The hydroperoxide moiety of aliphatic lipid hydroperoxides is not affected by hypochlorous acid

Article abstract of DOI:10.1016/j.chemphyslip.2014.09.005

The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and spectrometric analysis revealed that the hydroperoxide moiety of 15S-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HpETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) is not affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride system.

Full text of DOI:10.1016/j.chemphyslip.2014.09.005

Chemistry and Physics of Lipids 184 (2014) 42–51
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
The hydroperoxide moiety of aliphatic lipid hydroperoxides is not
affected by hypochlorous acid
Josefin Zschaler a,b,*, Juergen Arnhold a,b
a
Institute for Medical Physics and Biophysics, Medical Faculty, Leipzig University, Härtelstraße 16-18, 04107 Leipzig, Germany
Translational Centre for Regenerative Medicine (TRM), Leipzig University, Philipp-Rosenthal-Str. 55, 04103 Leipzig, Germany
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 27 May 2014
Received in revised form 16 August 2014
Accepted 19 September 2014
Available online 24 September 2014
The oxidation of polyunsaturated fatty acids to the corresponding hydroperoxide by plant and animal
lipoxygenases is an important step for the generation of bioactive lipid mediators. Thereby fatty acid
hydroperoxide represent a common intermediate, also in human innate immune cells, like neutrophil
granulocytes. In these cells a further key component is the heme protein myeloperoxidase producing
HOCl as a reactive oxidant. On the basis of different investigation a reaction of the fatty acid
hydroperoxide and hypochlorous acid (HOCl) could be assumed. Here, chromatographic and
spectrometric analysis revealed that the hydroperoxide moiety of 15S-hydorperxoy-5Z,8Z,11Z,13E-
eicosatetraenoic acid (15-HpETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) is not
affected by HOCl. No reduction of the hydroperoxide group due to a reaction with HOCl could be
measured. It could be demonstrated that the double bonds of the fatty acid hydroperoxides are the major
target of HOCl, present either as reagent or formed by the myeloperoxidase-hydrogen peroxide-chloride
system.
Keywords:
Hypochlorous acid
Lipid hydroperoxides
Arachidonic acid
Myeloperoxidase
Lipoxygenase
ã 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
1970). In case of the reaction of linoleic acid hydroperoxide with
HOCl, singlet oxygen was also generated, however, to a much lower
Animal and plant lipoxygenases are able to oxidize unsaturated
fatty acids to aliphatic hydroperoxides, which can be further
metabolized to an array of biologically active substances such as
leukotrienes, lipoxins, and oxo-modified fatty acids (Stables and
Gilroy, 2011). These fatty acid hydroperoxides can also undergo
non-enzymatic homolytic decomposition into toxic electrophilic
yield as with hydrogen peroxide (Miyamoto et al., 2006). In
contrast, no chemiluminescence of singlet oxygen was observed in
the reaction of tert-butyl hydroperoxide with HOCl despite the
detection of different reaction products (Arnhold et al., 1996;
Miyamoto et al., 2006; Panasenko et al., 1997). Other indications
for a possible involvement of lipid hydroperoxides in HOCl-driven
reactions came from experiments where effects of previously
accumulated lipid peroxidation products on HOCl-induced oxida-
tion of unsaturated phospholipids were examined (Panasenko and
Arnhold, 1999; Panasenko et al., 1997). In these investigations, a
higher yield of peroxidation products was measured with
increasing concentrations of hydroperoxides. In all experiments
with lipid hydroperoxides as well as tert-butyl hydroperoxide the
question raises, was really the hydroperoxide moiety involved in
the initiating reaction with HOCl or were the observed changes in
the product spectrum caused by other reactive groups present in
the lipid material.
2
+
components in the presence of transition metal ions such as Fe
+
and Cu or ascorbic acid (Jian et al., 2009; Lee and Blair, 2000).
Hypochlorous acid (HOCl) is a reactive product of the heme
protein myeloperoxidase found in neutrophilic granulocytes and to
a lesser extent in monocytes (Arnhold and Flemmig, 2010). This
species is involved in pathogen defense and regulation of immune
reactions. HOCl is well known to react with hydrogen peroxide
under formation of singlet oxygen as evidenced by infrared
chemiluminescence at 1268 nm (monomol emission of singlet
oxygen) or emission in the red spectral region (dimol emission of
singlet oxygen) (Held et al., 1978; Kanofsky, 1989; Khan and Kasha,
The confirmation of a direct reaction between a fatty acid
hydroperoxide group and HOCl would be important for physio-
logical reactions in neutrophilic granulocytes as during stimulation
of these cells both the 5-lipoxygenase, which oxidizes arachidonic
acid to 5S-hydroperoxy-6E,;8Z,11Z,14Z-eicosatetraenoic acid (5-
HpETE), and myeloperoxidase will be activated and therefore,
*
Corresponding author at: Institute for Medical Physics and Biophysics, Medical
Faculty, Leipzig University, Härtelstraße 16-18, 04107 Leipzig, Germany. Tel.: +49
41 97 15774.
3
E-mail address: josefin.zschaler@medizin.uni-leipzig.de (J. Zschaler).
http://dx.doi.org/10.1016/j.chemphyslip.2014.09.005
009-3084/ã 2014 Elsevier Ireland Ltd. All rights reserved.
0

J. Zschaler, J. Arnhold / Chemistry and Physics of Lipids 184 (2014) 42–51
43
reaction products of both enzymes would be present close
together. In order to evaluate a potential reaction between fatty
acid hydroperoxides and HOCl, 15S-hydroperoxy-5Z,8Z,11Z,13E-
eicosatetraenoic acid (15-HpETE), the 15-hydroperoxide of arach-
idonic acid, was generated from soybean lipoxygenase and
analyzed by an reverse-phase high performance liquid chroma-
tography (RP-HPLC) technique with UV detection as well as by
electrospray ionization mass spectrometry. Additionally, linoleic
acid was applied as an alternative substrate for this enzyme. The
HPLC technique allows by using appropriate standards a clear
assignment of the corresponding lipid hydroperoxide molecules
and to follow its changes upon reaction with a partner of interest.
We could demonstrate that HOCl, present either as reagent or
formed by the myeloperoxidase-hydrogen peroxide-chloride
system, reacts with double bonds of lipid molecules, but not with
the hydroperoxide moiety.
the synthesis in borate buffer at pH 9 and at neutral pH,
respectively. This is in accordance with the activity optimum of
sLOX at basic pH values. As minor product 15-HETE was measured
in low concentrations at basic pH (5.3 Æ 3.37
m
M 15-HETE, pH 9,
M 15-HETE, pH
n = 6, yield of 3.37%) and at neutral pH (1.37 Æ 0.77
m
7.4, n = 6, yield of 0.27%). Afterwards the known amount of
hydroperoxy fatty acids was diluted in the appropriate buffer and
incubated with a certain amount of HOCl at room temperature
ꢀ
À
(20 C) for 5 min. The OCl concentration was determined
À1
À1
spectrophotometrically using
1 N sodium hydroxide solution (Morris, 1966).
e
292 = 350 M cm
at pH 12 in
2.3. Mass spectrometric analysis of the reaction products from 15-
HpETE and HOCl
Fatty acid hydroperoxides diluted in acetonitrile or acetonitrile/
water/acetic acid (60/40/0.2, v/v/v) were assessed with an ion trap
mass spectrometer (amaZon SL, Bruker Daltonics, Bremen,
Germany) via an ESI spray source in negative ionization mode
2. Material and methods
2.1. Material
with a flow rate of 2 ml/min. A mass-range from 100 to 800 m/z was
detected with enhanced resolution. Drying gas temperature was
ꢀ
The chemicals used were obtained from the following sources:
set to 180 C, drying gas flow rate to 4 l/min, nebulizer gas to 7.3 psi,
HPLC-standards 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13
S)-HpODE), 15S-hydro(pero) xy-5Z,8Z,11Z,13E-eicosatetraenoic
acid (15(S)-H(p)ETE), 9S-hydroperoxy-10E,12Z-octadecadienoic
acid (9(S)-HpODE), 15-oxo-5Z,8Z,11Z,13E-eicosatetraenoic acid
ionization voltage to 4500 V and ion charge control (ICC) to
35,000 counts. For MS/MS analysis isolation width was set to
4.0 m/z, fragmentation cutoff to 105 m/z and fragmentation
amplitude to 0.4 V.
(
(
15-oxo-ETE) from Cayman Chemical (distributed by Biomol,
LC-ESI-MS/MS was performed according to Kortz et al. (Kortz
Hamburg, Germany), HPLC solvents from Carl Roth (Karlsruhe,
Germany), arachidonic acid, linoleic acid and all other chemicals
from Sigma (Taufkirchen, Germany). The enzymes used were
obtained from the following sources: human neutrophil myelo-
peroxidase (MPO) from Planta (Vienna, Austria), lipoxidase
et al., 2013). In brief, 200
HpETE = 0.033–0.33 ng/ml) were injected on a Strata-X column
m, Phenomenex, Aschaffenburg,
ml of the sample solution (c15-
(L. Â I.D. 20 Â 2 mm, 25
m
Germany) for on-line solid phase extraction and chromatographi-
cally separated on a Kinetex C18 column (L. Â I.D. 100 Â 2.1 mm,
(
(
soybean) Type I-B (sLOX) and glutathione peroxidase from Sigma
Taufkirchen, Germany).
2.6 mm, Phenomenex, Aschaffenburg, Germany). A 5500 QTrap
mass spectrometer (AB Sciex, Darmstadt) in negative ionization
mode was applied for MS/MS analysis with multiple reaction
monitoring experiments for 15-HpETE (m/z 335.28/113.1) and
monochlorohydrins of 15-HpETE (m/z 387.28/113.1).
2.2. Synthesis of 15-HpETE with sLOX, chromatographic analysis and
reaction with HOCl
The enzymatic synthesis of 15-HpETE was performed with
soybean lipoxygenase (sLOX) in different buffer systems at pH
2.4. Reduction of HOCl-treated 15-HpETE with the glutathione
peroxidase system
7.4 or pH 9 (50 mM phosphate buffer at pH 7.4, 50 mM borate buffer
at pH 9). Thereby, 500
m
M of arachidonic acid was incubated with
Equimolar concentrations of 15-HpETE (250
were incubated at room temperature (20 C) for 5 min in 50 mM
m
M) and HOCl
ꢀ
ꢀ
3695 U sLOX at room temperature (20 C) in a volume of 1 ml for
5
min. The reaction was stopped by separation of the enzyme by
borate buffer, pH 9. Afterwards, reduced L-glutathione (500 mM or
ꢀ
centrifugation through a filter (30 kDa) at 4 C. The enzymatic
product was assigned as AA-OOH, hydroperoxides of arachidonic
acid. In some experiments linoleic acid was used and the product
was assigned as La-OOH, hydroperoxides of linoleic acid. The
amount of conjugated dienes was determined by UV–spectroscopy
1 mM) and 2 U glutathione peroxidase was added and further
incubated for 15 min. To stop the reaction, the enzyme was
separated by centrifugation through a filter (30 kDa). The filter was
previously equilibrated with the buffer system. Glutathione was
dissolved shortly before usage in 10 mM EDTA. Control experi-
ments were also performed without addition of EDTA, however, no
differences occurred.
À1
À1
using an extinction coefficient of
e236 = 27,000 M cm , as
specified from the manufacturer as coefficient for the synthetic
standards of 15-HpETE. The identity of the synthesis product was
confirmed by RP-HPLC after dilution in HPLC solvent. The
separation was performed by using a C18 column (Supelcosil
2.5. Modification of 15-HpETE by the myeloperoxidase-hydrogen
peroxide-chloride system
LC-18-DB, L.  I.D. 25 cm  4.6 mm, 5
acetonitrile/water/acetic acid (60/40/0.2, v/v/v) at a flow rate of
ml/min and the eluate was monitored at 234 nm and 280 nm. The
mm) isocratically eluted with
15-HpETE (100 mM) was incubated in the presence of the
1
peroxidase system composed of 200 nM or 400 nM MPO, 100
m
M
À
HPLC consisted of a Shimadzu liquid chromatographic system
equipped with a Shimadzu LC-10ATvp isocratic solvent delivery
system, Shimadzu SPD-10Avp dual wavelength absorbance detec-
H
2
O
2
and 140 mM Cl . The reaction was performed in phosphate
ꢀ
buffer (50 mM, pH 7.4) at room temperature (20 C) some
ꢀ
experiments were also done at 37 C. Hydrogen peroxide was
ꢀ
tor, Shimadzu CTO-10ASvp column oven (35 C) and Rheodyne
added in small amounts to prevent the inactivation of MPO. The
injector with 20
quantified by comparison of the peak area with that of known
amounts of synthetic standards. An average 15-HpETE concentra-
m
l loop volume. The amount of 15-HpETE was
2 2
concentration of H O was measured spectrophotometrically
À1
À1
using
e240 = 43.6 M cm
(Beers and Sizer, 1952). During the
was
10 min incubation of 15-HpETE with the peroxidase H O
2 2
tion of 370.26 Æ 21.77
m
M
(n = 6, yield of 74.05%) and
added in 20 steps every 30 s. Afterwards the samples were
incubated for further 5 min. To stop the reaction of the
2
10.01 Æ28.29 M (n = 6, yield of 42.00%) was determined for
m

4
4
J. Zschaler, J. Arnhold / Chemistry and Physics of Lipids 184 (2014) 42–51
myeloperoxidase the sample was diluted four-fold in HPLC solvent
acetonitrile/water/acetic acid (60/40/0.2, v/v/v)) and centrifuged
through a filter (30 kDa). The filter was previously equilibrated
fatty acids with a hydroxyl or a hydroperoxy moiety was possible.
This was important to assess the ability of the fatty acid
hydroperoxide as reaction partner of HOCl.
(
with the HPLC solvent.
Afterwards, the enzymatic product of 15-HpETE synthesis was
incubated with different amounts of HOCl at pH 9 (50 mM borate
buffer) for 5 min and was examined by analytic RP-HPLC with UV-
detection at 234 nm (Fig. 1A). The 15-HpETE peak decreased with
increasing HOCl concentration. Equimolar ratio of 15-HpETE and
HOCl resulted in a strong decrease of 15-HpETE by 84% compared
to the untreated 15-HpETE sample. Instead two new peaks
3. Results
3.1. Reaction of 15-HpETE or 13-HpODE with HOCl as assessed by RP-
HPLC
The enzymatic synthesis of hydroperoxides from arachidonic
r
emerged with lower t at 7.4 and 7.9 min. The proportion of the
acid (AA) and linoleic acid (LA) was performed with soybean
lipoxygenase (sLOX). Thereby sLOX selectively produce 15S-
hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HpETE) and
two peaks relative to 15-HpETE depended on the amount of HOCl.
Further peaks were not obvious. Importantly, any changes in 15-
HETE (t = 13.5 min) were not measured. Changes in all peak
r
1
3S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE), re-
intensities are related to the applied HOCl concentration (Fig. 1B).
It is obvious that the 15-HpETE concentration was reduced to a
larger degree in comparison to the formation of the two new peaks.
Apparently further products have to be formed that are not
detected by this HPLC approach.
spectively (Brash et al., 1987; Hamberg and Samuelsson, 1967).
The identity of the synthesis product was confirmed by reverse-
phase high performance liquid chromatography (RP-HPLC). Under
our conditions, intense product peaks at 234 nm were found at a
retention time (t
r
) of 14.6 min for 15-HpETE and 14.0 min for 13-
15-HpETE was also incubated with HOCl at pH 7. Here the
reduction of the 15-HpETE content after reaction with HOCl was
with 66% lower at an equimolar 15-HpETE: HOCl ratio as compared
to the reaction at pH 9. Also at pH 7 two peaks with lower retention
HpODE as revealed by the corresponding hydroperoxide standards.
A less intense peak was detected at 13.5 min corresponding for 15-
HETE. With the utilized chromatographic method the separation of
Fig. 1. Reaction of 15-HpETE with HOCl. Hydroperoxides or arachidonic acid (AA-OOH) was synthesized using arachidonic acid and sLOX in 50 mM borate buffer at pH 9 and
ꢀ
quantified by RP-HPLC. (A) 15-HpETE (380
m
M) was incubated with 380
m
M, 190
mM, 95
mM, 63
mM and 48
m
M HOCl at room temperature (20 C) for 5 min (50 mM borate
buffer, pH 9). For chromatographic analysis the samples diluted in solvent were isocratically eluted with acetonitrile/water/acetic acid (60/40/0.2, v/v/v) at a flow rate of 1 ml/
min. Peaks: 15-HETE (t = 13.5 min),15-HpETE (t = 14.6 min). The peak changes are related to the used HOCl concentration (B). Similar experiments were also performed at pH
(C). Thereby, the pH shift was done after 15-HpETE synthesis by addition of 1 N HCl. The chromatograms are representative examples of three independent experiments of
5-HpETE incubated with HOCl.
r
r
7
1

J. Zschaler, J. Arnhold / Chemistry and Physics of Lipids 184 (2014) 42–51
45
Fig. 2. Reaction of 13-HpODE with HOCl. Hydroperoxides of linoleic acid (La-OOH) was synthesized using 500
and quantified by RP-HPLC. (A) 400
m
M linoleic acid and 3695 U sLOX in 50 mM borate buffer at pH
ꢀ
9
m
M of 13-HpODE was incubated with HOCl in different ratios at room temperature (20 C) for 5 min. For chromatographic analysis the
samples diluted in solvent were isocratically eluted with acetonitrile/water/acetic acid (60/40/0.2, v/v/v) at a flow rate of 1 ml/min. Peaks: 13-HpODE (t
r
= 14.0 min), 9-HpODE
(t
r
= 14.4 min). The peak changes are related to the used HOCl concentration (B).
time (t
r
of 7.4 and 7.9 min) emerged in dependence on the used
double bonds that form a diene conjugate. 13-HpODE was
incubated with HOCl at pH 9 (50 mM borate buffer) and examined
by analytic RP-HPLC with UV-detection at 234 nm (Fig. 2A).
Without HOCl a double peak of 13-HpODE and the synthesis by-
HOCl amount. However, the relative proportion of these peaks
relative to 15-HpETE was lower compared to the experiment at pH
9. When these peak changes are related to the applied HOCl
concentration (Fig. 1C) it can be seen that also at pH 7 the changes
of the 15-HpETE peak area are more intense than the increase of
the peaks at 7.4 and 7.9 min.
The two novel peaks have apparently an intact conjugated diene
structure due to their absorption at 234 nm. Because of the absence
of any formation of 15-HETE it can be excluded that HOCl converts
the hydroperoxide of 15-HpETE to 15-HETE. Apparently, the
detected changes in the product spectrum are caused by
interaction of HOCl with double bonds in 15-HpETE.
r
product 9-HpODE is present with a t of 14.0 and 14.4 min,
respectively. After addition of HOCl a drastic decrease of this
double peak was monitored. In particular this becomes obvious in
relation of the 13-HpODE peak area to the used HOCl concentration
(Fig. 2 B). No further peaks emerged. Thus, the decrease of the 13-
HpODE peak is probably due to a reaction of HOCl with a double
bond resulting in the loss of the conjugated diene structure and in
disappearance of the absorption at 234 nm.
Summarizing, the measured decrease of 15-HpETE and 13-
HpODE is most likely due to a reaction of HOCl with the double
bonds, but not by a reaction with the hydroperoxide moiety.
To specify this conclusion, HOCl was also incubated with 13-
HpODE. This hydroperoxide of linoleic acid contains only two
Fig. 3. Reaction of 15-HpETE with HOCl as assessed by ESI-MS. Ethanol of the synthetic available 15-HpETE solution was evaporated by vacuum and the remaining neat oil
was dissolved in borate buffer (50 mM, pH 9). 300
m
M of 15-HpETE was incubated in the absence (A) and presence of 300
m
M (B) or 600
m
M (C) HOCl at room temperature
ꢀ
(
20 C) for 5 min. For spectrometric analysis the samples were diluted in acetonitrile and measured in the negative ion mode with an ion trap ESI-MS. The spectra are
representative examples of three independent experiments.

4
6
J. Zschaler, J. Arnhold / Chemistry and Physics of Lipids 184 (2014) 42–51
Additional experiments were also performed in the presence of
further HOCl reaction partners, in particular methionine and
taurine (data not shown). Interestingly, at equimolar concentration
of methionine and taurine the intensity of the HOCl-derived 15-
intermediates through elimination of HCl from chlorohydrins as
in Fig. 3. Evidences for the occurrence of dichlorohydrins of 15-
HpETE were not detected in the measured eluates. The chlorohy-
drin formation was further evaluated by a direct LC-ESI-MS/MS
analysis. Thereby the mass transitions for 15-HpETE and 15-
HpETE + HOCl were monitored. Interestingly, the LC separation
resulted in a comparable chromatogram with lower retention
r
HpETE peaks (t of 7.4 and 7.9 min) were clearly decreased. That
means HOCl shows a stronger affinity to the thioether group of
methionine or the amino group of taurine than to 15-HpETE. This is
in accordance with the assumption that the double bonds of 15-
HpETE are target sites of HOCl. The reaction of HOCl with aliphatic
double bonds is well described and leads to the formation of
chlorohydrins (Spalteholz et al., 2004; Winterbourn et al.,1992). To
what extent the reaction of 15-HpETE with HOCl is accompanied
by the formation of chlorohydrins and whether the hydroperoxide
moiety participates was examined in the following experiments.
peaks for 15-HpETE + HOCl (t
r
of 4.8 and 4.9 min) compared to 15-
HpETE (t of 6.0 min). For 15-HpETE the characteristic fragment ion
r
was measured with m/z 113.1 Da, which was also applied for 15-
HpETE + HOCl. Apparently this fragment arise from cleavage of the
double bond allylic to the hydroperoxide as shown in a
comprehensive analysis of fragment ions of lipid hydroperoxides
from MacMillan and Murphy (MacMillan and Murphy, 1995).
Based on these results the chlorohydrin peaks and the 15-
HpETE peak eluted by RP-HPLC were further evaluated by MS/MS
analysis. The resulting fragment spectra are shown in Fig. 5.
The collision induced fragmentation of 15-HpETE (Fig. 5A) and
of the corresponding monochlorohydrin peaks (Fig. 5B and C)
resulted in a characteristic fragmentation pattern. For 15-HpETE
the isolation mass was set to 317 m/z because the dehydrated form
of 15-HpETE occurred in higher abundance. For the monochlor-
ohydrin species of 15-HpETE the isolation mass was set to 388 m/z
(isolation width 4 m/z) to ensure an equal isolation of both isotopes
species.
3.2. Reaction of synthetic and enzymatically synthesized 15-HpETE
with HOCl as assessed by ESI-MS and LC-ESI-MS/MS
The formation of chlorohydrins can be easily detected by the
mass changes of the fatty acids. Therefore the incubation products
of HOCl with the synthetic standards 15-HpETE was measured
with ion trap mass spectrometry (MS) (Fig. 3). The electrospray
ionization (ESI) was chosen as soft ionization technique due to the
instability of the hydroperoxide moiety.
The fatty acid hydroperoxide 15-HpETE has a monoisotopic
mass of 336.5 Da (Fig. 3). Consequently, in the negative ion mode a
peak at m/z 335.3 Da can be found as negative ion of 15-HpETE, and
a more prominent peak with a mass of m/z 317.3 Da was obvious
due to the elimination of a water molecule apparently from the
hydroperoxide moiety of 15-HpETE yielding a keto-acid or an
epoxy-acid (MacMillan and Murphy, 1995). Applying equimolar
concentrations of HOCl the peaks at m/z 335.3 Da and m/z 317.3 Da
decreased considerably, whereas several new peaks with higher
masses were formed. The intense peak couple with m/z 387.3 and
For 15-HpETE different product ions were observed formed by
loss of H O and CO from the carboxylate anion (273 m/z), by loss of
2 2
3
89.3 Da can be assigned to monochlorohydrins of 15-HpETE due
to mass shift of 52 Da. Additionally, the intensity ratio of 3 to
between these mass peaks corresponds well to the isotope ratio
1
3
5
37
of Cl and Cl. An excess of HOCl in relation of the 15-HpETE
concentration resulted also in a dichlorohydrin formation of 15-
HpETE with peaks at m/z 439.2, 441.2 and 443.2 Da. The intensity
ratio of these three peaks corresponds to 9:6:1 as expected from
the natural abundance of chlorine isotopes. Further minor peaks
around m/z 369.3 Da and 421.2 Da can be assigned to mono- and
dichlorohydrins of 15-HpETE, which eliminated a water molecule.
The less intense peaks at m/z 351.3 Da and 403.3 Da are apparently
attributed to the formation of epoxide intermediates through
elimination of HCl from chlorohydrins under slightly basic
conditions (Spalteholz et al., 2004; Winterbourn et al., 1992).
Furthermore, the reaction of 15-HETE and HOCl was also measured
with ESI-MS leading to a comparable chlorohydrin formation of 15-
HETE as for 15-HpETE (data not shown).
To get a clear connection between the chromatographic
analyzed peaks with lower retention time and the formation of
chlorohydrins of 15-HpETE, the analytic RP-HPLC was coupled with
ESI-MS measurements. On the one hand, this was performed by
collection of aliquots from the eluate after RP-HPLC analysis
corresponding to retention time of 7.4 and 7.9 min. These aliquots
were subsequently analyzed with an ESI quadrupole ion trap
Fig. 4. Reaction of 15-HpETE and HOCl as assessed by coupling of liquid
chromatography and ESI-MS. 15-HpETE was synthesized using arachidonic acid
and sLOX in 50 mM borate buffer at pH 9 and quantified by RP-HPLC. 15-HpETE
ꢀ
(
225
m
M) was incubated with 225
m
M HOCl at room temperature (20 C) in 100
m
l
(
Fig. 4A). On the other hand, the reaction mixture was analyzed
for 5 min (50 mM borate buffer, pH 9). (A) For chromatographic analysis the samples
diluted in solvent were isocratically eluted with acetonitrile/water/acetic acid (60/
40/0.2, v/v/v) at a flow rate of 1 ml/min. Aliquots of the eluate were collected and
measured in negative ionization mode with ESI-MS. Left and right inset:
characteristic MS spectra of the two peaks at 7.4 and 7.9 min are shown
respectively. (B) For direct online-SPE-LC-ESI-MS/MS analysis 15-HpETE + HOCl
and 15-HpETE were diluted 1:1000 and 1:10000 in methanol/water (62/38, v/v),
respectively, and measured in negative ionization mode. Chromatograms for 15-
HpETE and 15-HpETE + 52 Da (HOCl) are shown.
with a further instrument, a triple quadrupole linear ion trap mass
spectrometer (Fig. 4B).
The mass spectrometric analysis of the two HPLC peaks (t
.4 and 7.9 min) clearly assigns them as monochlorohydrins of 15-
HpETE due to an intense peak couple at m/z 387.2 and 389.2 Da
Fig. 4 A and inset). Furthermore, a less intense peak at m/z 351.3 Da
can be apparently attributed to the formation of epoxide
r
of
7
(

J. Zschaler, J. Arnhold / Chemistry and Physics of Lipids 184 (2014) 42–51
47
Fig. 5. Reaction of 15-HpETE and HOCl as assessed by ESI-MS/MS. 15-HpETE was synthesized using arachidonic acid and sLOX in 50 mM borate buffer at pH 9 and quantified
ꢀ
by RP-HPLC.15-HpETE (250
mM) was incubated with 250
m
M HOCl at room temperature (20 C) in 100
ml for 5 min (50 mM borate buffer, pH 9). For chromatographic analysis
the samples diluted in solvent were isocratically eluted with acetonitrile/water/acetic acid (60/40/0.2, v/v/v) at a flow rate of 1 ml/min. Aliquots of the eluate were collected
and measured in negative ionization mode with ESI-MS/MS. The isolation width was set to 4.0 m/z, fragmentation cutoff to 105 m/z and fragmentation amplitude to 0.4 V. (A)
MS/MS spectra of 317 m/z (15-HpETE-H O as isolation mass). (B) MS/MS spectra of 388 m/z (isolation mass) of the sample corresponding to the peak at 7.4 min. (C) MS/MS
2
spectra of 388 m/z (isolation mass) of the sample corresponding to the peak at 7.9 min. (D) Possible fragmentation pattern of 15-HpETE and 15-HpETE-epoxide intermediates
yielding characteristic ions shown in the corresponding spectra and marked with *.
two H
2
O (299 m/z) or by loss of two water molecules and CO
2
(315 m/z) or by loss of two water molecules and CO
measured comparable to the 15-HpETE fragmentation spectra. In
both monochlorohydrin forms of 15-HpETE (t of 7.4 and 7.9 min)
the ion with a mass of 113 m/z was detected. The common
appearance of this fragment in 15-HpETE and both monochlor-
ohydrin products indicates the preservation of the structure
between C13 and C20 in 15-HpETE and the investigated
chlorohydrin samples. This is in accordance with the assumption
that the monochlorohydrin forms of 15-HpETE detected by RP-
HPLC have an intact conjugated diene group.
In the MS/MS spectra of the peak at 7.4 and 7.9 min two further
characteristic ions at 205 m/z (more abundant for the peak at
7.4 min) and 217 m/z (more abundant for the peak at 7.9 min) were
detected (Fig. 5B and C). Apparently these fragments arise from a
similar rearrangement and cleavage of the corresponding mono-
chlorohydrin structure as for 15-HpETE. The fragment with a mass
of 205 m/z may be formed by cleavage of the ÀÀ C13QC14 ÀÀ
2
(271 m/z) were
(
255 m/z). Furthermore, two characteristic fragment ions were
detected also previously described (MacMillan and Murphy, 1995).
The ion at 113 m/z results apparently from cleavage of the double
bond ÀÀ C13QC14 ÀÀ adjacent to the point of attachment of the
hydroperoxide group and loss of water. This fragment was also
detected in the LC-ESI-MS/MS analysis. Another fragment ion at
r
219 m/z results from charge driven allylic fragmentation of
dehydrated 15-HpETE after 1,5-sigmatropic proton rearrangement
resulting in an intermediate with a triene structure between
C8 and C13. The cleavage occurs between C14 and C15.
The collision induced fragmentation of the corresponding
monochlorohydrin peak (387 m/z) resulted in an abundant
formation of the product ion 351 m/z due to elimination of HCl
and formation of an epoxide intermediate. In addition, product
ions arising from 351 m/z by loss of H
and CO from the carboxylate anion (289 m/z), by loss of two H
2
O (333 m/z), by loss of H
2
O
O
2
2

4
8
J. Zschaler, J. Arnhold / Chemistry and Physics of Lipids 184 (2014) 42–51
To improve the resolution of the chromatogram, especially in
that part where the HOCl dependent fatty acid peaks are located, a
lower amount of acetonitrile was used in the solvent. As a
consequence the retention times of 15-H(p)ETE (15-HETE t
r
= 31.1
min and 15-HpETE t = 36.6 min) and of the HOCl modified fatty
r
acid peaks (Fig. 6) were increased. Both fatty acids show a reaction
with HOCl. For 15-HpETE the simultaneous decrease of the 15-
HpETE content and the appearance of two new peaks (t
r
of 14.1 and
15.5 min) reappeared (Fig. 6A). This was also the case for the 15-
HETE/HOCl incubation (Fig. 6B). A comparable decrease of the 15-
HETE content was measured and two new peaks with a lower
retention time emerged. Whereby one of them is a double peak
r r
(t = 11.8 and 12.2 min) the other one is a single peak (t = 13.4 min).
The HOCl modified peaks of 15-HpETE and 15-HETE can be clearly
distinguished according their retention time. This is a further
indication that the products of the reaction of HOCl with 15-HpETE
differ clearly from 15-HETE in their chemical identity. Additionally
it shows, that during the reaction of 15-HpETE with HOCl the
hydroperoxide moiety does not participate. Therefore it can be
assumed that only the double bonds of 15-HpETE and 15-HETE are
the target for HOCl.
In a further experiment 15-HpETE and 15-HETE was incubated
together with HOCl, whereby HOCl was added in equimolar
concentration to the sum of 15-H(p)ETE (Fig. 7).
After incubation of 15-H(p)ETE with HOCl a drastic decrease of
the 15-H(p)ETE peaks occurred. Furthermore, four peaks with
r
lower retention times were observed with comparable t to the
single incubation of 15-H(p)ETE with HOCl. That means also in the
presence of both fatty acids their appropriate HOCl dependent
peaks can be identified.
Fig. 6. Reaction of 15-HpETE or 15-HETE with HOCl. Ethanol of the synthetic
available 15-HpETE or 15-HETE solution was evaporated by vacuum and the
remaining neat oil was dissolved in PBS at pH 7.3. 100 mM HOCl was incubated with
equimolar concentration of 15-HpETE (A) or 15-HETE (B) at room temperature
ꢀ
3.4. Reduction of HOCl-treated 15-HpETE with the glutathione
peroxidase system
(
20 C) for 5 min, respectively. For chromatographic analysis the samples diluted in
solvent were isocratically eluted with acetonitrile/water/acetic acid (50/50/0.2, v/v/
v) at a flow rate of 1 ml/min. Peaks: 31.1 min 15-HETE/36.6 min 15-HpETE.
Glutathione peroxidase (GpX) is able to reduce the hydroperoxy
moiety of 15-HpETE to a hydroxyl group (15-HETE). This
characteristic of GpX was utilized to reduce the products of the
15-HpETE/HOCl reaction. Therefore 15-HpETE was incubated with
HOCl and further treated with a mixture GpX and glutathione
(GSH) or GSH alone (Fig. 8). As already shown in Fig. 1, the
incubation of 15-HpETE with HOCl yielded two new peaks with tr
at 7.4 min and 7.9 min. Upon application of GpX and GSH, these
combined with release of water. In this fragment the epoxide could
reside at position ÀÀ C5QC6 ÀÀ or ÀÀ C8QC9 ÀÀ assuming that both
possible monochlorohydrin structures of 15-HpETE with an intact
conjugated diene group yielding this fragment. The ion at 217 m/z
seems to be formed by a cleavage of the bond between C14 and
2
C15 of the epoxide intermediate under H O release. This
fragmentation pattern requires a 1,5-sigmatropic proton rear-
rangement as described for 15-HpETE. This could only be expected
in a monochlorohydrin species of 15-HpETE, where the chlorohy-
drin is formed at the double bond at ÀÀ C5QC6 ÀÀ .
peaks shifted to lower retention (t = 6.9/7.3 min). Applying only
r
GSH, three peaks with t at 6.9 min, 7.3 min and 7.9 min emerged.
r
The peak around t of 7.3 min is apparently composed of two
r
Therefore it may be hypothesized that the peak at 7.4 min
represent the monochlorohydrin of 15-HpETE, where the chloro-
hydrin is formed at the double bond at ÀÀ C8QC9 ÀÀ yielding a
fragmentation pattern with a prominent fragment at 205 m/z. The
peak at 7.9 min could represent the monochlorohydrin of 15-
HpETE, where the chlorohydrin is formed at the double bond at
ÀÀ C5QC6 ÀÀ yielding a fragmentation pattern with both ions at
205 m/z and 217 m/z.
3.3. Reaction of synthetic 15-HpETE in comparison to 15-HETE with
HOCl as assessed by RP-HPLC
Whether the hydroperoxy moiety is involved in the reaction of
15-HpETE with HOCl, was further investigated by a direct
comparison of the reaction of HOCl with synthetic standards 15-
HpETE and 15-HETE, respectively. Therefore the ethanol solvent of
Fig. 7. Reaction of 15-H(p)ETE with HOCl. A mixture of the two standards 15-HpETE
and 15-HETE containing ethanol solutions was evaporated by vacuum and the
remaining neat oil was dissolved in PBS at pH 7.3. 200
00 M of 15-HpETE and 100
m
M HOCl was incubated with
15-HpETE and 15-HETE was evaporated by vacuum and the
ꢀ
1
m
m
M of 15-HETE at room temperature (20 C) for 5 min.
remaining neat oil was dissolved in PBS (pH 7.3). Then equimolar
concentrations of HOCl and 15-HpETE or 15-HETE were incubated
for 5 min and afterwards analyzed by RP-HPLC (Fig. 6).
For chromatographic analysis the samples diluted in solvent were isocratically
eluted with acetonitrile/water/acetic acid (50/50/0.2, v/v/v) at a flow rate of 1 ml/
min. Peaks: 31.0 min 15-HETE/36.5 min 15-HpETE.

J. Zschaler, J. Arnhold / Chemistry and Physics of Lipids 184 (2014) 42–51
49
applied at physiological pH (pH 7.4) (Table 1). As control sample
15-HpETE was equally treated like the enzyme samples.
A small decrease of the original used 100
mM 15-HpETE to
8
8
mM was apparently related to the additional centrifugation step
À
through a 30 kDa filter. When MPO, H
together a reduction of the 15-HpETE content from previous
7.5 M to 41 M and the appearance of two new peaks with
2 2
O and Cl were present
8
m
m
lower retention time (t
r
= 7.4 and 7.8 min) was obvious. Thus, the
À
whole MPO-H
2
O
2
-Cl system caused the same alterations on 15-
HpETE as the reactant HOCl. The incubation of 15-HpETE with H
2 2
O
alone did not induce any significant changes in the 15-HpETE
content, only a slight reduction to 81
ingly, in the presence of MPO, either alone or together with Cl or
m
M was detected. Interest-
À
2 2
H O , the amount of 15-HpETE was significantly diminished, but to
a lower extent than in the presence of the complete MPO system.
Fig. 8. Reaction of 15-HpETE with HOCl und further reduction using GpX and GSH.
Hydroperoxides or arachidonic acid (AA-OOH) was synthesized using arachidonic
À
The recovery of 15-HpETE was about 70
and only 59 M by MPO + H
mM by MPO and MPO + Cl
acid and sLOX in 50 mM borate buffer at pH 9 and quantified by RP-HPLC. 250
mM of
m
2
O
2
. However, no new peaks appeared
1
5-HpETE was incubated with equimolar concentration of HOCl at room
neither for chlorohydrins nor 15-HETE in the chromatograms.
Control experiments of the reaction of 15-HpETE with the MPO-
ꢀ
temperature (20 C) for 5 min and then mixed with 2 U GpX and 1 mM GSH or
00 M/1 mM GSH alone. These samples were incubated for further 15 min. The
5
m
À
ꢀ
2
H O
2
-Cl were also performed at 37 C, however no temperature
reaction was stopped by separation of the enzyme by centrifugation through a filter.
For better comparison also the samples without GpX were centrifuged through a
filter. For chromatographic analysis the samples diluted in solvent were isocratically
eluted with acetonitrile/water/acetic acid (60/40/0.2, v/v/v) at a flow rate of 1 ml/
min. Peaks: 13.4 min 15-HETE/14.6 min 15-HpETE. The chromatogram is
representative example of three independent experiments.
influence could be determined (data not shown).
4
. Discussion
a
The reaction of HOCl with aliphatic fatty acid hydroperoxides
was investigated by RP-HPLC and ESI-MS. We could not detect any
products originating from a direct interaction of the hydroperoxide
moiety with HOCl. Upon incubation of 15-HpETE with HOCl, no
increase in the amount of the corresponding hydroxy fatty acid
(15-HETE) could be detected. However, the reaction of HOCl with
unsaturated fatty acid hydroperoxides, like 15-HpETE, resulted in
formation of chlorohydrins. The chromatographic analysis of 15-
HpETE products using RP-HPLC revealed two new chlorohydrins
r
closely overlapping peaks. The third peak (t = 7.9 min) could be
comparable to the second peak of the HOCl derived 15-HpETE
signal.
Taking into account that GpX and GSH convert the hydro-
peroxy group into a hydroxyl group, which can also be seen in the
increase of the 15-HETE signal (Fig. 8), it is apparent that under
mild oxidizing conditions a stepwise transformation of the HOCl
modified 15-HpETE signal into the HOCl modified 15-HETE signal
occurred. This is also supported by the fact that the HOCl modified
(t = 7.4 and 7.8 min) with an intact conjugated diene configuration.
r
These new products have still the hydroperoxide group because
15-HETE peaks have a lower retention time than the correspond-
the reaction of synthetic 15-HETE with HOCl resulted in products
with different retention times. Furthermore, the hydroperoxide
group of the chlorohydrin products could be reduced into the
hydroxyl form using the glutathione peroxidase system. Different
reaction conditions were tested, the chlorohydrin formation takes
place both in phosphate buffer (pH 7.4) and borate buffer (pH 9)
also no differences were determined performing the reaction at
ing 15-HpETE peaks. Glutathione alone mediates also the
conversion, however, with a lower efficiency as the GpX/GSH
system.
3.5. Modification of 15-HpETE by myeloperoxidase
ꢀ
The heme-containing myeloperoxidase (MPO) is an important
37 C (data not shown). During the incubation of linoleic acid
enzyme of neutrophil granulocytes producing HOCl under
hydroperoxide with HOCl singlet oxygen has been detected
(Miyamoto et al., 2006). Apparently, singlet oxygen should
À
inflammatory conditions. The MPO-H
2
O
2
-Cl system was now
Table 1
Reaction of 15-HpETE with the MPO-H
incubated with 200 nM or 400 nM MPO with or without the addition of final 100
À
2
O
2
-Cl system. 100
m
M of 15-HpETE, synthesized using arachidonic acid and soybean lipoxygenase and quantified by RP-HPLC, was
À
mM H
2
O
2
, supplied in small amounts, in the presence or absence of 140 mM Cl . The reaction
ꢀ
was performed in phosphate buffer (50 mM, pH 7.4) at room temperature (20 C). After 10 min incubation the reaction was stopped by four-fold dilution in HPLC solvent
acetonitrile/water/acetic acid (60/40/0.2, v/v/v)). Afterwards the samples were centrifuged through a filter (30 kDa) and were analyzed by HPLC. The mean values and
(
standard deviations were calculated from three to five independent experiments. A statistical hypothesis test was performed using a two-tailed t-test (unequal sample sizes,
unequal variances, p ꢁ 0.001 = ***).

5
0
J. Zschaler, J. Arnhold / Chemistry and Physics of Lipids 184 (2014) 42–51
originate not from the direct reaction between both partners.
Another candidate reaction for the formation of singlet oxygen is
the interaction of two peroxyl radicals according to the Russell
mechanism (Russell, 1957).
targets in lipid samples. Oxidized lipid material contains also
different carbonyls, carbohydrates, cyclic endoperoxides, radical
species and other oxidant products (Frankel, 1987). Some of these
products can be targeted by HOCl.
The formation of chlorohydrins upon the reaction of HOCl with
unsaturated fatty acids or phospholipids containing double bonds
in their acyl chains is well described (Arnhold et al., 2001;
Spalteholz et al., 2004; Winterbourn et al., 1992). Possibly all four
double bonds of 15-HpETE are the target site for HOCl. In case of
chlorohydrin formation on double bonds at ÀÀ C5QC6 ÀÀ or
Primarily chlorohydrin formation of fatty acids in phospholi-
pids are implicated in pathophysiological activities due to the
alteration of membrane integrity and the enhanced formation of
lysophospholipids from unsaturated phosphatidylcholines under
the influence of hypochlorous acid (Arnhold et al., 2002). Products
of MPO and eosinophil peroxidase (EPO) are also known to
inactivate the formation of leukotrienes and prostaglandins in
neutrophil and eosinophil granulocytes (Goetzl, 1982; Henderson
et al., 1982; Paredes and Weiss, 1982). Whether the formation of
chlorohydrin derivatives of the eicosanoids or direct interaction of
MPO metabolites with 5-lipoxygenase or prostaglandin H synthase
is the reason for this inactivation, cannot be clarified at the
moment. However, a reaction of the hydroperoxide group of
arachidonic acid metabolites with HOCl can be excluded.
ÀÀ C8QC9 ÀÀ ,
the
diene
configuration
at
ÀÀ C11QC12 ÀÀ C13QC14 ÀÀ remains intact and these chlorohydrin
can be detected by HPLC at 234 nm. Chlorohydrin formation on
double bonds at ÀÀ C11QC12 ÀÀ or ÀÀ C13QC14 ÀÀ disrupts the
diene conjugate structure and these chlorohydrins are not
detectable at 234 nm. This is also reflected by the fact that the
decrease of the 15-HpETE peak at 14.6 min was about twice the
increase of the summary peak area of both chlorohydrin peaks at
7.4 and 7.9 min. To further assess the position of the chlorohydrin
group after reaction of 15-HpETE with HOCl fragmentation spectra
were obtained of the monochlorohydrin species of 15-HpETE
eluted at lower retention time by RP-HPLC. Fragmentation pattern
5. Conclusion
A possible reaction of fatty acid hydroperoxides with HOCl was
assumed in different studies. Thereby the occurrence of fatty acid
radicals and the subsequent reduction of the hydroperoxide group
were hypothesized. But these assumptions have been disproved for
a reaction of 15-HpETE with HOCl. Both the chromatographic and
spectrometric analysis of the reaction products revealed that no
reduction of the hydroperoxide moiety occurs. Instead, chlorohy-
drin of 15-HpETE were formed in consequence of the incubation of
15-HpETE and HOCl. Therefore double bonds of 15-HpETE are a
more preferred target during the reaction of 15-HpETE and HOCl.
r
analysis revealed that apparently the peaks at t of 7.4 and 7.9 min
represent the monochlorohydrins of 15-HpETE, where the
chlorohydrin is formed at the double bond at ÀÀ C8QC9 ÀÀ and
ÀÀ C5QC6 ÀÀ , respectively. The equimolar incubation of synthetic
15-HpETE with HOCl leads to the formation of monochlorohydrins
of 15-HpETE. Only a surplus of HOCl leads to the detection of
dichlorohydrins, although in a lower extent. In the presence of
further reaction partners for HOCl, like the thioether group of
methionine or the amino group of taurine, the chlorohydrin-
forming reaction can be suppressed. Therefore, the double bonds of
15-HpETE are a minor attractive target site of HOCl in comparison
Conflict of interest
to other HOCl targets. Rate constants for the reaction of HOCl with
7
À1 À1
methionine (3.8 Â 10 M
s
, pH 7.4 (Pattison and Davies, 2001))
The authors have no conflicts of interest to declare. The authors
alone are responsible for the content and writing of the paper.
3
À1 À1
or taurine (3.5 Â10 M
s
, pH 4.7 (Marquez and Dunford, 1994))
are much higher than rate constants for the reaction of HOCl with
aliphatic double bonds in phospholipids. Values for the later
Transparency document
À1 À1
constants are reported to be 9 M
s
(Pattison et al., 2003) or
À1 À1
0
.5 M
s
(Panasenko et al., 1997).
The Transparency document associated with this article can be
found in the online version.
The modification of 15-HpETE was also possible through the
À
complete MPO-H
2
O
2
-Cl system. Thereby, the formation of the
characteristic two peaks with lower retention time was detected
similar to the incubation of 15-HpETE with HOCl. That means that
MPO system is also able to form chlorohydrins of 15-HpETE via the
Acknowledgements
The authors would like to thank Dr. Uta Ceglarek and Juliane
Dorow (Institute of Laboratory Medicine, Clinical Chemistry and
Molecular Diagnostics, Medical Faculty, Leipzig University) for
their help in acquiring the LC-MS/MS data. The authors gratefully
acknowledge the financial support provided by the Sächsische
Aufbaubank from a funding of the European Regional Development
Fund (ERDF) (SAB Project Nr: 100116526) and the German Federal
Ministry of Education and Research (BMBF 1315883).
production of HOCl. Interestingly, further control reactions of 15-
À
À
HpETE with several components of the MPO-H
2
O
2
-Cl system
revealed that already the presence of MPO or MPO + Cl dimin-
ished the yield of 15-HpETE. However, there was no formation of
chlorohydrins or 15-HETE. Thus, any two-electronic reduction of
15-HpETE to 15-HETE by MPO can be excluded. Ferric MPO is well
known to reduce different organic hydroperoxides to the
corresponding hydroxides (Furtmuller et al., 2000). However,
the interaction of hydroperoxides of arachidonic acid with MPO
was not investigated by these authors. Maybe some 15-HpETE was
non-specifically bound to the enzyme and removed during the
centrifugation step. The further decrease in the 15-HpETE yield in
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