(Received for publication, April 24, 1995)
From the
The free radical oxidation of arachidonic acid esterified to
glycerophospholipids is known to generate complex metabolites, termed
isoprostanes, that share structural features of prostaglandins derived
from prostaglandin H
Oxygen free radicals are generated in vivo by a variety
of enzymatic and nonenzymatic
reactions(1, 2, 3) . These reactive oxygen
species are thought to play an important role in tissue damage
characteristic of many diseases including atherosclerosis, inflammatory
diseases, cancer, aging, and ischemia reperfusion
injury(4, 5, 6) . While the exact biochemical
mechanisms relating free radical generation to the pathology are
unclear, most points of view consider profound alterations in tissue
biochemistry as a result of lipid peroxidation, DNA damage, or
irreversible alteration of
proteins(7, 8, 9, 10, 11) .
Although lipid peroxidation leads to a large number of products, some
of which are stable species derived from polyunsaturated fatty acyl
substituents of phospholipids in membrane bilayers, the measurement of
low molecular weight products such as pentane, malonyldialdehyde, and
4-hydroxynonenal have been the most widely measured oxidation products
for studies of lipid peroxidation(12, 13) . It is
likely, however, that these low molecular weight products result from
extensive rearrangement of the initial oxidized phospholipid species
and, as such, may not reflect initial oxidation events taking place at
the lipid bilayer membrane level.
Recently compounds that are
isomeric to prostaglandins (isoprostanes) were discovered to be
generated by free radical-mediated processes and whose formation was
not catalyzed by the enzyme prostaglandin H
synthase(14, 15) . F
Since the
nonenzymatic, free radical-induced formation of
F
HPLC analysis of the external standards was run immediately
after the analysis of oxidized eicosanoids.
Reverse phase HPLC separation of the oxidized
1-hexadecanoyl-2-arachidonoyl-GPCho revealed the presence of a large
number of products. The major component in the mixture remained
unreacted 1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine, as
indicated by the absorbance profile at 205 nm and elution at 78 min (Fig. 1A). The elution of conjugated trienes was
indicated by absorbance at 270 nm (Fig. 1B), and many
components absorbing at this wavelength were detected having
significantly less lipophilicity than the starting material. The UV
spectra of several components eluting between 44 and 54 min were
suggestive of the characteristic vibronic UV absorption of conjugated
trienes, as illustrated by the component eluting at 53 min in this HPLC
separation (Fig. 1B, inset). The UV absorption
profile at 235 nm (data not shown) revealed elution of several
conjugated diene oxidized products in this same general area of the
chromatogram.
Figure 1:
Reverse phase HPLC separation of
1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine (16:0a/20:4-GPC) oxidized by
Cu
Figure 2:
Reverse phase HPLC of the free acids
obtained following saponification of the oxidized phospholipids in
fraction 53 by gradient system B (see ``Experimental
Procedures''). A, elution of components absorbing at 270
nm. A single component eluted at 15.3 min, which yielded the
ultraviolet absorption spectrum of the conjugated triene (inset). B, equal aliquots of each half-minute
fraction collected during the HPLC run were taken to dryness, dissolved
in Ca
Figure 3:
A, tandem mass spectrometry of m/z 335, the major ion obtained by electrospray ionization of
HPLC fraction (system B) (RRI = 2.54) eluting between 15 and
15.5 min (Fig. 2). B, electron ionization mass spectrum
of the major component obtained following catalytic reduction and
derivatization to the pentafluorobenzyl ester trimethylsilyl ethers
obtained by GC/MS. This mass spectrum was indicative of derivatized
5,12-dihydroxyeicosanoic acid.
Figure SI:
Scheme I.
The response of Indo-1-labeled
neutrophils to LTB
Figure 4:
Increase in fluorescence in human
neutrophils containing Indo-1 following addition of
B
There was sufficient quantity of the
isoleukotriene eluting with a relative retention index of 2.54 (Fig. 2A) for a dose-response study (Fig. 5). An
EC
Figure 5:
Dose-response curve for the elevation of
intracellular free calcium caused by the B
Free radical oxidation of arachidonic acid esterified to
glycerophospholipids is known to result in a host of oxidized
intermediates. The recent discovery of the isoprostanes as free radical
oxidation products of arachidonic acid and arachidonate-containing
glycerophospholipids has emphasized that complex rearrangements of
intermediate peroxy radical or hydroperoxides, formed as initial
oxidation products, can take place. Described here is a new family of
free radical-generated eicosanoids derived from arachidonoyl
phospholipids. Several dihydroxy eicosanoids were observed, including
several that contain a conjugated triene structural feature similar to
that observed in the enzymatic products of 5-lipoxygenase. Detailed
structural studies of an abundant, biologically active component (RRI
= 2.54) revealed this eicosanoid to be a
5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. Therefore, we have
termed this conjugated triene dihydroxy eicosanoids as a
``B
Several free radical mechanisms are possible by
which such dihydroxy-conjugated trienes could be formed from
arachidonic acid esterified to phospholipids. It is most likely that
two separate radical abstraction events removing the bisallylic
hydrogen atoms on carbon atoms 7 and 10 occurred and the addition of
molecular oxygen resulted at carbon atoms 5 and 12 of the arachidonic
acid backbone. One interesting possibility is an intermediate formation
of a leukotriene A
Several
B
A number of LTB
B
synthase. Furthermore, certain
isoprostanes have been found to exert biological activity through
endogenous receptors on cell surfaces. Using mass spectrometry and
ancillary techniques, the free radical oxidation of
1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine was studied in the
search for products of arachidonic acid isomeric to the leukotrienes
that are derived from 5-lipoxygenase-catalyzed metabolism of
arachidonic acid. Several conjugated triene metabolites were
chromatographically separated from known 5-lipoxygenase products and
structures characterized as 5,12-dihydroxy-6,8,10,14-eicosatetraenoic
acid esterified to the glycerophosphocholine backbone. We have termed
these products as B
-isoleukotrienes. Following
saponification some, but not all, B
-isoleukotrienes were
found to exert biological activity in elevating intracellular calcium
in Indo-1-loaded human polymorphonuclear leukocytes. This activity
could be blocked by a leukotriene B
receptor antagonist. An
EC
of approximately 30 nM was determined for one
unique B
-isoleukotriene with a relative retention index of
2.54. We have shown that free radical processes can lead to the
formation of biologically active isoleukotrienes in
glycerophosphocholine liposomes, and we propose that
B
-isoleukotrienes may also be formed in membrane
glycerophospholipids as a result of lipid peroxidation during tissue
injury. Such B
-isoleukotrienes could then mediate events of
tissue damage through activation of leukotriene B
receptors
on target cells.
-isoprostanes
(isomeric of prostaglandin F
) were readily identified
esterified to phospholipids in the liver of rats that had been treated
with carbon tetrachloride in a model of free radical
hepatotoxicity(16) . One such free radical product, 8-epi
prostaglandin F
was also found to have significant
biological activity as the free acid. This isoprostane was found to
cause a potent constriction of the renal artery of the rat and reduced
glomerular filtration rate in the rat kidney(17) . It was also
found to have activity in other organs including the lung, where it
constricted the pulmonary artery, but was a more potent constrictor of
the bronchial airway in the isolated perfused lung(18) .
Interestingly, these biological activities could be prevented or even
reversed by a thromboxane A
receptor
antagonist(17) , suggesting that this F
-isoprostane
was exerting its pharmacological effect through the endogenous receptor
for thromboxane. These findings have led to the suggestion that
isoprostanes may serve as lipid mediators of free radical-induced
damage at the tissue level(15, 17) .
-isoprostanes in vivo result from a free radical
attack on arachidonoyl-containing phospholipids in vivo, it
became of interest to investigate whether or not other complex
molecules esterified to phospholipids could be formed by free radical
reactions, in particular complex molecules structurally related to the
leukotrienes. Leukotrienes are normally thought to be derived only from
the 5-lipoxygenase pathway of arachidonic acid metabolism. To clarify
whether or not such compounds could be formed, we investigated the free
radical oxidation 1-hexadecanoyl-2-arachidonoyl-glycerophosphocholine
liposomes and report that hydroxyl radical generated by a modified
Fenton reaction (19) led to the formation of numerous products
including several isomers of leukotriene B
(isoleukotrienes) that exhibited biological activity via the
leukotriene B
receptor.
Materials
Leukotriene B and other
eicosanoid standards were purchased from Cayman Chemical Co. (Ann
Arbor, MI) and used without further purification.
1-Hexadecanoyl-2-arachidonoyl glycerophosphocholine was purchased from
Avanti Polar Lipids (Alabaster, AL). Radiolabeled
1-hexadecanoyl-2[
C
]arachidonoyl
glycerophosphocholine (57 mCi/mmol) was purchased from DuPont NEN. The
LTB
receptor antagonist LY223982 was a kind gift from Eli
Lilly. All solvents used were of the highest available purity. Hydrogen
peroxide (30%, w/v), copper(II) chloride, digitonin, ammonium acetate,
EGTA, Trizma (Tris base), and phosphate-buffered saline tablets were
purchased from Sigma or Aldrich. Sepralyte octadecylsilyl solid phase
extraction packing material (40 µm) was purchased from Analytichem
International (Harbor City, CA). Pentafluorobenzyl bromide,
diisopropylethylamine, and 5% rhodium adsorbed on alumina powder were
purchased from Aldrich. Bis-trimethylsilyl trifluoroacetamide was
purchased from Supelco (Bellefonte, PA). Indo-1 acetoxymethyl ester was
obtained from Calbiochem.
Free Radical Oxidation of
1-Hexadecanoyl-2-arachidonoyl-GPCho
Ten micromoles of
1-hexadecanoyl-2-arachidonoyl-GPCho()
in
CHCl
solution (10 mg/ml) was placed in an 8-ml screw cap
glass tube.
1-Hexadecanoyl-2[
C
]arachidonoyl-GPCho
(5 µCi) was also added to some reactions. The solvent was
evaporated at room temperature under a stream of N
gas. The
phospholipid was immediately resuspended in 4.8 ml of 50 mM
PBS, pH 7.3, by vortexing and then sonicating for 5 s at maximum power.
H
O
(30%, w/v), and 6 mM CuCl
were added to the solution resulting in final concentrations of
600 mM and 100 µM, respectively. The solution was
capped and heated to 37 °C on a gently shaking water bath for 3 h.
For some experiments reactions were allowed to continue at 5 °C
overnight before extraction. Progress of oxidation was monitored by UV
absorbance at 235 nm for conjugated dienes and 270 nm for conjugated
trienes in a 0.1% aliquot.
Solid Phase Extraction
The reaction mixture was
loaded onto 2 g of reverse phase 40-µm silica particles packed in a
low pressure glass column. The column was preconditioned with 30 ml of
methanol followed by 30 ml of PBS under slight pressure. Salts and
remaining HO
were eluted with 30 ml of water.
Oxidized GPCho was eluted with 15 ml of methanol into a 50-ml
pear-shaped flask. The remaining 1-hexadecanoyl-2-arachidonoyl-GPCho
was eluted with an additional 10 ml of methanol.
Stannous Chloride Reduction
Hydroperoxides or
endoperoxides formed during the oxidation reaction were reduced with
stannous chloride to prevent further rearrangement and degradation of
these products. Stannous chloride (100 mM) was added to the
methanol fraction containing the oxidized GPCho to a final
concentration of 1 mM. The flask was placed on a rotary
evaporator and the solvent removed to near dryness. The remaining
liquid in the flask was rinsed into a test tube (1 ml) and
injected directly onto the reverse-phase HPLC system.
Reverse Phase HPLC of Oxidized GPCho
A Beckman
(Berkeley, CA) ODS 5-µm 4.6 mm 25-cm column with a Waters
(Marlborough, MA) ODS Guard-pak precolumn was used to separate the
oxidized GPCho. The solvent system (system A) consisted of 85%
methanol, 1 mM ammonium acetate at 1.5 ml/min for 25 min,
followed by a linear gradient to 100% methanol, 1 mM ammonium
acetate over a 50-min period. Either a photodiode array detector
(Hewlett-Packard 1090A) or a linear 206 scanning UV detector (Linear
Instruments, Reno, NV) were used to continuously record UV spectra,
scanning from 205 nm to 320 nm with a 1-nm step size. A fraction
collector was used to collect effluent at 1-min intervals. For those
experiments in which radiolabeled 1-hexadecanoyl-2-arachidonoyl GPCho
was used, 10% of the oxidized GPCho products were separated on HPLC
with a Flo-One Beta (Radiomatic, Riviera Beach, FL) radiochromatography
detector connected to the effluent stream after the UV detector.
Electrospray Ionization Mass Spectrometry
Reverse
phase HPLC fractions containing the conjugated triene chromophore were
analyzed using electrospray ionization (ESI) mass spectrometry and
tandem mass spectrometry. Flow injection was used to introduce 2 µl
of the HPLC fractions at a flow rate of 10 µl/min with 85%
methanol, 1 mM ammonium acetate as the mobile phase. The Sciex
API III (Perkin-Elmer Sciex, Toronto, Canada) was
operated in negative ion mode with an orifice voltage of -105 V
in order to collisionally decompose the GPCho acetate adducts to
[M - 15]
ions. Product ion spectra
were obtained using a collision energy of 30 eV and collision gas
thickness (argon) of 220
10
molecules/cm
. Negative ion ESI mass spectra of the
oxidized fatty acids obtained from hydrolysis of the oxidized GPCho
were obtained using an orifice voltage of -60 V. For all ESI
analyses, the curtain gas flow was 1.2 liter/min, the nebulizer
pressure was 40 p.s.i., the turbospray flow was 7 liters/min, and the
turbospray temperature was 400 °C.
Saponification of Oxidized GPCho and Reverse Phase
HPLC
Fractions from the first HPLC separation (system A)
identified as containing components with a conjugated triene
chromophore were saponified by addition of 0.5 ml of 1 N sodium hydroxide at room temperature for 1 h. The fractions were
then acidified with 50 µl of 88% formic acid, and the methanol
evaporated under vacuum. The fractions were reconstituted in 1 ml of
30% methanol, 70% water (0.05% acetic acid), pH 5.7, for injection into
the HPLC system. Spectra of peaks eluting from the Beckman ODS 5-µm
4.6 mm 25-cm column with a Waters ODS Guard-pak precolumn were
continuously collected using a photodiode array detector. The HPLC was
operated with a three-step gradient (system B) starting at 40% B and
ramping to 55% B in 6 min, then to 61% B in 15 min, and to 100% B in 5
min, where B = methanol:acetonitrile (35:65) and A =
0.05% acetic acid, adjusted to pH 5.7 with ammonium hydroxide. The flow
rate was 1 ml/min, and fractions were automatically collected at 0.5
min/tube. The elution of each component relative to prostaglandin
B
and LTB
external standards was calculated by
the following equation for relative retention index
(RRI).
Measurement of Intracellular Calcium
Levels
Changes in neutrophil intracellular calcium levels were
determined by measuring the fluorescence of Indo-1 as described
previously (20) with minor changes. The intracellular calcium
levels were calculated as described (21) using a dissociation
constant of 250 nM for Indo-1/Ca complex. In
some cases, the cells were preincubated with 10 µM LTB
receptor antagonist, LY223982, prior to the
addition of agonists.
Gas Chromatography/Mass Spectrometry
The remainder
of the HPLC system B fractions containing the hydrolyzed fatty acids
were analyzed both by electron capture negative ion mass spectrometry
and by electron impact ionization mass spectrometry using a Finnigan
SSQ mass spectrometer (Finnigan Corp., San Jose, CA) interfaced with a
capillary gas chromatograph column. For electron capture GC/MS, a
portion of the fractions were derivatized as pentafluorobenzyl esters
and trimethylsilyl ethers as described previously(22) . Prior
to electron ionization GC/MS, samples were hydrogenated by bubbling
hydrogen gas through a methanol solution of the sample for 30 min using
rhodium adsorbed on alumina as the catalyst (1 mg). The samples
were then derivatized as pentafluorobenzyl esters trimethylsilyl
ethers.
/H
O
for 3 h at 37 °C (2
mM phospholipid in 50 mM PBS) and reduced with 1
mM SnCl
. A, elution of components
detected at 205 nm revealed the major component as unreacted starting
material at 78 min. B, elution of components absorbing at 270
nm. Fraction 53 contained an oxidized phospholipid displaying the
ultraviolet spectrum suggestive of a conjugated triene (inset). Other conjugated trienes eluted between 44 and 54
min. C, a major oxidized phospholipid component in fraction 53
yielded an ion at m/z 798.6 [M -
15]
by electrospray ionization mass spectrometry
that was collisionally activated and decomposed to yield fragment ions
including the carboxylate anions for hexadecanoate (m/z 255)
and the carboxylate anion for arachidonate plus two additional oxygen
atoms (m/z 335).
Aliquots of each fraction collected during the HPLC
separation were analyzed by electrospray ionization (ESI) mass
spectrometry, which yields abundant [M -
15] ions for the lipid glycerophosphocholine
molecular species(23) . For example, fraction 53 yielded
abundant [M - 15]
ions at m/z 798.6 and 814.7. These [M - 15]
ions were then selected for subsequent tandem mass spectrometry.
Collision-induced decomposition of the ion at m/z 798.6 from
fraction 53 yielded two carboxylate anions at m/z 255
(characteristic for hexadecanoate) and m/z 335 (corresponding
to the addition of two oxygen atoms to the arachidonate carboxylate
anion) consistent with the carboxylate anion for a
dihydroxyeicosatetraenoic acid (Fig. 1C). The ion at m/z 480 corresponded to the loss of the sn-2
substituent as a neutral ketene, confirming that the dioxygenated
arachidonoyl moiety was esterified at sn-2. The ions at m/z 317 and 195 likely resulted from secondary fragmentation
of m/z 335. Tandem mass spectrometric analysis of HPLC
fractions between 44 and 54 min revealed elution of several
dioxygenated eicosatetraenoic acids esterified at the sn-2
position of phosphatidylcholine.
Conjugated Triene Eicosanoids
The oxidized GPChos in the
fractions between 44 and 54 min (Fig. 1A) were
individually saponified and the liberated oxidized fatty acids were
purified by the second reverse phase HPLC, system B. Components were
observed in several fractions that had UV spectra with maximum
absorption at 270 nm and vibronic bands 10 nm on either side at 260 and
280 nm, characteristic of a conjugated triene (Table 1). Each
HPLC fraction (system B) was tested for its ability to elicit an
increase in intracellular calcium from human polymorphonuclear
leukocytes loaded with the fluorescent dye Indo-1 to screen for
components with biological activity. HPLC separation of the free acids
liberated from phospholipid fraction 53 (system A) is shown in Fig. 2A with the elution of a single component at 15.3
min (RRI = 2.54) absorbing at 270 nm (Fig. 2A, inset). The 0.5-ml fraction collected between 15 and 15.5 min
was also found to have the highest level of activity in elevating
intracellular calcium in the human neutrophil (Fig. 2B). Other closely eluting molecules maximizing
in adjacent fractions were also present in this sample; however, these
components did not have the characteristic UV chromophore of a
conjugated triene (data not shown) but may have biological activity.
/Mg
free Hanks' balanced
salt solution containing 0.05% bovine serum albumin and added to
neutrophils previously loaded with Indo-1 (1 µM). The
increase in [Ca
] was calculated as
described (21) .
Negative ion ESI/MS of the HPLC fractions (system B) containing
conjugated triene free acids consistently revealed [M -
H] ions at m/z 335, consistent with the
carboxylate anion expected for dioxygenated arachidonic acid.
Collision-induced decomposition of m/z 335 from these
fractions yielded numerous product ions that were similar but often not
identical to those observed following collision-induced decomposition
of LTB
(data not shown). Collision-induced decomposition of m/z 335 from the component eluting at 15.3 min (Fig. 2A) yielded the MS/MS spectrum shown in Fig. 3A with characteristic ions at m/z 59,
129, 195, and 317, all of which are also observed upon
collision-induced decomposition of m/z 335 from
LTB
, 6-trans-LTB
, and
6-trans-12-epi-LTB
. This collision-induced
decomposition mass spectrum differed significantly from that of other
dihydroxy eicosanoids such as 8,15-, 5,6-, or
5,15-dihydroxyeicosatetraenoic acid isomers (data not shown). This mass
spectral data was consistent with a 20-carbon fatty acid containing
four double bonds and two hydroxyl substituents. The covalent backbone
of the molecule was established following catalytic reduction of this
metabolite using hydrogen and Rh/Al
O
followed
by derivatization to the pentafluorobenzyl ester trimethylsilyl ether.
The electron ionization mass spectrum (Fig. 3B) clearly
revealed the presence of a saturated 20-carbon fatty acid derivative
with two hydroxyl groups as trimethylsilyl ethers. The
-cleavage
ions m/z 555 and 215 supported assignment of a 12-hydroxy
substituent, and m/z 369 and 401 supported assignment of a
5-hydroxy substituent(24) . A conjugated triene could only
exist between the hydroxyl substituents at carbon atoms 5 and 12,
placing the triene at carbons 6, 8, and 10. While the position of an
isolated double bond at carbon 14 was not unambiguously assigned, this
was the original position of the
-6 double bond in arachidonic
acid and this portion of the molecule was likely not altered by the
oxidation process. The structure of this free radical product was thus
established as 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, a
B
-isoleukotriene. The oxidized phospholipid from which this
dihydroxyeicosanoid was obtained was therefore
1-hexadecanoyl-2-[5,12-dihydroxy-6,8,10,14-eicosatetraenoyl]glycerophosphocholine
(see Fig. SI).
Pharmacological Activity of
B
As shown in Table 1, several conjugated triene eicosanoids were products of
the free radical oxidation of 1-hexadecanoyl-2-arachidonoyl-GPCho.
Several of these eicosanoids were found to stimulate an increase in
intracellular free calcium ion in the human neutrophil. The absolute
quantity used in the neutrophil assay differed for each sample since an
equal proportion (16%) of each HPLC fraction was tested for biological
activity and the absolute yield of each eicosanoid free radical product
varied. In order to compare activities in the absence of complete
dose-response curves, the biological activity in Table 1is
expressed as the natural logarithm of the increase in intracellular
calcium concentration (nM) per nanomolar concentration of
eicosanoid tested. Some compounds displayed no activity in this assay.
For example, the component with the HPLC relative retention index at
1.66 elicited no increase in intracellular free calcium ion when tested
at a concentration of 28.2 nM (54 pmol). In contrast, the
component that eluted with a relative retention index of 1.77 caused an
increase of 626 nM [Ca-Isoleukotrienes
]
when tested with 38 pmol.
(7 pmol) and the isolated isoleukotriene
with an RRI = 1.86 (68 pmol) is shown in Fig. 4. Both
compounds elicited a large increase in free intracellular calcium
within the human neutrophil, although LTB
was more than
10-fold more potent in this response. In contrast, the dual
lipoxygenase product
(5S,12S)-dihydroxyeicosatetraenoic acid in much
larger quantity (90 pmol) elicited only a 43 nM increase in
intracellular free calcium ion (data not shown).
-isoleukotriene (final concentration, 34 nM) with
HPLC relative retention index of 1.86 and LTB
(final
concentration, 3.4 nM). Following addition of LY223982 (10
µM), the same agonists were
tested.
Further
investigation of the pharmacologic effect of leukotrienes on the human
neutrophil revealed that elevation of intracellular calcium ion could
be blocked by administration of 10 µM LTB receptor antagonist LY223982(27) . As shown in Fig. 4, both responses from the component with relative
retention index = 1.86 and LTB
were completely
attenuated by the LTB
receptor antagonist LY223982. The
LTB
receptor antagonist also blocked the biological
response elicited by eicosanoids with relative retention index of 2.54
and 1.77 (data not shown).
of 30 nM was calculated for this eicosanoid
product. This is approximately 100-fold less than that observed for
LTB
itself, which has an EC
of 0.3-1
nM(25, 26) .
-isoleukotriene
free acid eluting between 15 and 15.5 min in Fig. 2(RRI
= 2.54). The calculated EC
is approximately 30
nM.
-isoleukotriene'' as a free radical product of
lipid peroxidation that resembles enzymatically produced
LTB
.
-like structure as a non-free radical
intermediate present in the phospholipid. Hydrolysis of such a
conjugated triene epoxide with water would yield isoleukotrienes as
5,12-dihydroxyeicosatetraenoic acids.
-isoleukotrienes were found to be potent agonists
stimulating the elevation of intracellular free calcium ion in the
human neutrophil. Furthermore, this action could be blocked by the
LTB
receptor antagonist LY223982(27) . While the
potency of the B
-isoleukotriene that was studied in
greatest detail (RRI = 2.54) was found to be somewhat less than
that for LTB
itself, it is similar to that found for other
chemotactic substances such as the peptide
fMLP(28, 29, 30) . This
B
-isoleukotriene and others described here, if formed in vivo, would have sufficient potency to play an important
role in activating or priming neutrophils for a respiratory burst or
priming neutrophils for leukotriene production by the 5-lipoxygenase
pathway. Such oxidized products of arachidonate formed in membrane
glycerophospholipids as a result of lipid peroxidation could be
released as free acids (31) and activate nearby cells. As such,
these isoleukotrienes would serve as mediators of tissue response to
lipid peroxidative events.
isomers have
been synthesized and studied as competitive substrates for the
LTB
receptor(32, 33, 34) , as
well as for biological activity including causing an elevation of
intracellular calcium (25, 35) and
chemotaxis(33, 34, 36, 37, 38) .
A wide variation in activity was observed for such isomers, suggesting
certain structural features of LTB
are important for
binding and biological activity, which include a cis configuration of the double bond at carbon-6 and stereochemistry
of the 12-hydroxyl substituent. The geometry of the double bonds in the
conjugated triene moiety is also quite important for receptor
recognition and it is noteworthy that the trans, cis, trans configuration of the double bonds at carbons 6, 8, and
10 confers tighter binding than those isomers having the all trans configuration in the conjugated triene moiety(39) .
However, many of the synthetic isomers possess profound activity in
many systems. The exact stereochemistry of the biologically active
B
-isoleukotrienes is currently under investigation.
-Isoleukotrienes are a family of free radical-generated
eicosanoids derived from arachidonoyl glycerophospholipids. These free
radical products of lipid peroxidation resemble enzymatically produced
LTB
in both structure and biological activity. As such they
could serve as lipid mediators of cellular free radical damage in
tissues exerting an effect by way of the LTB
receptor.
, leukotriene B
; GC, gas chromatography;
RRI, relative retention index.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.