1.
Separation of lipid classes by SPE
Extractions on aminopropyl bonded phase columns were performed on the SPE device (Baker), which allows to
processed more samples simultaneously. Columns were conditioned by 4 ml of hexane. The 100 l of
chloroform solution with a sample or a mixture of standards was applied on the columns. The first fraction,
containing all neutral lipids, was obtained after washing the column by 4 ml of mobile phase A– chloroform and
2-propanol (2:1, v/v).The second extraction phase containing free fatty acid was eluted with 4 ml of the mobile
phase B– 2% acetic acid in diethyl ether (v/v). The last fraction comprising phospholipids was eluted by 4 ml of
the mobile phase C – methanol-water (9:1, v/v). All three fractions were dried by stream of nitrogen, then
dissolved in chloroform, and stored at -20°C for further analyses – derivatization and GC/FID measurement and
also for HPLC/ESI/MS analyses.
The PL and NL recovery of the SPE method: The PL recovery of the SPE method was calculated from the
addition of internal standards mentioned in chapter 2.1 (covers 5 particular PCs and 4 PEs). Standards were
added in two levels (conc. 10µg/ml and 50µg/ml) to the matrix – extract of Pyrrhocoris apterus fat body. Each
level of standards was repeated ten times, and the result represents mean of these repetitions. Nonpolar lipids in
our case are represented by TG and DG and the phospholipid fraction by PE and PC. These classes were chosen
because of their major abundance in insect biological samples.
2.
Characterization of FA acyls by GC/FID
GC analyses were performed on a GC VARIAN 3400 equipped with split/splitless injector and flame ionization
detector (FID) (USA). A 30 m x 0.22 mm BPX 70 (0.25 m) column from SGE (SciTech, Prague, Czech
Republic) was used with an oven temperature program from 120 to 260 C at a rate of 10 C/min. The injector
and the FID detector were set at 260 and 280 C, respectively. The helium carrier gas flow-rate was 30 cm/s,
with a split flow-rate of 15 ml/min.
3.
Determination of the PL classes by phosphorus analysis
The protocol involved the following steps:
1) Scraping off the silica gel of the individual spots of SP, PC, PS, PI, PE and CL from the HPTLC plates after
developing, drying and visualization by iodine into 5 ml vials
2) Adding HClO4 (0.5 ml of 70 %) to the scraped silica gel in vials, combustion for 20 min. in an electric heater
set at 180  C temperature
3) Cooling 15 min., adding of water (4 ml), mixing
4) Adding of (NH4)2MoO4 (0.5 ml, 2.5 % in water), mixing
5) Adding of freshly prepared aqueous acetic acid (0.5 ml, 10 %), mixing, warming 5 min. in boiling water bath
6) Quick cooling, leaving stand until next day at room temperature
7) Spectrophotometric measurements at 820 nm
The calibration was based on spectrophotometric measurement of the KH 2PO4 standard solutions (0, 0.025, 0.05,
0.1 and 0.2 mol/ml concentrations of KH2PO4 were used), processed in the same way as described above for
phospholipids. For each series of measurements, blank spots of the same size as those of the phospholipids were
scraped off and processed.
1
The standards of PC- and PE - dioleoyl (20 g per spot) were applied together with samples on HPTLC plates
and subjected to the same procedure of phosphorus analysis.
4.
Figure S1
HPTLC separation of particular lipid classes
1, 3 - lipid extract from Pyrrhocoris apterus female thoraces
2, 4 - lipid extract from Pyrrhocoris apterus male thoraces
5 - lipid standard mixture
The concentration levels of SM varied from 2.5 to 9.0, CL - 1.7 to 7.1, PS 1.8 to 9.5 and PI 2.8 to 7.5 mmol/mg of muscle tissue (n=15). No significant differences were recorded
between the sexes.
2
5.
Figure S2
HPLC ESI MS total ion current chromatogram of P. apterus thoracic muscle of PL SPE fraction. For better
clearance is only the part of chromatogram (from 7.76 to 25.27 min) presented.
3
6.
Figure S3
GC FID chromatograms of total lipid extract of P. apterus thoracic muscle and fatty acid methyl esters of
purchased standards of fatty acids with 18 carbons and one double bond (all from Sigma-Aldrich (Germany).
Additionally some more standards are present in chromatograms. The total lipid extract is on the top and
particular fatty acid methyl esters are marked by Arabic number and the standards are marked by letters, as
follows in table:
double
bonds position
mark
lenght
IS
C26
12.754
1
C16
10.441
2
C16
3
C17
4
cis/trans rt
1
9 cis
10.942
11.301
No methyl ester - contamination 11.852
5
C18
12.086
6
C18
1
9 cis
12.349
7
C18
2
12,10 cis
12.823
8
C18
3
15, 13, 11 cis
13.398
a
C18
1
9 trans
12.241
b
C18
1
11 trans
12.288
c
C18
1
5 cis
12.317
d
C20
5 9, 11, 13, 15, 17 cis
15.161
e
C18
1
9 cis
12.347
f
C20
3
14, 16, 18 cis
14.458
g
C18
1
11 cis
12.416
h
C20
1
11 cis
13.749
i
C18
1
12 cis
12.461
j
C20
1
13 cis
13.813
k
C18
1
15 cis
12.618
l
C18
4
9, 11, 13. 15 cis
13.676
The updated data was measured due laboratory
innovation by autosampler AOC – 20S,
(Shimadzu)
with
(Shimadzu)
and
injector
AOC
chromatographic
–
20i,
column
BPX-70 (SGE) coupled with flame ionization
detector
GC-2014
(Shimadzu).
The
temperature was set to 120°C and than
increased by rate 10°C in minute to 230°C and then hold for 4 minutes.
4
5