Epoxy-Keto Derivative of Linoleic Acid Stimulates
Aldosterone Secretion
Theodore L. Goodfriend, Dennis L. Ball, Brent M. Egan, William B. Campbell, Kasem Nithipatikom
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Abstract—Plasma levels of aldosterone are not always predictable from the activity of renin and the concentration of
potassium. Among the unexplained are elevated levels of aldosterone in some obese humans. Obesity is characterized
by increased plasma fatty acids and oxidative stress. We postulated that oxidized fatty acids stimulate aldosteronogenesis. The most readily oxidized fatty acids are the polyunsaturated, and the most abundant of those is linoleic acid. We
tested oxidized derivatives of linoleic acid for effects on rat adrenal cells. One derivative, 12,13-epoxy-9-keto-10(trans)octadecenoic acid (EKODE), was particularly potent. EKODE stimulated aldosteronogenesis at concentrations from 0.5
to 5 ␮mol/L, and inhibited aldosteronogenesis at higher doses. EKODE’s stimulatory effect was most prominent when
angiotensin and potassium effects were submaximal. The lipid’s mechanism of action was on the early pathway leading
to pregnenolone; its action was inhibited by atrial natriuretic peptide. Plasma EKODE was measured by liquid
chromatography/mass spectrometry. All human plasmas tested contained EKODE in concentrations ranging from 10⫺9
to 5⫻10⫺7 mol/L. In samples from 24 adults, levels of EKODE correlated directly with aldosterone (r⫽0.53, P⫽0.007).
In the 12 blacks in that cohort, EKODE also correlated with body mass index and systolic pressure. Those other
correlations were not seen in white subjects. The results suggest that oxidized derivatives of polyunsaturated fatty acids
other than arachidonic are biologically active. Compounds like EKODE, derived from linoleic acid, may affect adrenal
steroid production in humans and mediate some of the deleterious effects of obesity and oxidative stress, especially
in blacks. (Hypertension. 2004;43[part 2]:358-363.)
Key Words: fatty acids 䡲 oxidative stress 䡲 hypertension 䡲 aldosterone 䡲 obesity 䡲 adrenal gland
A
attention on the properties of oxidized derivatives of linoleic
acid. We showed that incubating linoleic acid (C18:2, n-6) with
rat hepatocytes produced one or more compounds that stimulated aldosterone production by rat adrenal cells. Rat hepatocytes
did not produce enough of the oxidized products to characterize,
so we used a cell-free method to generate a mixture of milligram
quantities of oxidized products of linoleic acid.11 We fractionated this mixture by high-pressure liquid chromatography
(HPLC) and bioassayed the fractions in rat adrenal cells. The
most active derivatives were further purified and their structures
determined by mass spectroscopy.11 We were then able to
confirm that at least one of the linoleic acid derivatives produced
in our cell-free system—the most active one—was produced by
rat hepatocytes in vitro. This paper reports some of the adrenal
effects and interactions of the most potent and most prevalent of
the oxidized products of linoleic acid formed in our system,
12,13-epoxy-9-keto-10(trans)-octadecenoic acid (EKODE), and
the results of a single comparison of plasma levels of EKODE
and aldosterone in 24 humans. EKODE is a derivative of linoleic
acid that has been described before, but its existence and effects
in mammals were unexplored.12
ldosterone production by adrenal cells in vitro can be
affected by more than 20 hormones, autacoids, ions, and
nutrients.1,2 It is probable, therefore, that regulation of aldosterone secretion in intact animals and humans is more
complex than classical schemes that involve primarily angiotensin II and potassium. In fact, several authors have invoked
unknown factors to explain levels of aldosterone that do not
comply with predictions based on classical regulators of the
adrenal.3–5 We encountered one such inexplicable situation
when we found that aldosterone levels were higher in some
subjects with visceral obesity and hypertension than in lean
normotensive subjects, even when we took into account the
levels of renin activity and potassium.6
Obesity and hypertension are associated with relatively high
levels of fatty acids and increased oxidative stress.7–10 These
facts led us to postulate that oxidized derivatives of fatty acids
might drive aldosterone secretion and possibly explain the
relatively high levels of aldosterone in some subjects with
visceral obesity. The most readily oxidized fatty acids are
polyunsaturated, and the most prevalent polyunsaturated fatty
acid in humans is linoleic acid. Therefore, we focused our
Received September 29, 2003; first decision October 27, 2003; revision accepted December 8, 2003.
From the William S. Middleton Memorial Veterans Hospital and Departments of Medicine and Pharmacology of the University of Wisconsin (T.L.G.,
D.L.B.); Madison; Department of Medicine (B.M.E.), Medical University of South Carolina, Charleston; and Department of Pharmacology (W.B.C.,
K.N.), Medical College of Wisconsin, Milwaukee.
Correspondence to Theodore L. Goodfriend, MD, Associate Chief of Staff for Research, Veterans Hospital, 2500 Overlook Terrace, Madison, WI
53705. E-mail [email protected]
© 2004 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000113294.06704.64
358
Goodfriend et al
Oxidized Linoleic Acid and Aldosterone
359
Methods
EKODE Synthesis
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Linoleic acid was oxidized in two steps. First, linoleic acid was
incubated with soybean lipoxygenase Type V (Sigma, St. Louis, Mo)
to produce 13(S)-hydroperoxide, which was partially purified by
silicic acid chromatography eluted with acetone:hexane. The hydroperoxide was then exposed to ferric ion and cysteine, which
catalyzed rearrangements with free radical intermediates.12 The
stable products were extracted into chloroform and partially purified
by a second silicic acid column. The fractions were tested for their
effects on rat adrenals, and the active fractions were purified by
reversed-phase HPLC using a C-18 column eluted with a gradient of
65% to 75% methanol in water acidified with 0.1% acetic acid. The
column was monitored by ultraviolet absorbance at 234 nm. EKODE
eluted at a polarity index of 7.381 (67.7% methanol). It was
repurified over the same column using a shallower gradient, dried,
and stored as aliquots under argon at ⫺70°C.
The quantity of EKODE in the final extract and aliquots was
determined by 3 techniques: gravimetric measurement; absorbance at
234 nm assuming a molar extinction coefficient of 16 500;13 and
isotope dilution using a trace of 14C-labeled linoleic acid of known
specific activity in the synthetic protocol.
EKODE in plasma was extracted into hexane:isopropanol:1N
citric acid (10:40:1). We determined that citric acid did not cleave
the epoxide bond while it protonated the carboxyl group. To control
for recovery, we added deuterated EKODE (2D4 EKODE), 4 ng per
0.2 mL of plasma. Deuterated EKODE was produced from deuterated linoleic acid (generously supplied by Dr. Richard Adlof,
National Center for Agricultural Utilization Research, Peoria, Ill)
using the lipoxygenase/iron-cysteine reactions described above. The
quantity of EKODE in each extract was determined by liquid
chromatography/electrospray ionization mass spectrometry (LC/
MS). The molecular weight of EKODE is 310, so the chromatographic eluate was monitored at masses 311 and 315 m/z, representing the m⫹1 ions of EKODE and deuterated EKODE produced by
positive ion mass spectrometry.14
Bioassays were performed using rat adrenal glomerulosa cells
isolated from capsules by collagenase digestion.15 EKODE and other
lipids were dissolved in ethanol. Incubations were 2 hours at 37°C.
Incubation medium was 10% Medium 199 (Sigma #5017) in
HEPES-buffered balanced salt solution.15 The final concentration of
potassium was 3.6 mmol/L and of calcium was 0.184 mmol/L unless
otherwise noted. Ethanol concentrations never exceeded 1%. No
albumin was added to the incubation medium. Aldosterone in the
supernatant was measured by radioimmunoassay, using antibody
from ICN Biomedicals (Irvine, Calif) and labeled aldosterone from
Diagnostic Products Corp (Los Angeles, Calif). When the “early
pathway” of steroidogenesis was assessed, trilostane 10⫺5M was
added to the incubation mixture to inhibit 3-␤-hydroxysteroid
dehydrogenase, and pregnenolone was measured by radioimmuno-
Figure 1. Structural formula of 12,13-epoxy-9-keto-10(trans)octadecenoic acid.
assay using antibody from ICN and labeled pregnenolone from
Perkin-Elmer Life Sciences (Boston, Mass). EKODE had no effect
when added directly to radioimmunoassays of aldosterone or pregnenolone (data not shown).
The human subjects whose plasma was examined in this experiment were participating in a detailed study of obesity and hypertension, the design and results of which are described in other
publications.16,17 In brief, 24 adult subjects between the ages 24 and
49 were enrolled; 12 were white, 12 black. Half of each group were
men; half were women. Half of the subjects were lean normotensives, and half were obese hypertensives. Although the average
blood pressures of the obese subjects were higher than the lean, none
of the screening blood pressures exceeded 159/99 mm Hg, and none
of the subjects had taken antihypertensive medication for 2 weeks.
None of the subjects was a smoker at the time of the study. Adiposity
was reported as body mass index. Fat distribution was not assessed
at the time when plasma was drawn. The plasma samples we
measured were collected on the first day of the study, while subjects
were on their usual diets but had fasted overnight and were supine for
30 minutes before venipuncture. Plasma was separated immediately
and stored frozen. The clinical experiment and plasma measurements
were approved by the institutional review boards at the Medical
University of South Carolina and the University of Wisconsin.
Plasma aldosterone was measured by solid-phase radioimmunoassay using antibody-coated tubes from Diagnostic Products Corp.
Plasma renin activity was assessed by measuring angiotensin I by
radioimmunoassay after incubation under conditions described in the
kit purchased from Perkin-Elmer Life Sciences.
Statistical analysis of correlations within the clinical data were
assessed by Pearson and Spearman analyses. For the former,
EKODE values were entered as the logarithm.
Results
Effects of EKODE on Rat Adrenal
Glomerulosa Cells
The structural formula of EKODE is depicted in Figure 1.
Figure 2A shows the response of rat adrenal glomerulosa
cells to increased concentrations of EKODE in the presence
of different ambient concentrations of potassium. These data
are redrawn in Figure 2B as a family of dose-response curves
Figure 2. Interaction of EKODE and potassium in rat
adrenal glomerulosa cells. A, the effects of graded
doses of EKODE on cells incubated in different concentrations of potassium. B, same data as a family
of responses to graded doses of potassium in the
presence of different concentrations of EKODE. Cell
preparation, incubation, and aldosterone assays are
described in the text and Reference 15. Aldosterone
production is reported as nanograms per million
cells per hour, and the points are bracketed by SEs
of the means for triplicate incubation tubes within a
single experiment. Numbers labeling the curves are
the concentrations of potassium (A) or EKODE (B) in
the medium. In B, data for the lowest concentration
of EKODE (0.15 ␮mol/L) are omitted.
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Figure 3. Interaction of EKODE and angiotensin II in rat adrenal
glomerulosa cells. Cell preparation, incubation and aldosterone
assays are described in the text, the legend to Figure 2, and
Reference 15. A and B display the same data in different ways.
Numbers labeling the curves are the concentrations of angiotensin II (A) or EKODE (B) in the medium. In B, data for the lowest
concentration of EKODE (0.15 ␮mol/L) are omitted.
to potassium when different amounts of EKODE were present in the incubation medium. EKODE exerted its greatest
effect when the potassium concentration was low; potassium
effect was essentially unaffected by the presence of EKODE
until concentrations greater than 5 ␮M were tested. Effects of
higher concentrations of EKODE are described below.
Figure 3A and 3B show the interaction of EKODE and
angiotensin II. As in the case of potassium, EKODE’s effect
was visible only when angiotensin doses were submaximal.
EKODE did not add a stimulatory effect to the effects of high
doses of angiotensin. Similarly, EKODE did not add to
maximal stimulation by dibutyryl cAMP (dbcAMP) (see
below).
EKODE stimulated the “early pathway of aldosteronogenesis,” the pathway that includes signal transduction and the
steroidogenic steps between cholesterol and pregnenolone,
(Figure 4). When steroidogenesis was blocked at the level of
pregnenolone by trilostane, EKODE stimulated formation of
that intermediate to the same extent it stimulated the complete
pathway to aldosterone when no inhibitor was present. The
lack of EKODE effect on the late pathway is illustrated by the
data in Figure 5. When aldosteronogenesis was maximized by
addition of intermediates closer to aldosterone than pregnenolone, EKODE had no stimulatory effect.
It should be noted that the effect of EKODE is biphasic,
stimulating aldosterone production at concentrations between
0.5 and 5 ␮mol/L, and inhibiting at concentrations above that
(Figure 4 and 5). The inhibitory effect is evident when
steroidogenesis is maximally stimulated by steroid precursors, dbcAMP, high concentrations of potassium (Figure 5),
or angiotensin (data not shown). The inhibitory effect of high
concentrations of EKODE is also evident when steroidogenesis is blocked at the level of pregnenolone (Figure 4).
The stimulatory effect of EKODE was neutralized by
addition of atrial natriuretic peptide (ANP; Figure 6). Inhibitors of angiotensin receptors did not alter the response of rat
adrenal cells to EKODE (data not shown).
Figure 4. Comparison of EKODE effects on the early pathway
and overall pathway of aldosterone biosynthesis by rat adrenal
cells. Cell preparation and incubation are described in the text,
the legend to Figure 2, and Reference 15. The early pathway
was assessed by measuring pregnenolone in a medium containing trilostane, 10 ␮mol/L, an inhibitor of 3-␤-hydroxysteroid
dehydrogenase. Aldosterone and pregnenolone were measured
by radioimmunoassays with no significant cross-reactivity for
the other steroid.
EKODE Levels and Correlations in
Human Subjects
EKODE was detected in every human plasma sample examined. The mean level was 5⫻10⫺8 mol/L, the range was from
10⫺9 to 5⫻10⫺7 mol/L. Concentrations of EKODE correlated
directly with levels of aldosterone in plasmas from 24 human
Figure 5. Comparison of EKODE effects on the late pathway
and overall pathway of aldosterone biosynthesis by rat adrenal
cells and on aldosterone production stimulated by potassium
and dibutyryl cAMP (dbcAMP). Cell preparation and incubation
are described in the text, the legend to Figure 2, and Reference
15. The late pathway was assessed by measuring aldosterone
production in a medium containing trilostane (10 ␮mol/L) to
block the early pathway and steroid precursors beyond
prenenolone. Depicted are the results with progesterone (Progest) and corticosterone (Cortico), each added to a concentration of 3 ␮mol/L. Similar results were obtained when pregnenolone was added (data not shown). Also shown are the
effects of EKODE on cells stimulated with potassium
(7.2 mmol/L) and dbcAMP (1 mmol/L). Although EKODE did not
stimulate aldosterone production when these stimuli were present, high doses of the lipid were inhibitory to all agonists acting
on the early pathway.
Goodfriend et al
Oxidized Linoleic Acid and Aldosterone
361
Spearman correlation coefficient between EKODE and body
mass index was r⫽0.71, P⫽0.006. There was no statistical
relationship between EKODE and BMI in whites.
Discussion
Figure 6. Inhibition of EKODE by atrial natriuretic peptide (ANP).
Cell preparation was described in the text, the legend to Figure
2, and Reference 15. ANP (10⫺7 mol/L) was added at the start
of incubation.
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subjects. This is shown in Figure 7, where the EKODE values
are transformed to their logarithms. The simple correlation
coefficient for the group as a whole was r⫽0.57, P⫽0.004 by
Pearson analysis of the values and r⫽0.53, P⫽0.007 by
Spearman rank analysis.
We observed a racial difference in the correlations between
EKODE and other variables. The EKODE:aldosterone relationship was stronger among the 12 blacks than the 12 whites
(0.43 versus 0.32 by Spearman rank analysis), although
neither group displayed a statistically significant correlation
when analyzed separately. In addition, EKODE levels correlated directly with systolic blood pressure in blacks, (r⫽0.56,
P⫽0.05 by Spearman rank analysis) but not in whites. The
only significant relationship between EKODE levels and
adiposity was found among the black subjects in whom the
Figure 7. Plasma aldosterone and EKODE levels in 24 human
subjects. All samples were drawn in the morning from subjects
who had been eating their regular diet and who fasted overnight. Subjects were supine 30 minutes before venipuncture.
Results for blacks and whites are shown in the solid and open
circles, respectively. Aldosterone values are plotted linearly;
EKODE values were transformed to their logarithms. The Spearman rank order correlation coefficient, which is independent of
the transformation of the data, was r⫽0.53, P⫽0.007 for the
cohort as a whole.
Aside from the eicosanoids, oxidized derivatives of polyunsaturated fatty acids have not been extensively studied in
humans and other mammals. The reactions producing these
derivatives add oxygen to the fatty acid; they do not result in
energy-yielding “beta-oxidation.” Polyunsaturated fatty acids
are quite susceptible to reactions with oxygen, yielding
epoxides, ketones, hydroxylated derivatives, and a variety of
cleaved products.18 Some oxidized derivatives of linoleic acid
are called leukotoxins because they are formed by leukocytes
and are toxic to mammalian tissues.19
Oxidized derivatives of unsaturated fatty acids can be
formed nonenzymatically, as in the spoilage of vegetable oils.
Enzyme-catalyzed oxidation usually begins with the action of
a lipoxygenase, as the result of which molecular oxygen adds
to one side of a double bond to form a hydroperoxide. If the
starting material is linoleic acid (C18:2, n-6), and the lipoxygenase is 15-lipoxygenase, the initial product is 13hydroperoxy-octadecadienoic acid.20 (This numerical confusion results from the fact that the enzyme was named for the
carbon it usually attacks in 20 carbon fatty acids like
arachidonic.) The fatty acid hydroperoxide is readily rearranged and further oxidized to more stable products. These
steps involve free radicals and can be accelerated by free
radical generators such as ferric iron and cysteine.11,12
The principal product of our reaction scheme with linoleic
acid contains both epoxide and oxo- (ketone) oxygens in
addition to the carboxyl group, with one remaining double
bond. Its structure is 12,13-epoxy-9-keto-10(trans)octadeceneoic acid, (EKODE). This unusual compound was
not only one of the most prevalent products of our synthesis,
it proved to be one of the most active in adrenal cells. We
have found EKODE in the plasma of 26 humans, dozens of
rats and mice, and 14 Rhesus monkeys. However, we do not
know how or where the EKODE in plasma is formed. Some
of it may, in fact, originate in foodstuffs.
EKODE did not add to the stimulatory effect of high doses
of angiotensin II in vitro. This suggests that any regulatory
role it might have in humans would be greatest when renin
activity is low. When renin activity is suppressed by high
dietary sodium intake, for example, any stimulus of aldosterone secretion becomes particularly relevant to the pathogenesis of cardiovascular disease. We found that the correlation
between visceral obesity and plasma aldosterone was strongest when subjects ingested a high-salt diet and had very low
plasma renin activities.6 This observation may have relevance
to the relationship between obesity and blood pressure, but it
would relate to our current in vitro results only if visceral
obesity increased EKODE production and EKODE increased
aldosterone production.
EKODE stimulates production of corticosterone by rat
adrenal zona fasciculata cells, in addition to its effect on
aldosterone production by the zona glomerulosa.21 In both
zones, the lipid’s effect appears to be on the early pathway of
steroidogenesis, a series of reactions that includes signal
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February 2004 Part II
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transduction and produces pregnenolone. This is consistent
with inhibition of EKODE by ANP, a peptide inhibitor of
signal transduction.22 A likely mechanism of action for
EKODE is to increase intracellular calcium (M. Payet,
personal communication, 2003).
The biphasic shape of the EKODE dose-response curve is
unusual and puzzling. Inhibition of the adrenal by high
concentrations may help explain the inappropriately low
aldosterone levels in some dire clinical conditions where
levels of nonesterified fatty acids are high.23
A stimulation of aldosterone production in rat adrenal cells
can be seen at concentrations as low as 0.5 ␮mol/L (Figure 2
to 4), a concentration found in some human plasma. This
raises the possibility that EKODE is a regulator of aldosterone production in vivo. This postulate is supported by the
correlation between plasma EKODE levels and plasma aldosterone in a single cohort of 24 human subjects. On the other
hand, maximal stimulation by EKODE in vitro requires 10⫺6
mol/L, a concentration higher than any we have found in
humans to date, casting doubt on a postulated role for
EKODE as an important regulator of aldosterone in all
humans. Furthermore, some circulating EKODE is bound to
albumin, although the extent and strength of that binding is
unknown. Despite these doubts, EKODE may affect aldosterone production in those people in whom it circulates at high
concentrations, or in whom it is formed in the adrenal gland
itself.
Lipoxygenase activity in the adrenal cortex has been
shown to be important for the steroidogenic response to
angiotensin II, but this importance is attributed to oxidized
derivatives of arachidonic acid.24 Perhaps oxygenated derivatives of linoleic and other 18-carbon fatty acids also behave
as autacoids in the adrenal.
It should be noted that other oxidized derivatives of
linoleic acid produced by our cell-free reaction sequence
share the adrenal properties of EKODE. Principal among
these other derivatives is 13-keto-9,11-octadecadienoic acid,
and even linoleic acid itself displays some activity in the
adrenal gland (data not shown). The biological properties of
oxidation products of all unsaturated fatty acids deserve
further study. Gamma-linolenic acid (C18:3, n-6) and docosahexaenoic acid (C22:6, n-3) have been shown to inhibit
aldosterone production in hypertensive rats.25,26 Although
those inhibitory effects were originally attributed to the
unmodified fatty acid, it is possible that oxidized products of
the fatty acids were also active.
Oxidative reactions involving polyunsaturated fatty acids
are part of a phenomenon called oxidative stress, in which
reactive oxygen species accumulate and alter molecules
outside of their normal metabolic sequence. Reactive oxygen
species have a very short lifespan, measured in seconds or
fractions of a second. Oxidative stress has been implicated in
the pathogenesis of hypertension.8,27–30 Hypertension is a
condition that lasts for years, so it is difficult to see how
evanescent atoms or molecules could cause it. Oxidized
derivatives of fatty acids might be among the long-lived
products of oxidative stress that would serve as downstream
mediators affecting blood pressure. After intravenous infusion into Rhesus monkeys, EKODE decayed from the plasma
at a rate measured in minutes (Goodfriend, unpublished
observations, 2003). That would make EKODE a more
durable player in blood pressure regulation than the oxidants
themselves. If EKODE proves to be an effective stimulus of
aldosterone secretion in humans, then the longevity of that
steroid would further extend the duration of reactive oxygen’s
effect on blood pressure. Considering aldosterone’s growing
list of extrarenal effects, EKODE might participate through
aldosterone in the pathogenesis of other cardiovascular diseases.31 Also plausible is the possibility that EKODE is
merely a marker for oxidative stress, not a mediator of its
effects.
We saw positive correlations between obesity and
EKODE, EKODE and aldosterone, and aldosterone and
blood pressure in blacks. That would support our original
hypothesis about the possible link forged by EKODE to
connect obesity and hypertension. However, all of those
connections were not found in the white subjects. In the same
cohort, the black subjects displayed more evidence of oxidative stress, measured as isoprostanes, than the white subjects
when they received an intravenous infusion of lipid.17
Perspectives
Our findings suggest that oxidation products of polyunsaturated fatty acids other than arachidonic acid exert effects on
humans that may contribute to regulation of blood pressure
by their effects on aldosterone secretion. The effects may be
greater in blacks than whites. At the least, these fatty acid
derivatives, readily demonstrated in human plasma, merit
study as possible mediators or markers of oxidative stress and
its sequelae.
Acknowledgments
This research was supported by the Department of Veterans Affairs
(T.G., D.B.), a grant from the American Heart Association (T.G.,
D.B.), and from the National Institutes of Health (W.B., K.N., B.E.).
We acknowledge the expert assistance of Marilyn Isbell in performing the plasma EKODE determinations.
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Hypertension. 2004;43:358-363; originally published online January 12, 2004;
doi: 10.1161/01.HYP.0000113294.06704.64
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