Figure S1 Mueller et al. Oxylipin analysis methods
Figure S1 legend
Figure S1
Mechanisms of oxygen-18 exchange in [18O]water. Mechanisms a and b are
employed to prepare internal standards in Box 2 of the main text. a) carboxyl-oxygens can be
exchanged in the presence of a strong acid catalyst such as anhydrous HCL. b) under very
mild, neutral conditions, both oxygens can be exchanged using an enzyme (E) such as an
esterase or a lipase as catalyst. c) another option is the alkaline procedure, in which the acids
are first methylated (Me) and subsequently hydrolysed in [18O]water. Note that in this
mechanism almost complete exchange can only be achieved by repeating the reaction
sequence. Stars indicate oxygen-18 labeled oxygen atoms. Mechanism c is exploitable with
the alkaline exchange procedure described below.
Supplementary protocols and examples:
Acid oxygen exchange conditions for selected compounds:
Typically, weak acids with relatively high pKs values are more readily labeled than strong
acids with a low pKs. Water-free oxylipins (0.5 mg) are dissolved in 50 µL of a commercial
premade 4 M HCl solution in 1,4-dioxane (Aldrich: 34,554-7). Oxygen-18 water (25 µL) is
added. Incubations should be performed in 0.3 mL glass vials with a screw cap sealed with a
teflon disk in a heating block. After incubation, the cap is removed and replaced by a GCseptum. Thereafter, the sample is dried at 90°C under vacuum by penetrating the septum with
a needle connected to a vacuum line. An incubation period of 2 h at 70°C or 20 h at 40°C is
sufficient to incorporate theoretical amounts of oxygen-18 into fatty acids. In the case of JA,
initiate the labeling with water-free MJ; an incubation period of 2 h at 90°C is sufficient to
reach almost equilibrium (unlabeled JA should,ideally be below 0.5 % and triply labeled JA
above 70%). The keto group oxygen-18 label needs to be re-exchanged (see below) since this
oxygen is labile under weak acidic conditions or even in the injector port of the GC under
certain conditions. Salicylic acid is a strong acid that can be labeled by incubation with the
labeling solution at 90°C for 12 h (yielding about 1% unlabeled salicylic acid). The mixture is
then dried and the procedure is repeated in order to yield labeled standard containing less than
0.2 % unlabelled salicylic acid. The labeling procedure also works well with other
compounds. For instance, amino acids can be labeled by incubation with [O18] water (50 µL)
and 4 M HCl/dioxane (25 µL) at 90°C for 12 h. It should be noted that the protocol works
equally well with methyl esters since ester hydrolysis occurs rapidly and quantitatively under
the acid labeling conditions.
Re-exchange of keto group oxygen-18 labels in JA
Triply labeled JA can be readily converted to doubly labeled JA since the carboxylic acid
group are stable under alkaline conditions while the keto group oxygen rapidly exchanges
with the oxygen of water. Triply labeled JA is dissolved in 30 µL of MeOH and this is
carefully transferred to a fresh reaction tube. A 1 M solution of KOH in unlabeled water (20
µL) is added and mixed with the pipette (do not vortex) and the solution is incubated at 40°C
for 1 h. Thereafter, 0.5 mL of water is added. The sample is then extracted with 1 mL of
hexane/diethyl ether (2:1, v/v) containing 20 µL acetic acid. The sample is centrifuged and the
organic layer is dried under a stream of nitrogen. It is important that acetic acid is removed
completely before dissolving the lipid in an anhydrous solvent (i.e. acetonitrile) for long term
storage.
Enzymatic exchange procedure for OPDA
OPDA as well as a great variety of other oxylipins can be labeled under neutral and mild
conditions by using pancreatic lipases or liver esterases. Another advantage of the enzyme
procedure is that oxylipins with keto groups are only labeled in the carboxylic acid group.
Both enzymes work well with OPDA. However, JA is a poor substrate for these enzymes. A
fresh stock suspension of the enzyme is prepared by suspending 20 mg of the enzyme powder
in 1 mL anhydrous ethylene glycerol prior incubation. Anhydrous buffer salts are prepared by
drying 2 µL of a 1 M Tris-HCl buffer (pH 7.4) containing 150 mM CaCl2 in a glass vial at
90°C for 10 min under vacuum. A methanolic solution of OPDA (100 µg) is added to the
residue. After drying of the sample under a stream of nitrogen, 50 µL of the enzyme stock
solution and 25 µL of the labeled water are added. The mixture is incubated at 40°C. The
exchange reaction reaches almost equilibrium after 4 h and is completed after 8 to 12 h.
Thereafter, the mixture is extracted with 1 mL hexane/diethyl ether (2:1, v/v) containing 10
µL of acetic acid. The sample is centrifuged and the organic layer is dried under a stream of
nitrogen. It is important that acetic acid is removed completely before dissolving the lipid in
an anhydrous solvent (i.e. acetonitrile) for long term storage.
An alkaline procedure for oxygen-18 exchange
Uses and principle: This method can be used for acid labile oxylipins that are stable under
strong alkaline conditions and which are not substrates for hydrolases. However, this
procedure is time consuming and more expensive than the other methods given in Box 2 of
the main text. Under alkaline conditions carboxyl oxygens do not exchange with the oxygen
label in [18O]water. In order to incorporate the label, oxylipins are first methylated. The
resulting methyl esters are then hydrolysed with [18O]NaOH in [18O]water. During hydrolysis
one oxygen-18 atom is incorporated into the carboxyl group yielding a singly labeled
oxylipin. In order to exchange both oxygen atoms esterification and hydrolysis have to be
repeated several times.
Protocol: The oxylipin (100 µg) is methylated with TMS-diazomethane (see experimental
procedures) and dissolved in 20 µL of dioxane. A 1 M [18O]NaOH solution in [18O]water is
prepared by dissolving sodium methoxide in [18O]water. A 50 µL aliquot of this solution is
incubated with the oxylipin methyl ester (100 µg in 20 µL of dioxane) at room temperature
for 1 h. Thereafter, the solution is diluted with 2 mL of 50 mM citric acid in normal water and
extracted with 2 mL of Et2O. Remaining water in the Et2O phase is removed by incubating the
solution with 2,2-dimethoxypropane (acetone dimethyl acetal) for 5 min. The sample is dried
under vacuum and methylated with TMS-diazomethane. The incorporation of oxygen-18 into
the oxylipin is determined by GC-MS. The procedure is repeated until unlabeled oxylipin is
below the limit of detection. After three rounds of hydrolysis there should be approximately
72% of doubly labeled compound (see below).
Formulas to calculate oxygen-18 incorporation into target oxylipins
1. Acid and enzyme catalysed isotope exchange
Strong acids (pathway a in Fig. S1) and enzymes (pathway b in Fig. S1) serve as a catalysts
that catalyse exchange of oxygen-16 against oxygen-18 until equilibrium with respect to the
oxygen-18 label in the target compound and water is reached.
The theoretical atom % oxygen-18 incorporation into oxylipins can be calculated by the
following formulas in which x is the atom % 18O of the 18O water used.
unlabeled oxylipin:
RC16O16O- (%) = (100 – x)2/100 (0.04 % with x = 98%)
singly labeled oxylipin:
RC18O16O- (%) = 2 * x * (100 – x)/100 (3.92 % with x = 98%)
doubly labeled oxylipin:
RC18O18O- (%) = x2/100 (96.04 % with x = 98%)
After reaching the equilibrium, there should be 0.04% unlabeled, 3.92% singly labeled and
96.04% doubly labeled oxylipin when working with 98 atom % oxygen-18 water.
2. Alkaline isotope exchange
The theoretical atom % oxygen-18 incorporation into the oxylipin can be calculated after each
round of hydrolysis (pathway c in Fig. S1) with the following formula in which x is the atom
% 18O of the water/hydroxyl ions in the reaction, z1 is % of unlabeled acid, z2 is % of singly
labeled acid and z3 that of doubly labeled acid before the hydrolysis step (at the beginning z1
= 100%, z2 = 0% and z3 = 0%):
unlabeled oxylipin:
RC16O16O- = z1 + (z2/2) * ((100 – x)/100) – (z1 * x/100)
singly labeled oxylipin:
RC18O16O- = (z1 * 98/100) + z2 + (z3 * (100 – x)/100) – ((z2/2) *
(100– x)/100) – (z2/2 * x/100)
doubly labeled oxylipin:
RC18O18O- = z3 + ((z2/2) * 98/100) – (z3 * (100 – x)/100).
After the first hydrolysis round there should be 2% of unlabeled, 98% of singly labeled and
0% of doubly labeled compound when working with 98 atom % [18O]water. After three
rounds of hydrolysis there should be 0.58% of unlabeled, 27.44% of singly labeled and 72.03
of doubly labeled compound.