Clinical Chemistry 43: 2403-2407, 1997;
(Clinical Chemistry. 1997;43:2403-2407.)
© 1997 American Association for Clinical Chemistry, Inc.
Basal concentrations of free and esterified monohydroxylated fatty acids in human blood platelets
Michel Guichardanta,
Chantal Thevenon,
Jean Francois Pageaux and
Michel Lagarde
INSERM. U352, Biochimie et Pharmacologie INSA-Lyon, 20 Ave. Albert Einstein, 69621 Villeurbanne, France.
a Author for correspondence. Fax + 33 4 72 43 85 24; e-mail Michel.Guichardant{at}insa-lyon.fr
 |
Abstract
|
|---|
Monohydroxylated fatty acids (HO-FA), namely 12-hydroxyeicosatetraenoic
and 12-hydroxyheptadecatrienoic acids, are enzymatically formed in
response to platelet activation. Different techniques, including gas
chromatography (GC) and liquid chromatography–mass spectrometry
(LC-MS), have been described to measure HO-FA in activated cells, but
they are not well-adapted to resting cells. Measurements of free and
esterified HO-FA at basal concentration require the prevention of
platelet activation. For this purpose, such an activation was minimized
by adding various inhibitors to the anticoagulant. Platelet recovery
was greater in the protected group than in controls (473 x
109 ± 4.0 x 109 platelets/L vs 410
x 109 ± 4.53 x
109 platelets/L, respectively) (mean ± SEM, n =
9, P <0.05). Lipids were extracted and immediately
hydrogenated to avoid fatty acid autoxidation occurring during the
workup. Unesterified and esterified HO-FA were analyzed by GC-MS, and
the former were lower in the protected group (1.52 ± 0.84
pmol/109 platelets) than in the unprotected one (12.63
± 10.52 pmol/109 platelets) (mean ± SEM, n = 9,
P <0.05). Interestingly, only traces of HO-FA were
detected in both the triglyceride and sterol ester fractions, and they
were also weakly esterified in phospholipids.
|
Introduction
|
|---|
Monohydroxylated fatty acids are synthesized from a wide range of
polyunsaturated fatty acids and mainly derive from the lipoxygenase
pathway activation (1)(2). They can also be
formed via the cyclooxygenase pathway for 12-hydroxyheptadecatrienoic
acid (HHT) and via autoxidation mechanisms involving oxygen free
radicals that contribute in the genesis of a variety of human diseases
and tissue injuries (3)(4).1
These
monohydroxylated fatty acids can be incorporated into blood cells and
esterified into their phospholipids (5)(6)(7)(8).
Numerous approaches have been proposed for measuring these compounds,
including HPLC (9)(10)(11), gas
chromatography–mass spectrometry (GC-MS)
(12)(13), and liquid chromatography–mass
spectrometry (LC-MS) (14)(15). However, none
of them has been adapted to determine the basal concentrations of
unesterified and esterified monohydroxylated fatty acids, but rather
for their measurement in activated cells.
In the present study, unesterified and esterified monohydroxylated
fatty acids have been measured in nonactivated platelets. For this
purpose two anticoagulants were compared, the difference being the
presence of a cocktail of complementary inhibitors, namely the strong
calcium chelator EDTA, the cyclooxygenase inhibitor aspirin (ASA), and
the adenylyl cyclase activator prostaglandin E1
(PGE1). Platelets were then isolated from their plasma, and
their lipids immediately hydrogenated to prevent further artifactual
monohydroxylated fatty acid formations. Monohydroxylated fatty acids
were then analyzed by GC-MS.
|
Materials and Methods
|
|---|
materials
All chemicals were analytical grade and obtained from
Sigma-Fluka-Aldrich Chemical Co. PGE1 was from Cayman
Chemical. Analytical-grade organic solvents and silica gel thin-layer
chromatography (TLC) were purchased from Merck. The capillary column
was from Hewlett Packard.
synthesis of 2-ricinoleoyl phosphatidylcholine
2-Ricinoleoyl phosphatidylcholine (2-ricinoleoyl-PC), used as an
internal calibrator, was synthesized according to the method published
by Isaacson et al. (16) and modified as follows. Both
carboxyl and hydroxyl groups were protected with
dimethyl-tert-butylsilyl chloride, and then a mild alkaline
hydrolysis of the dimethyl-tert-butylsilyl ester according
to Morton and Thompson (17). The resulting
butyldimethylsilyl ether ricinoleic acid was converted into its
anhydride as previously described by Selinger and Laidot
(18). Soybean lysophosphatidylcholine was then acylated
with this anhydride under mild conditions with 4-pyrrolidinopyridine
according to Mason et al. (19). The 2-ricinoleoyl-PC was
then deprotected as previously described (17) and purified
by TLC by using the mixture chloroform:methanol:methylamine (60:20:5 by
vol, 400 mL/L in water) and finally quantified by GC with
diheptadecanoyl phosphatidylethanolamine as an internal calibrator.
platelet isolation
Blood was taken from healthy donors onto citric acid, trisodium
citrate, and dextrose as anticoagulant (ACD), with and without a
mixture of 10-3 mol/L EDTA, 2 x
10-4 mol/L ASA, and 10-6 mol/L
PGE1. All the donors gave informed consent. We called
platelets isolated from blood taken onto ACD without inhibitors
"unprotected platelets" and platelets isolated from blood taken
on the anticoagulant containing the mixture of inhibitors "protected
platelets." Blood was then centrifuged at 200g for 17
min. The supernatant (platelet-rich plasma) was acidified to pH 6.4
with 0.15 mol/L citric acid and spun down for 10 min at 900g
(20). The pellets were resuspended into Tyrode HEPES
buffer, pH 7.35, and platelets were counted with a thrombocoulter
counter.
lipid extraction
Lipids were extracted twice with a mixture of chloroform:ethanol
(2:1 by vol) containing 2 nmol of 2-ricinoleoyl-PC and 1 nmol of
ricinoleic acid used as internal calibrators, and the water phase was
acidified to pH 3 before the second extraction step. Lipids were
immediately hydrogenated by hydrogen bubbling for 6 min in the presence
of PtO2 used as a catalyst. This step is important to
avoid further polyunsaturated fatty acid oxidation that would lead to
artifactual monohydroxylated fatty acid formation. The catalyst was
eliminated by centrifugation and the different lipid fractions were
separated by silica gel TLC with the solvent mixture hexane:diethyl
ether:acetic acid (60:40:1 by vol). Monohydroxylated fatty acids and
phospholipids were scraped off and extracted by diethyl ether and by
the mixture chloroform:ethanol (2:1 by vol), respectively.
Phospholipids were treated at 100 °C with 5% KOH methanolic
solution for 1 h and the free monohydroxylated fatty acids were
purified by TLC as stated above. Finally, they were converted into
methyl ester, trimethylsilyl ether (TMS) derivatives as previously
described (13).
gc-ms
The methyl ester, hydrogenated, TMS derivatives were then injected
into a gas chromatograph–mass spectrometer, a Hewlett Packard
apparatus model 6890. The column was a 30 m x 0.25 mm i.d. model
HP-5MS, coated with 50 mL/L phenylmethyl siloxane as stationary phase.
The carrier gas was helium at a flow rate of 1 mL/min and samples were
injected with a splitless injector with a head column pressure of 54.5
kPa (7.9 psi). The temperature of the injection port was 260 °C and
the transfer line was kept at 270 °C. The temperature program was
initially set at 57 °C for 5 min, increased at 20 °C/min until
200 °C, and then increased at 4 °C/min until 270 °C. Electron
impact (EI) ionization was done at 25 eV. Compounds were quantified
with the selected ion monitoring (SIM) mode with two specific ions
resulting from the breakdown of the molecule at both parts of the O-TMS
group, including the O-TMS, and selected for each derivative. The ion
m/z = 73, which corresponds to the TMS group, was only
used as a qualifier.
statistical analysis
Data were compared by using a Student–Fischer paired
t-test.
|
Results
|
|---|
Platelets were isolated from blood taken on classical ACD
anticoagulant (unprotected platelets) and also on ACD containing a
mixture of inhibitors of activation (protected platelets) as described
in Materials and Methods. In the presence of these
inhibitors, the recovery of platelets isolated from their plasma was
significantly improved (P <0.05). The platelet count was
473 x 109 ± 4.0 x 109/L in the
protected group and 410 x 109 ± 4.53 x
109/L in the unprotected one (mean ± SEM, n =
9).
After hydrogenation, the mass spectra of the monohydroxylated fatty
acid derivatives showed two main specific fragments corresponding to
the breakdown at both parts of the carbon carrying the derivatized
hydroxyl group, including this group. The example of the
15-hydroxyeicosatetraenoic acid (15-HETE) derivative fragmentation is
shown in Fig. 1
. The peak at m/z = 73 represents the loss of
the TMS group. The quantification of the methyl ester, hydrogenated,
TMS derivatives was performed as described in Materials and
Methods.
The total amount of free monohydroxylated fatty acids, which can be
calculated by the sum of 12-OH-17:0, 12-OH-20:0, and 15-OH-20:0, was
significantly lower (P <0.05) in the protected group
(1.52 ± 0.84 pmol/109 platelets) than in the
unprotected one (12.63 ± 10.52 pmol/109 platelets)
(mean ± SEM, n = 9). The difference was also significant
(P <0.05) for both the 12- and 15-OH-20:0 (Fig. 2
). Although not statistically tested, the amount of 12-OH-17:0
was much higher in the unprotected group (1.74 ± 1.16
pmol/109 platelets) (mean ± SEM, n = 9) than in
the protected one where it was undetectable. 13-Hydroxyoctadecanoic
acid (13-HODE) and 5-HETE were also lowered in the protected group, but
this decrease did not reach significance (results not shown). The same
tendency can be observed for the total monohydroxylated fatty acids
esterified in platelet phospholipids (23.48 ± 22.82
pmol/109 platelets in the protected group vs 47.74 ±
65.41 pmol/109 in the unprotected one, Fig. 3
) (mean ± SEM, n = 9), but this does not reach
significance. It is noteworthy that in protected platelets the ratio
between the amount of monohydroxylated fatty acids esterified in
platelet phospholipids and the amount of unesterified monohydroxylated
fatty acids was about fourfold greater than that observed in
unprotected platelets. This suggests that the monohydroxylated fatty
acids produced in the unprotected platelets were poorly esterified in
the phospholipid fraction.
View larger version (23K):
[in this window]
[in a new window]
|
Figure 2. Basal concentration of unesterified monohydroxylated fatty
acids detected in human platelets in both the protected (black
bars) and unprotected (hatched bars) groups.
Results are expressed in pmol/109 platelets and all
values are the mean ± SEM (n = 9). * P <0.05 as
compared with the protected group.
|
|
View larger version (20K):
[in this window]
[in a new window]
|
Figure 3. Basal concentration of esterified monohydroxylated fatty
acids measured in human platelet phospholipids in both the protected
(black bars) and unprotected (hatched bars)
groups.
Results are expressed in pmol/109 platelets and all
values are the mean ± SEM (n = 9). P <0.05 as
compared with the protected group.
|
|
In both groups, different monohydroxylated fatty acid isomers, such as
11-, 14-, and 17-OH-22:0 derived from 22-carbon polyunsaturated fatty
acids, presumably from 22:6n-3, were measurable. It remains that both
the 12- and the 15-OH-20:0 were the major compounds detected in
platelet phospholipids. In contrast, only traces of monohydroxylated
fatty acids were detected in both the triglyceride and the sterol ester
fractions (results not shown).
|
Discussion
|
|---|
Our results indicate that platelet activation was prevented when
blood was taken on anticoagulant containing a cocktail of complementary
inhibitors. Indeed, the platelet recovery was significantly improved in
the protected group. Such an activation occurring during venipuncture
and during the platelet isolation process has already been established
(21). The protecting effect is due to the different
inhibitors added to the anticoagulant. EDTA chelates the calcium
required for the phospholipase A2 activation, and therefore
less arachidonic acid is available for both the cyclooxygenase and
lipoxygenase pathways (22). EDTA also blocks the
iron-promoted peroxidation (23)(24). On the
other hand, PGE1 also inhibits the phospholipase
A2 activity via adenylyl cyclase activation and the
increased cAMP concentration, which contribute to hinder platelet
activation (25). Finally, ASA was also used to block any
arachidonic acid-induced platelet activation, as it is known to inhibit
cyclooxygenase (26) by acetylation of the enzyme
(27). This inhibition was effective, since we were unable
to detect any HHT in protected platelets. Therefore, these inhibitors
minimized platelet activation during venipuncture and during their
isolation and prevented the formation of microaggregates that could
have been lost during the centrifugation steps.
Occasional appearance of very high concentrations of hydroxy compounds
led us to suspect that autoxidation during the workup can still be a
problem (28). The hydrogenation step introduced just after
the lipid extraction reduces the double bonds and eliminates the
possibility of further artifactual oxidation during the sample workup
and analysis. This hydrogenation also provides a simplification of the
mass spectra and increases the detection sensitivity. After
hydrogenation and derivatization as methyl ester and TMS, the
fragmentation pattern shows two main ions resulting from the breakdown
of the molecule at both parts of the O-TMS group including the O-TMS.
These fragments are specific to the hydroxyl group position on the
chain and are used to measure the hydroxylated fatty acids with the SIM
detection mode. The saturated hydroxylated derivatives elute from the
column according to their carbon chain length and the position of the
derivatized hydroxyl group. For example, the 12-hydroxy fatty acid for
a given chain length has the shortest retention time (20.37 min) and
the 15-hydroxy the longest (20.61 min). The EI mode used for
quantification of the monohydroxylated fatty acids is relatively more
confident than the negative ionization mode, which is based only on a
single ion at m/z = M-181. Therefore, three criteria
were used to validate a proper peak recognition: (a) the
chromatographic retention time of the two main ions used for the
quantification must have the same retention time as that found for the
commercial calibrator injected under the same conditions,
(b) the ion at m/z = 73 (loss of TMS) used
as a qualifier ion must be present at the same retention time, and
(c) the relative abundance of these three ions must be in
the same order of magnitude as that observed in the spectrum of the
corresponding calibrator molecule. However, the hydrogenation step,
which enhances the sensitivity and avoids artifacts as previously
mentioned, does not define precisely the polyunsaturated fatty acid
precursor. It remains that the 12-OH-20:0 and 15-OH-20:0 correspond
mainly to 12-HETE and 15-HETE, respectively, arachidonic acid being the
most abundant fatty acid with 20 carbons in blood cells. Interestingly,
the amount of the presumed 12-HETE and 15-HETE in both the unesterified
and esterified compartments of platelets were quite similar. Platelets
produced mainly 12-HETE, suggesting that 15-HETE may be a marker of
surrounding leukocyte activation occurring in whole blood
(2) and made available to platelets during cell–cell
interactions (29). In contrast to what we observed with
such 12- and 15-hydroxy derivatives, 5-hydroxy-20:0 and 13-hydroxy-18:0
were not significantly higher in the unprotected platelets. This
differs from the atherosclerotic situation where both 15-HETE and
13-HODE were enhanced in rabbit arteries (30).
In both protected and unprotected platelets, the total amount of
unesterified monohydroxylated fatty acids was lower compared with that
of monohydroxylated fatty acids esterified in phospholipids. However,
assuming ~400 nmol of phospholipids per 109
platelets, we may calculate that <1 of 10 000 phospholipid acyl
chains was a monohydroxylated fatty acid. Their relative abundance in
membrane lipids is in good agreement with previous data from Spector et
al. (2) who reported such an estimation. Moreover, in
platelets, monohydroxylated fatty acids seem preferentially esterified
in the phospholipid fraction, as only traces were detected in both the
triglyceride and sterol ester fractions. In addition, the observation
that monohydroxylated fatty acids produced in greater amounts in
unprotected platelets were weakly esterified in phospholipids supports
previous works reporting low rates of 12-HETE incorporation in cell
phospholipids (7)(8).
Finally, this GC-MS procedure allowed us to measure the overall
esterified and unesterified monohydroxylated fatty acids in human blood
platelets at picomole concentrations. This may be a reliable and
sensitive way to approach the in vivo specific oxidation of arachidonic
acid as well as the result of oxidative stress.
|
Acknowledgments
|
|---|
This work was funded by INSERM and a grant from the Région
Rhône-Alpes. We thank Patricia Postal for her skilled technical
assistance.
|
Footnotes
|
|---|
1 Nonstandard abbreviations: HHT,
12-hydroxyheptadecatrienoic acid; GC-MS, gas chromatography–mass
spectrometry; ASA, aspirin; PGE1, prostaglandin
E1; 2-ricinoleoyl-PC, 2-ricinoleoyl phosphatidylcholine;
TLC, thin-layer chromatography; ACD, citric acid, trisodium citrate,
and dextrose (anticoagulant); TMS, trimethylsilyl ether; EI, electron
impact; SIM, selected ion monitoring, HODE, hydroxyoctadecanoic acid;
and HETE, hydroxyeicosatetraenoic acid. 
|
References
|
|---|
-
Hamberg M, Samuelsson B. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Natl Acad Sci U S A 1974;71:3400-3404.
[Abstract/Free Full Text]
-
Spector AA, Gordon JA, Moore SA. Hydroxyeicosatetraenoic acids (HETEs). Prog Lipid Res 1988;27:271-323.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Halliwell B. Oxidants and human disease: some new concepts. FASEB J 1987;1:358-364.
[Abstract]
-
Jaurgens G, Hoff HF, Chisolm GM, Esterbauer H. Modification of human serum low density lipoprotein by oxidation—characterization and pathophysiological implications. Chem Phys Lipids 1987;45:315-336.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Stenson WF, Parker CW. Metabolism of arachidonic acid in ionophore-stimulated neutrophils. Esterification of a hydroxylated metabolite into phospholipids. J Clin Invest 1979;64:1457-1465.
-
Stenson WF, Nickells MW, Atkinson JP. Esterification of monohydroxy fatty acids into the lipids of a macrophage cell line. Prostaglandins 1983;26:253-264.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Legrand AB, Wang J, Sobo G, Blair IA, Brash AR, Oates JA. Incorporation of 12(S)-hydroxyeicosatetraenoic acid into phosphatidylcholine signaling pathway. Biochim Biophys Acta 1996;1301:150-160.
[Medline]
[Order article via Infotrieve]
-
Vernhet L, Hichami A, Hamon L, Cochet MF, Legrand A. Incorporation of 12(S)-hydroxyeicosatetraenoic acid into phospholipids and active diacylglycerols in rat liver epithelial cells: effects on DNA synthesis. J Lipid Mediat Cell Signal 1996;13:233-248.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Croset M, Lagarde M. Stereospecific inhibition of PGH2-induced platelet aggregation by lipoxygenase products of icosaenoic acids. Biochem Biophys Res Commun 1983;112:878-883.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Brash AR, Hawkins DJ. High performance liquid chromatography for chiral analysis of eicosanoids. Methods Enzymol 1990;187:187-195.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Borgeat P, Picard S, Vallerand P, Bourgoin S, Odeimat A, Sirois P, Poubelle PE. Automated on-line extraction and profiling of lipoxygenase products of arachidonic acid by high performance liquid chromatography. Methods Enzymol 1990;187:98-116.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Guido DM, McKenna R, Mathews WR. Quantitation of hydroperoxy-eicosatetraenoic acids and hydroxy-eicosatetraenoic acids as indicators of lipid peroxidation using gas chromatography–mass spectrometry. Anal Biochem 1993;209:123-129.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Guichardant M, Lagarde M. Studies on platelet lipoxygenase specificity towards icosapolyenoic and docosapolyenoic acids. Biochim Biophys Acta 1985;836:210-214.
[Medline]
[Order article via Infotrieve]
-
Ikeda M, Kusaka T. Liquid chromatography–mass spectrometry of hydroxy and nonhydroxy fatty acids as amide derivatives. J Chromatogr 1992;575:197-205.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Guichardant M, Lagarde M, Lesieur M, De Maack F. Thermospray–mass spectrometric analysis of underivatized monohydroxy fatty acids: application to stimulated platelets. J Chromatogr 1988;425:25-34.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Isaacson Y, Sherbourne CD, Gross RW, Stenson WF. The synthesis and molecular dynamics of phospholipids having hydroxylated fatty acids at the sn-2 position. Chem Phys Lipids 1990;52:217-226.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Morton DR, Thompson JL. Total synthesis of 3-oxa-4,5,6-trinor-3,7-inter-m-phenylene prostaglandins. 2. Conjugate addition approach. J Org Chem 1978;43:2102-2106.
-
Selinger Z, Laidot Y. Synthesis of fatty acid anhydrides by reaction with dicyclohexylcarbodiimide. J Lipid Res 1966;7:174-195.
[Abstract]
-
Mason JT, Broccoli AV, Huang C-H. A method for the synthesis of isomerically pure saturated mixed-chain phosphatidylcholines. Anal Biochem 1981;113:96-101.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Lagarde M, Bryon PA, Guichardant M, Dechavanne M. A simple and efficient method for platelet isolation from their plasma. Thromb Res 1980;17:581-588.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Samuelsson B, Granström E, Green K, Hamberg M, Hammarström S. Prostaglandins. Annu Rev Biochem 1975;44:669-695.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Rittenhouse-Simon S, Russel FA, Deykin D. Mobilization of arachidonic acid in human platelets: kinetics and calcium dependency. Biochim Biophys Acta 1977;488:370-388.
[Medline]
[Order article via Infotrieve]
-
Van Kuijk FJGM, Thomas DW, Stephens RJ, Dratz EA. Gas chromatography–mass spectrometry methods for determination of phospholipid peroxides; I. Transesterification to form methyl esters. Free Radic Biol Med 1985;1:215-225.
-
Van Kuijk FJGM, Thomas DW, Stephens RJ, Dratz EA. Gas chromatography–mass spectrometry method for determination of phospholipid peroxides; II. Transesterification to form pentafluorobenzyl esters and detection with picogram sensitivity. Free Radic Biol Med 1985;1:387-393.
-
Siess W, Lapetina EG. Functional relationship between cyclic AMP-dependent protein phosphorylation and platelet inhibition. Biochem J 1990;271:815-819.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Weis HJ. Platelet physiology and abnormalities of platelet function. N Engl J Med 1975;293:531-541.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Roth GJ, Majerus PW. The of effect of aspirin on human platelets. J Clin Invest 1975;56:624-632.
-
Thomas DW, Van Kuijk FJGM, Dratz EA, Stephens RJ. Quantitative determination of hydroxy fatty acids as an indicator of in vivo lipid peroxidation: gas chromatography–mass spectrometry methods. Anal Biochem 1991;198:104-111.
[Web of Science][Medline]
[Order article via Infotrieve]
-
Lagarde M, Gualde N, Rigaud M. Metabolic interactions between eicosanoids in blood and vascular cells. Biochem J 1988;257:313-320.
-
Wang T, Powell WS. Increased levels of monohydroxy metabolites of arachidonic acid and linoleic acid in LDL and aorta from atherosclerotic rabbits. Biochim Biophys Acta 1991;1084:129-138.
[Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
|
|
|
|
 
C. Calzada, E. Vericel, B. Mitel, L. Coulon, and M. Lagarde
12(S)-Hydroperoxy-eicosatetraenoic acid increases arachidonic acid availability in collagen-primed platelets
J. Lipid Res.,
September 1, 2001;
42(9):
1467 - 1473.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
