Thermodynamic and structural characterization of cis-trans isomerization of 12-(S)-hydroxy-(5Z, 8E,10E)-heptadecatrienoic acid by high-performance liquid chromatography and gaschromatography-mass spectrometry

Article abstract of DOI:10.1016/S0009-3084(98)00066-8

It is shown that 12-(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid (5-cis-HHT) - a physiological metabolite of arachidonic acid-is acid-catalyzed converted into a less polar substance with its maximum UV-absorption at λ(max)=232 nm and a molar absorptivity of about ε=26 600±200 M^-1 cm^-1. Using a reversed-phase high-performance liquid chromatographic (HPLC) method this equilibrium reaction (K(c)=1.78±0.05 at pH 1.10 and 298 K) could be thermodynamicly characterized as a pH dependent, exergonic and exothermic reaction according to kinetics of a first order reaction (at pH 1.10 and 298 K: Δ(R)G°=-1.42±0.07 kJ mol^-1, Δ(R)H°=-3.50±0.9 kJ mol^-1, Δ(R)S°=-7.0±3.0 J mol^-1*K, Δ(R)H(f)(≠)=100.0±4.0 kJ mol^-1). Kinetic data for several pH-values and temperatures are presented. These data and structural characterization by gaschromatography-mass spectrometry (GC/MS) lead to the conclusion that 5-cis-HHT is isomerized to 12-(S)-hydroxy-(5E,8E,10E)-heptadecatrienoic acid (5-trans-HHT). Copyright (C) 1998 Elsevier Science Ireland Ltd.

Full text of DOI:10.1016/S0009-3084(98)00066-8

CPL
CHEMISTRY AND
PHYSICS OF LIPIDS
Chemistry and Physics of Lipids
95 (1998) 181 188
ELSEVIER
Short communication
Thermodynamic and structural characterization of cis-trans
isomerization of 12-(S)-hydroxy-(5Z, 8E,10E)-heptadecatrienoic
acid by high-performance liquid chromatography and
gaschromatography-mass spectrometry
Harald John a,b Werner Schlegel b,,
a
Klinik und Poliklinikfiir Geburtshilfeund Frauenheilkunde, WestfgilischeWilhelrns-UniversitiitMiinster, Dornagkstr. 11,
D-48149 Miinster, Germany
bAnorganisch-ChernischesInstitut, Analytische Chernie, Westfiilische Wilhelrns-UniversitiitMiinster, D-48149 Munster, Germany
Received 11 May 1998; received in revised form 10 June 1998; accepted 2 July 1998
Abstract
It is shown that 12-(S)-hydroxy-(5Z,8E, lOE)-heptadecatrienoic acid (5-cis-HHT)--a physiological metabolite of
arachidonic acid--is acid-catalyzed converted into a less polar substance with its maximum UV-absorption at
2max = 232 nm and a molar absorptivity of about ~= 26600 __+200 M - 1 cm - 1. Using a reversed-phase high-perfor-
mance liquid chromatographic (HPLC) method this equilibrium reaction (Kc = 1.78 __+0.05 at pH 1.10 and 298 K)
could be thermodynamicly characterized as a pH dependent, exergonic and exothermic reaction according to kinetics
of a first order reaction (at pH 1.10 and 298 K: ARG°= - 1.42 + 0.07 kJ mol 1, ARHO= _ 3.50__+ 0.9 kJ mol-l,
ARS° = - 7.0 ± 3.0 J mol- l'K, ARHf~ = 100.0 + 4.0 kJ mol- 1). Kinetic data for several pH-values and tempera-
tures are presented. These data and structural characterization by gaschromatography-mass spectrometry (GC/MS)
lead to the conclusion that 5-cis-HHT is isomerized to 12-(S)-hydroxy-(5E,8E, lOE)-heptadecatrienoic acid (5-trans-
HHT). © 1998 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: 12-(S)-Hydroxyheptadecatrienoic acid (HHT); Cis-trans isomerization; High-performance liquid chro-
matography; Gas chromatography-mass spectrometry
1. Introduction
*
Corresponding author. Tel.: +49 251 8356114; fax: +49
Arachidonic acid (AA) is an ubiquitous es-
251 8356114
sential unsaturated fatty acid. AA is converted
0009-3084/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(98)00066-8

182
H. John, W. Schlegel/ Chemistry and Physicsof Lipids 95 (1998) 181-188
prostaglandin H 2 (PGH2) by the cyclooxygenase
enzyme complex (Hamberg and Samuelsson, 1974;
Hamberg et al., 1974). PGH2 is metabolized via
thromboxane synthase to thromboxane A2, mal-
acid was obtained from Cayman (Ann Arbor,
MI). Acetonitrile (ultragradient grade) and water
(HPLC grade) were obtained from J.T. Baker
(Deventer, The Netherlands). Tri-sodium citrate
ondialdehyde
and
12-(S)-hydroxyheptadeca-
5.5 hydrate, trifluoracetic acid (TFA), ethanol
trienoic acid (HHT). This synthesis is stereospecific
for the 5Z,8E, IOE-HHT isomer (5-cis-HHT) and
appears predominantely in lung microsomes (Wlo-
dawer and Hammarstrrm, 1978) and platelets
and petrolether were provided by Merck (Darm-
stadt, Germany) and were of reagent grade. Solid-
phase extraction (SPE) columns (Chromabond C 18
ec, 500 mg in glass) were obtained from Macherey-
Nagel (Diiren, Germany).
(
Yoshimoto et al., 1977). There is still little infor-
mation available about the physiological role of
HHT, its enzymatic metabolization or chemical
derivatization in vivo (John et al., 1998). In order
to clarify the physiological function of HHT,
fundamental knowledge of its chemical properties
and behaviour is required. The relevance of chem-
ical transformation might be discussed with respect
to the physiological function of HHT. Further-
more, these properties may be important for the
determination of HHT. When analyzing HHT, this
substance is often extracted under acid conditions,
since analyses of prostaglandins and HHT are done
after acidification of the samples. In addition
HPLC and thinlayer chromatographic measure-
ments of underivatized fatty acids are performed
with acid solvents in order to protonate the car-
boxylicfunctions of the analytes (Pruimboom et al.,
2
.2. Kinetic studies on 5-cis-HHT conversion
Aliquots of ethanolic solutions of physiologi-
cal 5-cis-HHT (10 /Lg) were dried in glass tubes
under a gentle stream of nitrogen. The residues
were redissolved in temperature adjusted mix-
tures comprising of 60% v/v acetonitrile and
40% v/v 0.061 M citrate buffer. These solutions
were stored at ll°C (pH 1.10), at 25°C (pH
1
.10, 1.24 and 1.56) and at 38°C (pH 1.10) in
waterbathes. After several periods of reaction
time (0-18 h, dependent on pH and tempera-
ture) aliquots of 20 /~1 were chromatographed
using the HPLC method described below and
peak areas were integrated.
1992; McKinnon et al., 1993). Following these
procedures HHT is also stored and treated in acid
solutions for variable periods of time and at differ-
ent temperatures. Therefore, this report describes
the chemical behaviour of the physiological 5-cis-
HHT especially under acid conditions in vitro. The
appearance of a HHT-derivative--defined as
HHT-D--could be observed in a time and pH
dependent manner. The acid-catalyzed conversion
of 5-cis-HHT into HHT-D--not discussed in the
literature so far--is illustrated with respect to
kinetic and thermodynamic data. The physiological
potency of HHT remains to be investigated, how-
ever.
2
.3. Purification of 5-cis-HHT and HHT-D
HPLC eluates corresponding to 5-cis-HHT
and HHT-D were collected in glass tubes, di-
luted with water (resulting in 30% v/v acetoni-
trile) and extracted using solid-phase extraction
on C18 ec material (John et al., 1998). SPE elu-
ates were evaporated to dryness and used for
GC/MS measurements.
2.4. Chromatographic methods
The HPLC equipment consisted of a Rheo-
dyne 7725i syringe loading injector (Cotati, CA,
USA), a pump system 322, a diode-array detec-
tor 440 (DAD) and the Kroma 2000 HPLC
software from Kontron Instruments (Neufahrn,
Germany).
2
. Materials and methods
.1. Materials
2-(S)-Hydroxy-(5Z,8E, 10E)-heptadecatrienoic
2
1

H. John, W. Schlegel / Chemistry and Physics of Lipids 95 (1998) 181-188
183
The isocratic HPLC analyses (CH3CN-HzO, pH
.5, 55:45, v/v) were carried out at 30°C on a
25 x 4.6 mm I.D. column packed with Nucleosil
20 C18 (particle size 5 am), and a pre-column
26 x 6.0 mm I.D.) of the same material which
was obtained from Bischoff (Leonberg, Ger-
many). The temperature of the RP column was
adjusted to 30°C in a Precitherm PFV water bath
HHT-D--measured from 190 to 300 nm by
DAD--was identical to that one of HHT and its
UV-absorption maximum was detected at 232 nm
as well (results not shown). These spectroscopic
data suggest the existance of the same chro-
mophoric group for 5-cis-HHT and HHT-D.
In order to characterize the kinetics of this
conversion concentration-time curves were mea-
sured for all combinations of pH and tempera-
tures. A typical curvature is shown in Fig. 2a.
These concentration profiles are typical for an
equilibrium reaction. As demonstrated by the to-
tal peak area, ZA, sum of peak area of 5-cis-
HHT, A(HHT), and peak area of HHT-D,
A(HHT-D), the molar absortivity of HHT-D at
232 nm, e(HHT-D), is smaller than that one of
5-cis-HHT, e(HHT)= 33000 M-l cm-1 (Hecker
and UUrich, 1988). e(HHT-D) was determined by
plotting EA-A(HHT) versus Ao(HHT)-A(HHT)
resulting in straight lines with the slope ct (results
not shown). Ao(HHT) is the initial peak area of
5-cis-HHT. ~ was defined as:
3
1
1
(
(
Boehringer, Mannheim, Germany). The flow rate
was 1.0 ml rain-~. HHT and HHT-D were de-
tected at 232 nm. For GC/MS analyses purified
5
-cis-HHT and HHT-D were converted into their
bis-(trimethylsilyl)-derivatives. The GC (Varian
400, Varian, Walnut Creek, CA, USA) equip-
3
ment consisted of a cold inlet system, Gerstel 502
with a 25 m HPU two capillary column (Hewlett-
Packard, Waldbronn, Germany) which terminates
at the MS-EI source, 70 eV, (Finnigan MAT
8230, Finnigan, San Jose, CA). Helium was used
as carrier gas. The injection temperature rose
from 60 up to 310 °C and the oven temperature
rose from 80 up to 300°C at 8°C min-t for 24
rain.
=
~(HHT - D)/e(HHT) at 232 nm.
(1)
The ratio of molar absorptivities, ~t, was calcu-
lated by the mean and standard deviation of all
measurements to be 0.806 +0.006 resulting in
e(HHT-D) = 26600 + 200 M - ~cm - 1.
3
. Results and discussion
The investigation of the conversion of physio-
logical 5-cis-HHT under acid conditions requires
complete dissolution of HHT in the solvent. As
acid buffer solutions effect a partition equilibrium
of HHT between the vial surface and the aqueous
solution the addition of acetonitrile was used to
avoid this phenomenon (John, H., unpublished
data). In addition the sample matrices comprising
of 60% v/v acetonitrile allowed direct chromato-
graphic analyses. 5-cis-HHT was detected at 232
nm according to its conjugated double-bonds and
its ~-hydroxy-group (Fig. 3).
Furthermore, Fig. 2, a shows the period of half
change, z~/z, that is defined as the period of reac-
tion time producing half the amount of the equi-
librium concentration of HHT-D. zt/2 was
determined graphically.
The influence of pH and temperature on the
kinetics were measured using the following combi-
nations: at 298 K and close to 106 Pa (chemical
standard conditions) for pH 1.10, 1.24 and 1.56
and at pH 1.10 for 284, 298 and 311 K, respec-
tively. As shown in Table 1 the period of half
change decreased strongly with the increasing re-
action temperature and the decreasing pH-values.
The reaction order and the rate constants for
the conversion of 5-cis-HHT were calculated us-
ing kinetic data on the hypothesis of a first order
equilibrium reaction. Plots of In{(Ao(aHHT)-
Aeq(HHT))/(A(HHT)-Aeq(HHT))} versus the re-
action time yielded in straight lines (regressions of
first order) proving the correctness of the hypoth-
When analyzing the HHT-solutions by HPLC-
technology after increasing periods of reaction
time decreasing 5-cis-HHT concentrations (tR =
5.98 _+0.02 rain) cou!d be observed for all tested
pH-values and temperatures corresponding to in-
creasing concentrations of a new product (tR =
6.98 _+0.02 min)--defined as HHT-D (Fig. 1). As
indicated by its higher retention time HHT-D is
less polar than 5-cis-HHT. The UV-spectrum of

8
4
H. John, W. Schlegel/ Chemistry and Physics of Lipids 95 (1998) 181-188
0
~F i2~
t"-~.
"N '~.
"" ~
~
.1
m
0
0
~
"~'
o
~
~
•'13 ,.o ,.o
+I +I+I +I
=
~"
~
~
~
~
~
'~
0
0
0
0
0
+
I+I +I+I+I
I
~
o
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+I
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+
T
Nu~
,
ge e
+
I
.... +I o
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mtltlm~l
0
+I +I0.+IO
..
=
E
8
+~+~+~+l+~ .~ e ~
.Q.
°
~
0
I~
~
~Io

H. John, W. Schlegel / Chemistry and Physics of Lipids 95 (1998) 181-188
185
Cq
¢
¢3
t"q
o
O
r,¢')
0
.00
2.50
5.00
9.01
retention time
Fig. 1. HPLC analyses of reaction time dependent conversion of 5-cis-HHT into HHT-D. Nucleosil 120 C18, 125 x 4.6 mm I.D.,
pm particle size, CH3CN-H20 55:45 v/v, pH 3.5, 1 ml min ~ 1, 30oc, 232 nm, tR(5-cis-HHT) = 5.98 _+0.02 min, tR(HHT-D) =
.98 _+0.02 rain, reaction times: h 0.013 h, 2:1.5 h, 3:2.5 h, 4:4.5 h, 5:6.5 h, 6:13 h, 7:16 h and 8:18.5 h at 284 K and pH 1.10
5
6
esis (Fig. 2,b). Data of Table 1 present the slopes,
m, of these plots and underline the high accuracies
of regressions. The slopes, m, of these plots are
described by"
ARG, and the rate constants could be calculated.
The results are summarized in Table 1. Kc rose
from 1.63 _+0.05 at 311 K (pH 1.10) to 1.86 _+0.05
at 284 K (pH 1.10) indicating an exothermic
reaction. The equilibrium concentration of HHT-
D is about 60% higher than that one of 5-cis-HHT
indicating a thermodynamic more stable product
under the test conditions. The negative standard
Gibbs free energy of reaction, ARG° = - 1.42 _+
0.07 kJ mol -I (298 K, pH 1.10), indicates an
exergonic process.
m = (1 + Kg- 1)*k1
with
(2)
Kc = kl/k- I"
(3)
Kc is the equilibrium constant of the conversion of
-cis-HHT and k 1 and k_ 1 are the rate constants
5
of the forward and reverse reaction, respectively.
According to these results the conversion of 5-cis-
HHT is described by the Eqs. (4) and (5):
k_L
An Arrhenius plot was used to calculate the heat
of activation, ARH# , and the frequency factors, A,
for the forward (f) and reverse (r) reactions (pH
1.10). Frequency factors and heats of activation
were obtained as follows: Ar=2.89.1017 h -I,
ARH ~ = 100 _+4.0 kJ mol- l (forward reaction)
and At=6.70.1017 h 1, ARHr~ = 103.4_+4.0 kJ
mol - 1 (reverse reaction). The standard entropy of
reaction, ARS°, of the 5-cis-HHT conversion was
calculated using Eq. (6):
5
-- cis -- HHT k~- HHT - D
(4)
Kc = [HUT - D]/[5 - cis -HH~]
Aeq(HHT -- D)/Aeq(HHT).~
=
(5)
With respect to these equations the equilibrium
constant, K~, the Gibbs free energy of reaction,

1
86
H. John, W. Schlegel / Chemistry and Physics of Lipids 95 (1998) 181- 188
ARH° = AR GO + TAR S°
ARS°
=
R ln(Af/Ar)
(6)
(7)
resulting in - 7.0 __ 3.0 J mo-~,K (pH 1.10). The
standard enthalpy of reaction, ARH°, was ob-
tained using Eq. (7):
ARH° was negative ( - 3.50 + 0.9 kJ mol- ] at pH
1.10) proving an exothermic reaction. Assuming
that the frequency factors are independent on the
pH-values the heats of activation for the forward
and reverse reaction for all tested pH-values (pHi)
were calculated according to Eqs. (8) and (9):
4O
R T ln{k(pHi)/k(pH 1.10)} = AARH#
(8)
(9)
ARH#(pHi) = ARH#(pH 1.10) + AARH#
The standard enthalpies of reaction for tested
pH-values were characterized by Eq. (10):
2
0
ARH°(pHi) = ARHf~ (pHi) - ARH b~ (pHi)
(10)
The results are listed in Table 1. Using these data
gives the standard entropies of reaction:
ARS° = (ARGO
-
-
ARH°)/T
(11)
o
I
I
1
I
I
as demonstated in Table 1.
0
"Cli2
1
2
3
4
With respect to the quantity and negative value
of ARH° the cyclization of 5-cis-HHT between C5
and C9 to a cyclopentene-derivative (ARH° ~ +
reaction time (h)
8
4 kJ mol-1) must be excluded. Our findings
i
i
i
i
i
i
i
i
!
i
i
show equal values when compared with the gen-
eral isomerizations of ¢is (c) double-bonds to the
more stable trans (t) conformations. The standard
enthalpies and standard entropies are also in the
same range: e-2-pentene to t-2-pentene: ARH°=
v
g
<
,
I
-
- 4 kJ mol- 5, ARSO= _ 6 J mol- 1,K; c - 2-
hexene to t-2-hexene: ARH°= --2 kJ mo1-1,
ARS°= --6 J mol-]*K and c-3-hexene to t-3-
hexene: ARH° = -- 6 kJ mol- 5, ARSO= _ 5 J
mol-~,K (Aylward and Finlay, 1974).
g
The thermodynamic results of our study may
also point to a cis-trans isomerization of 5-cis-
HHT to 5-trans-HHT under physiological condi-
tions. In order to evaluate the structure of
HHT-D this substance was isolated and purified
by HPLC and concentrated by solid-phase extrac-
tion. GC/MS measurements were carried out after
trimethylsilylation of HHT and HHT-D using
"
1-
<
_
o
c
i
i
!
i
i
n
a
i
i
•
i
0
1
2
3
4
5
6
7
8
9
10 11
reaction time (h)
Fig. 2. (a) Peak area-time curves of the conversion of 5-cis-
HHT into HHT-D measured by HPLC (298 K, pH 1.10) -O--
5-cis-HHT, -V- HHT-D, -•-total of preak areasr]/2: period
N-methyl-N-(trimethylsilyl-)-2,2,2
trifluoroac-
of half change, Nucleosil 120 C18, 125 x 4.6 mm I.D., 5 pm
particle size, CH3CN-H20 55:45 v/v, pH 3.5, 1 ml min-],
etamide (MSTFA). The mass spectrum of the
HHT-D derivative is shown in Fig. 3. The molec-
ular peak at m/z = 424 proved that HHT-D has
been derivatized to its bis-(trimethylsilyl)-deriva-
tive indicating a free hydroxy- and a free car-
30°C, 232 nm. (b) Determination of rate constants for the
conversion of 5-cis-HHT Slopes, m, are defined by: m = (1 +
KZI*)k]. ~311 K, pH 1.10; • 298 K, pH 1.10; • 298 K, pH
1.24; • 298 K, pH 1.56; • 284 K, pH 1.10.

H. John, W. Schlegel / Chemistry and Physics of Lipids 95 (1998) 181-188
187
3
34
reported that cis-trans isomers are metabolized
differently by the same enzymes (Lawson and
Holman, 1981; Berdeaux et al., 1996). The con-
version of cis- into trans-isomers can be achieved
by isomerases (Chen et al., 1994). At least it has
been shown that enzymes inducing peroxidation
of polyunsaturated fatty acids are able to produce
both the cis- and the trans-isomers of hydroper-
oxides (Lodge et al., 1995). Therefore, further
studies on the physiological power of 5-trans-
HHT might be useful to clarify the physiological
role of HHT.
2
25
%1 .1 "
2
.5"
.0"
.5"
.0"
.5"
.0
4
O9
2
1
OSiMe2
60"
3
19
1
1.10
7"3
3
~
5
53
t2.4
0
4O"
~5
0
Z26
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21~
_
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~o i~o 1~o ~o
References
m/z
Fig. 3. Mass spectrum (70 eV) of bis-(trimethylsilyl)-derivative
of HHT-D; molecular weight, 424. GC/MS analysis was done
with a 25 m HPU 2 capillary column, MS-E1, 70 eV. Condi-
tions for GC: carrier gas: He, injection volume: 1 /~1,injection
temperature: 60-310°C, oven temperature, 80-300°C at 8°C
min- i for 24 min m/z 424 (M + ), 409 (M +-15, loss of CH3),
Aylward, G.H., Finlay, T.J.V., 1974. SI chemical data. In:
Aylward, G.H. Finlay, T.J.V. (Eds.), John Wiley and Sons,
Australia, pp. 92.
Berdeaux, O., Chardigny, J.M., Sebedio, J.L., et al., 1996.
Effects of a trans isomer of arachidonic acid on rat platelet
aggregation and eicosanoid production. J. Lipid Res. 37,
2244-2250.
Chen, L.-S., Jin, S.-J., Tserng, K.-Y., 1994. Purification and
mechanism fA3,AS-t-2,t-4-dienoyl-CoA isomerase from rat
liver. Biochemistry 33, 10527-10534.
3
53 (M+-71, loss of C5H10, 334 (M+-90, loss of Me3SiOH),
25 (M +-199, loss of CH2 CH--CH (CH2)3 COO-SiMe3), 73
Me3Si +) and 75 (Me2SiOH ÷).
2
(
Hamberg, M., Samuelsson, B., 1974. Prostaglandin endoper-
oxides. Novel transformations of arachidonic acid in hu-
man platelets. Proc. Nat. Acad. Sci. USA 71, 3400 3404.
Hamberg, M., Svensson, J., Wakabayashi, T., Samuelsson, B.,
boxylic group. The data of the MS profile exclude
hydration, dehydration, esterification or other re-
action processes that could change the molecular
weight or functionality of 5-cis-HHT. In compari-
son to the mass spectrum of 5-cis-HHT (John et
al., 1998) the same fragments differing in their
relative intensity can be noticed. These facts indi-
cate very similar structures of 5-cis-HHT and
HHT-D.
1974. Isolation and structure of two prostaglandin en-
doperoxides that cause platelet aggregation. Proc. Natl.
Acad. Sci. USA 71, 345-349.
Hecker, M., Ullrich, V., 1988. 12(S)-Hydroxy-5,8,10 (Z,E,E)-
heptadecatrienoic acid (HHT) is preferentially metabolized
to its 12-keto derivative by human erythrocytes in vitro.
Eicosanoids. 1, 19-25.
John, H., Cammann, K. and Schlegel, W., 1998. Development
and Review of Radioimmunoassay of 12-S-Hydroxyhep-
tadecatrienoic Acid. Prostaglandins and other Lipid Medi-
ators (accepted for publication in January 1998).
Lawson, L.D., Holman, R.T., 1981. Oxidation of the geomet-
ric and positional isomers of octadecenoic acid by rat heart
and liver mitochondria. Biochim. Biophys. Acta 665, 60-
In consequence of the discussed thermodynamic
(
ARH° and ARS°), kinetic (first order reaction),
spectroscopic (2.... e, UV-spectrum) and struc-
tural (free -OH and -COOH groups, less polarity)
data, it can be concluded that 5-cis-HHT is acid-
catalyzed converted into 5-trans-HHT.
Therefore, longer treatment or storage of 5-cis-
HHT under acid conditions have to be avoided in
order to prevent false analyses. Furthermore, our
findings may be relevant under physiological con-
ditions. Geometric isomers are known for their
different physiological potencies as described for
pheromones or cis-trans retinal--necessary for
the eye-function--(Stryer, 1995) and it has been
6
5.
Lodge, J.K., Sadler, P.J., Kus, M.L., Winyard, P.G., 1995.
Copper-induced LDL peroxidation investigated by 1H-
NMR spectroscopy. Biochim. Biophys. Acta 1256, 130-
1
40.
McKinnon, K.P., Madden, M.C., Noah, T.L., Devlin, B.D.,
993. In vitro ozone exposure increases release of arachi-
1
donic acid products from a human bronchial epithelial cell
line. Toxicol. Appl. Pharmacol. 118, 215-223.

188
H. John, W. Schlegel/ Chemistry and Physics of Lipids 95 (1998) 181 188
Pruimboom, W.M., Vollebregt, M.J., Zijlstra, F.J., Bonta,
I.L., Wilson, J.H.P., 1992. Eicosanoid production by
density-defined human peritoneal macrophages during infl-
ammation. Agents Actions, special conference issue. C96
Wlodawer, P., Hammarstr6m, S., 1978. Thromboxane syn-
thase from bovine lung-solubilizationand partial purifica-
tion. Biochem. Biophys. Res. Comm. 80, 525-532.
Yoshimoto, T., Yamamoto, S., Okuma, M., Hayaishi, O.,
1977. Solubilization and resolution of thromboxane syn-
thesizing system from microsomes of bovine blood
platelets. J. Biol. Chem. 252, 5871-5874.
9
8.
Stryer, L., Biochemistry, 4th ed. In: Stryer, L. (Ed.), W.H.
Freeman and Company, New York, pp. 318-320.