Biogenic Synthesis, Purification, and Chemical Characterization of
Anti-inflammatory Resolvins Derived from Docosapentaenoic Acid (DPAn-6) |
| |
Authors: | Bindi Dangi Marcus Obeng Julie M Nauroth Mah Teymourlouei Micah Needham Krishna Raman and Linda M Arterburn |
| |
Institution: | Martek Biosciences Corporation, Columbia, Maryland 21045 |
| |
Abstract: | Enzymatically oxygenated derivatives of the ω-3 fatty acids
cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and
cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, have
potent inflammation resolution activity (Serhan, C. N., Clish, C. B., Brannon,
J., Colgan, S. P., Chiang, N., and Gronert, K. (2000) J. Exp. Med.
192, 1197–1204; Hong, S., Gronert, K., Devchand, P. R., Moussignac, R.,
and Serhan, C. N. (2003) J. Biol. Chem. 278,
14677–14687). Our objective was to determine whether similar derivatives
are enzymatically synthesized from other C-22 fatty acids and whether these
molecules possess inflammation resolution properties. The reaction of DHA,
DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases produced oxylipins,
which were identified and characterized by liquid chromatography coupled with
tandem mass-spectrometry. DPAn-6 and DPAn-3 proved to be good substrates for
15-lipoxygenase. 15-Lipoxygenase proved to be the most efficient enzyme of the
three tested for conversion of long chain polyunsaturated fatty acids to
corresponding oxylipins. Since DPAn-6 is a major component of Martek
DHA-S™ oil, we focused our attention on reaction products obtained from
the DPAn-6 and 15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and
(10,17S)-dihydroxy-DPAn-6 were the main products of this reaction.
These compounds were purified by preparatory high performance liquid
chromatography techniques and further characterized by NMR, UV
spectrophotometry, and tandem mass spectrometry. We tested both compounds in
two animal models of acute inflammation and demonstrated that both compounds
are potent anti-inflammatory agents that are active on local intravenous as
well as oral administration. These oxygenated DPAn-6 compounds can thus be
categorized as a new class of DPAn-6-derived resolvins.Enzymatically formed oxygenation products of C-20 and C-22 long chain
polyunsaturated fatty acids
(LC-PUFAs),4 have
important biological roles in inflammation, allergies, and blood clotting and
are thus believed to have therapeutic potential in several chronic immune
diseases
(1–10)
Several biologically important products of
cis-5,8,11,14-eicosatetraenoic acid/arachidonic acid (ARA),
cis-5,8,11,14,17-eicosapentaenoic acid (EPA), and
cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) have been described
(4,
11,
12). Proinflammatory
oxylipins, such as leukotrienes and some prostaglandins, are derived from ARA,
an ω-6 fatty acid. Interestingly, the same fatty acid also serves as a
precursor to anti-inflammatory or proresolution molecules like lipoxins
(13,
14). Stable analogues of
lipoxins are being developed as drugs for asthma and other inflammatory airway
diseases (15,
16). Oxylipins derived from
ω-3 fatty acids, such as DHA and EPA, known as resolvins, are primarily
anti-inflammatory in nature
(17). EPA acts as a precursor
to the E-series resolvins that have shown potential in the treatment of
colitis, arthritis, and periodontitis
(18–20).
The resolvins of the D-series derived from DHA are useful as neuroprotective
agents. 10,17-Dihydroxy-4,7,11,13,15,19-docosahexaenoic acid (10,17-HDHA) or
neuroprotectin D1 is a resolvin that is formed endogenously in the human brain
and eye and is believed to exert its protective effect against cell
injury-induced oxidative stress
(21–23).The main enzymes responsible for the production of these oxygenated LC-PUFA
products are primarily lipoxygenases and, in addition, cyclo-oxygenases and
cytochromes P450. These enzymes produce oxylipins via transcellular activity,
often involving multiple cell types
(24). This activity mainly
results in mono-, di-, and tri-hydroxylation products of fatty acids that have
varying potencies, depending on the exact structure of the compound.
Lipoxygenases are non-heme, iron-containing dioxygenases that catalyze the
regioselective and enantioselective oxidation of polyunsaturated fatty acids
containing one or more cis,cis-1,4-pentadienoic moieties to give the
corresponding hydroperoxy derivatives
(25,
26). We thus considered that,
in addition to DHA and EPA, other C-22 PUFAs containing such methylene
interrupted double bonds may also be substrates for lipoxygenases and that
resulting products may have anti-inflammatory activity similar to DHA-derived
resolvins. DPAn-6 (cis-4,7,10,13,16-docosapentaenoic acid) is present
in algal oils, and recent studies have demonstrated that this fatty acid has
anti-inflammatory activities in vitro and, in conjunction with DHA,
also has anti-inflammatory activity in
vivo.5 Also, it
has been suggested that a combination of DHA and DPAn-6 could be a beneficial
natural therapy in neuroinflammatory conditions like Alzheimer disease.
Specifically, in a 3×Tg-AD mouse model of Alzheimer disease, DPAn-6 was
shown to reduce levels of early stage phospho-Tau epitopes, which in turn
correlated with a reduction in phosphorylated c-Jun N-terminal kinase, a
putative Tau kinase (27).
Although the precise mechanism of action of DPAn-6 in these inflammatory
milieus is not known, it suggests a possible role for oxylipin products of
DPAn-6 in resolution of inflammation. Also, another LC-PUFA, DPAn-3
(cis-7,10,13,16,19-docosapentaenoic acid) usually present along with
DHA and EPA in marine oils is known to be a potent inhibitor of platelet
aggregation
(28–30).
In addition, this LC-PUFA has a potent inhibitory effect on angiogenesis
through the suppression of VEGFR-2 (vascular endothelial-cell growth factor
receptor 2) expression. Angiogenesis is known to contribute to tumor growth,
inflammation, and microangiopathy, again pointing to the possibility that
anti-inflammatory activity of DPAn-3 might be mediated through resolvin-like
products as in the case of DHA and EPA
(31).The purpose of this research was to determine whether oxylipins are formed
from the C-22 LC-PUFAs, DPAn-6 and DPAn-3, by lipoxygenase activity; to
compare them to products formed from DHA; to chemically characterize products;
to purify key oxylipin products from the DPAn-6/15-lipoxygenase reaction; and
to test whether these compounds have resolvin-like anti-inflammatory activity.
This research also sets the stage for preparation and isolation of a wide
range of other C-22 oxylipins that could be evaluated as potential
anti-inflammatory compounds. |
| |
Keywords: | |
|
|