acetate 20:6:5, by vol; 253 lL for sphingolipid/sterols and in the present study, an experimentally determined cor-
127 lL for phospholipids), the sample centrifuged (as rection factor of 3.45 was applied to the ether-linked Et-
above) and the organic phase (upper phase) collected in a nGpl data (T. Mitchell, unpublished data). For ceramide the
4-mL vial. The original homogenate was re-extracted by characteristic ions were [M?H]? and [M-H2O]? for each
addition to the upper phase of MTBE/methanol/0.15 M species. The peak areas for both ions were exported from
ammonium acetate (20:6:5, by vol; 205 lL for sphingo- LipidviewTM, combined and corrected against the com-
lipid/sterols and 411 lL for phospholipids), followed by bined peak areas from the internal standard.
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Lipids
Table 1 ESI-MS settings for phospholipid and sphingolipid analysis of human brain occipital cortex
Lipid Ion mode Scan type DP EP CE CXP Range
Sterol Derivatization and GC–MS Settings After drying under nitrogen at 37 °C and reconstitution
in 30 lL toluene, derivatized samples were analyzed by an
The sterol fraction of the lipid extract (from both protocols) Agilent 7000B triple quadrupole mass selective detector
was dried down under nitrogen at 37 °C and the following interfaced with an Agilent 7890A GC system gas chro-
added: 1.5 mL 0.1 M hydrochloric acid, 1.5 mL 80 mM matograph, equipped with an automatic sampler and a
acetic acid, 250 lL 1 M sodium hydroxide and 250 lL computer workstation. The injection port and GC–MS
methanol (pH of final solution *4.75). Solid phase interface were kept at 280 °C. Separations were carried out
extraction columns (Waters Oasis Max 3 cc; Waters, Ry- on a fused silica capillary column (30 m 9 0.2 mm i.d.)
dalmere, NSW, Australia) were preconditioned with 2 mL coated with cross-linked 5 % phenylmethylsiloxane (film
methanol and 2 mL 40 mM acetate buffer (pH 4.5). The thickness 0.33 lM; Agilent, J and W). Helium was the
sample was then loaded and washed with 2 mL 30 % carrier gas with a flow rate of 1.2 mL/min (average
methanol 40 mM formic acid (pH 4.5). Sterol fractions velocity = 59 cm/sec). Derivatized samples (1 lL) were
were eluted with 1.8 mL hexane followed by 1.8 mL injected splitless into the GC injection port. Column tem-
acetone/hexane (30:70, by vol) and then dried down under perature was increased from 165 to 270 °C at 30 °C/min
nitrogen at 37 °C. Samples were then derivatized by the after 1 min at 165 °C, then temperature was raised to
addition of 20 lL acetonitrile and 20 lL Selectra-SIL 300 °C at 5 °C/min and held at 300 °C for 4 min. Selected-
BSTFA(N,O-bis(trimethylsilyl)trifluoroacetamide) con- reaction monitoring was performed using the PCI mode
taining 1 % TMCS (trimethylchlorosilane; United Chemi- using methane as the reagent gas (4 mL/min), Argon as the
cal Technologies, purchased from PM Separations, QLD, collision gas (1.5 mL/min) and 2.25 mL/min atomic He in
Australia) and incubation for 1 h at 37 °C. the collision cell to reduce chemical noise from metastable
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Lipids
Table 2 Target ion transitions used for GC–MS analysis of sterols in (a) and the Glass-Chloroform in the middle panel (b).
human brain occipital cortex Comparison of these representative spectra for each lipid
Compound Target ion transition class reveals no obvious difference between the two
m/z ? m/z (Collision energy) methods, in terms of ions present or the maximum ion
intensity (see Figs. 2, 3, 4, 5, 6; ‘‘a’’ vs. ‘‘b’’). This indi-
Squalene 327 ? 203 (5)
cates that the recovery levels were similar for both
(H)14-Demethyl-lanosterol 475 ? 399 (6)
methods.
14-Demethyl-lanosterol 469 ? 393 (4)
The lower panels (c) in Figs. 2, 3, 4, 5,and 6 show the
(H) Desmosterol 461 ? 371 (10)
quantified concentrations for each lipid class (nmol/g tissue
Desmosterol 455 ? 365 (8)
wet weight). The most abundant lipid species in the human
(H) Campesterol 474 ? 384 (8)
occipital cortex was ChoGpl (total *19,000 nmol/g tis-
Campesterol 471 ? 381 (8)
sue), followed by EtnGpl (*8,000 nmol/g tissue) and
24,25-Dihydrolanosterol 500 ? 395 (10)
PtdSer (*6,000 nmol/g tissue), CerPCho (*4,000 nmol/g
Stigmasterol 395 ? 255 (5)
tissue), with much lower levels of ceramide present
(H) b-Sitosterol 478 ? 75 (30) (*600 nmol/g tissue). No significant difference between
b-Sitosterol 471 ? 75 (25) Bead-MTBE and Glass-Chloroform methods was evident
Lanosterol 483 ? 393 (3) for the total levels of each lipid class (p [ 0.28).
(H) 24-Hydroxycholesterol 462 ? 372 (8) Looking in more detail firstly at the sphingolipids, we
24-Hydroxycholesterol 455 ? 365 (3) identified 13 ceramide species using both extraction
(H) 7-Ketocholesterol 480 ? 390 (20) methods (Fig. 2c). The most abundant species were
7-Ketocholesterol 473 ? 383 (10) Cer(d18:1/18:0) and Cer(d18:1/24:1). Only Cer(d18:1/
(H) 27-Hydroxycholesterol 462 ? 161 (15) 18:0) showed a significant increase (15 %) in concentration
27-Hydroxycholesterol 457 ? 161 (20) in the Bead-MTBE extraction (p = 0.027). For sphingo-
myelin, 15 species were identified (Fig. 3c). The major
species present were: CerPCho(d18:1/18:0), CerP-
Cho(d18:1/24:1) and CerPCho(d18:1/20:0). No significant
He. The ion source was maintained at 280 °C and the difference between extraction protocols was evident in the
quadrapoles at 150 °C. Table 2 lists the target ion transi- level of any CerPCho species (p [ 0.14).
tions that were monitored for each sterol species. In terms of the glycerophospholipids, 22 ChoGpl species
were present in the human occipital cortex (20 diacyl and 2
Statistical Analysis ether), with ChoGpl(16:0/18:1) and ChoGpl(16:0/16:0) the
most abundant species (Fig. 4c). No difference between the
Statistical analysis was performed using JMP 9.0 (SAS two methods was observed for ChoGpl analysis (p [ 0.11).
Institute Inc., NC, USA). All results are expressed as A total of 33 EtnGpl species were identified (18 diacyl and
means ± SEM. with p \ 0.05 set as the level of signifi- 15 ether species), with EtnGpl(18:0/22:6) by far the most
cance. Data was tested for normality using Shapiro–Wilk abundant species present (Fig. 5c). Only EtnGpl(O-16:1/
W test and homogeneity of variance using a 2-sided F Test. 22:5) and EtnGpl(O-18:2/20:4) showed a significant
Student’s t tests were used for comparisons between increase (27 % each) using the Bead-MTBE extraction
treatments (Bead-MTBE versus Glass-Chloroform) for method (p = 0.022 for both). Ether-linked EtnGpl species
each lipid species. were present at 35 % of total EtnGpl in human occipital
cortex. This value corresponds with the plasmalogen levels
present in normal human brains in previous studies, with
Results and Discussion values varying from 23 % in human anterior cingulate
cortex and primary visual cortex [3], up to 56 % of total
Data resulting from the quantification of human occipital EtnGpl in temporal cortex grey matter [32].
cortex lipids that were solvent-extracted using either the No significant difference between the two protocols was
Glass-Chloroform or Bead-MTBE methods are presented observed for the PtdSer analysis (p [ 0.38; Fig. 6c). Of the
in a similar format in all figures. The results for the 17 PtdSer species quantified, PtdSer(18:0/18:1) and Ptd-
sphingolipids, Cer and CerPCho, are shown in Figs. 2 and Ser(18:0/22:6) were present at the highest levels. Results
3 respectively. The glycerophospholipids, ChoGpl, EtnGpl for the lysoglycerophospholipids were consistent with their
and PtdSer, are shown in Figs. 4, 5 and 6 respectively. In diacyl counterparts, with 12 species of lyso-ChoGpl and 10
each of these figures, representative head group spectra species of lyso-EtnGpl quantified (results not shown). No
from the Bead-MTBE extraction are found in the top panel significant difference between extraction methods was
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Fig. 4 Comparison of choline glycerophospholipid (ChoGpl) species Fig. 5 Comparison of ethanolamine glycerophospholipid (EtnGpl)
identified in human occipital cortex using either the Bead-MTBE or species identified in human occipital cortex using either the Bead-
Glass-Chloroform method. ESI–MS precursor ion scan targeting MTBE or Glass-Chloroform method. ESI–MS neutral loss scan
184 m/z for: a Bead-MTBE extraction; b Glass-Chloroform extrac- targeting 141 Da for: a Bead-MTBE extraction; b Glass-Chloroform
tion; c Quantification of brain choline glycerophospholipid species extraction; c Quantification of brain ethanolamine glycerophospholipid
using both methods (Bead-MTBE black bars; Glass-Chloroform grey species using both methods (Bead-MTBE black bars; Glass-Chloroform
bars); n = 3 per group; error bars show ± SEM grey bars); n = 3 per group; error bars show ± SEM. *p \ 0.05
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phosphatidylserine) and sterols. This high-throughput Bead- 11. Fonteh AN, Harrington RJ, Huhmer AF, Biringer RG, Riggins
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Acknowledgments Adult brain tissue was received from the Syd- methanol. Clin Chim Acta 149:89–93
ney Brain Bank at Neuroscience Research Australia which is sup- 16. Fraser T, Tayler H, Love S (2008) Low-temperature improved-
ported by the National Health and Medical Research Council of throughput method for analysis of brain fatty acids and assess-
Australia, the University of New South Wales and Neuroscience ment of their post-mortem stability. J Neurosci Methods
Research Australia. This research was supported by a project grant 169:135–140
(APP1008307) from the Australian National Health and Medical 17. Fraser T, Tayler H, Love S (2010) Fatty acid composition of
Research Council (NHMRC) awarded to GMH and BG. TWM and frontal, temporal and parietal neocortex in the normal human
BG are supported by Australian Research Council Future Fellowships brain and in Alzheimer’s disease. Neurochem Res 35:503–513
(FT110100249 and FT0991986, respectively). GMH is supported by 18. Römisch-Margl W, Prehn C, Bogumil R, Röhring C, Suhre K,
an NHMRC Principal Research Fellowship (630434). Adamski J (2012) Procedure for tissue sample preparation and
metabolite extraction for high-throughput targeted metabolomics.
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