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Articles |
Departments of
1
Clinical Laboratory Science and
2
Social and Preventive Medicine, State University of New York at Buffalo, Buffalo, NY 14214.
a Address correspondence to this author at: Department of Social and Preventive Medicine, State University of New York at Buffalo, 270 Farber Hall, 3435 Main St., Buffalo, NY 14214. Fax 716-829-2979; e-mail rwbrowne{at}acsu.buffalo.edu
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Abstract |
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Methods: Hydroxy and hydroperoxy polyunsaturated fatty acid (PUFA) peroxidation products were analyzed in human blood plasma by reversed-phase HPLC after liquid-liquid extraction of total lipids and alkaline hydrolysis of lipid esters to liberate free PUFAs. An isocratic mobile phase containing 1 g/L acetic acid-acetonitrile-tetrahydrofuran (52:30:18, by volume) over 60 min duration, with ultraviolet absorbance detection at 236 nm by photodiode array, enabled the resolution and quantification of 13 regioisomeric hydroxy and hydroperoxy PUFAs.
Results: As little as 250 µL of human plasma was utilized with an analytical range of 0.033–1.6 µmol/L for each compound. Intra- and interassay CVs for all compounds detected in normal or oxidatively modified human plasma were 3.2–11% and 4.7–12%, respectively. Analytical recoveries were 87–103%. Analysis of human plasma exposed to artificial oxidation with Cu2+ ion and hydrogen peroxide, a free radical-generating reaction, showed marked increases in hydroxy and hydroperoxy PUFA concentrations.
Conclusion: Lipid-derived hydroxy and hydroperoxy PUFAs may be useful as clinical markers of lipid peroxidation and oxidative stress in the peripheral circulation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of lipid peroxidation has most often been accomplished
through semiquantitative analysis of total hydroperoxide content, total
conjugated dienes, or through measurement of LOOH breakdown products
(4)(5). Each of these techniques has
shortcomings. Total hydroperoxide methods such as iodometry and the
ferric iron oxidation/xylenol orange technique do not measure the
hydroxy derivative, which has been shown to be the predominant form
(6), and are subject to interference from non-lipid
peroxides present in either the sample or the reagents. Total
conjugated diene measurements are not specific for lipid peroxidation
because conjugated dienes are found in compounds such as isolinoleic
acid, which usually is present in human plasma in much higher
concentrations than oxidatively generated conjugated dienes
(7). Lipid peroxidation byproduct analyses, such as the
thiobarbituric acid reactive substances test, measure
malondialdehyde (MDA). MDA is a small three-carbon aldehyde generated
from the hydrolysis of certain LOOHs. However, MDA is not generated
exclusively by breakdown of LOOHs, nor does the thiobarbituric acid
reactive substances test measure MDA exclusively (8). The
test is therefore nonspecific. The most detailed analyses achieved to
date have been through use of gas chromatography–mass
spectrometry (9)(10)(11)(12). However, the hydroperoxide
group of LOOH is not stable at the temperatures required for gas
chromatographic analyses; therefore, direct measurement is impossible.
The typical strategy has been to analyze the trimethylsilyl derivative
of LOH before and after reduction of the sample with a strong reducing
agent such as sodium borohydride or triphenylphosphine (9).
The LOOH concentration is then calculated as the difference between
pre- and postreduction LOH concentrations. HPLC offers the advantage of
separating LOOHs and LOHs without derivatization. After HPLC
separation, detection schemes that include ultraviolet (UV)
absorbance of the conjugated diene at 234 nm, postcolumn
chemiluminescence, and electrochemical reduction have been described
(13)(14)(15)(16). Although chemiluminescence and electrochemical
techniques detect LOOHs at much lower concentrations than UV
absorbance, they do not detect to LOHs. This is a drawback
because it has been shown that the major lipid peroxidation products in
human plasma are the more stable LOHs (6). UV absorbance is
able to identify both LOOHs and LOHs because they both possess a
conjugated diene that absorbs strongly (
= 23 000–27 000)
near 234 nm. A general difficulty of chromatography applied to total
lipid peroxidation measurements is that LOOHs and LOHs occur
predominantly esterified in cholesterol esters, phospholipids, and
triglycerides, which are difficult to resolve in a single
chromatographic system. To overcome this and to provide a more accurate
estimation of total lipid peroxidation, alkaline hydrolysis of lipid
esters to yield free fatty acid hydroperoxides (FAOOHs) and hydroxy
fatty acids (FAOHs), followed by HPLC separation and direct UV
detection of the conjugated dienes has been described (16).
This method provides a substantial amount of information
regarding the amount of total lipid peroxidation, as well as the
identity of the PUFA precursor.
FAOOH and FAOH species found in biological material can vary in carbon chain length from 18 to 22 carbons and degree of unsaturation (2 to 6 double bonds). The position of the oxygenation site relative to the carbon chain also varies because each pair of double bonds allows for two theoretical peroxidation sites (17). In the case of linoleic acid, the major PUFA in biological systems, this produces two possible FAOOH and two possible FAOH regioisomers (i.e., 9- and 13-hydroperoxy-octadecadienoic acid and 9- and 13-hydroxy-octadecadienoic acid). As the number of double bonds increases so does the number of possible regioisomers. Increasing complexity is added in possible cis, trans (Z, E) isomers and enantiomers (R, S). Each of the above mentioned regioisomers can exist in cis-trans or trans-trans configuration, and normal phase HPLC systems have been described that resolve these isomeric forms (18) as well as chiral phase systems that resolve enantiomers (19).
We have described a method for the separation and analysis of mixtures
of synthetic hydroxy and hydroperoxy PUFAs by reversed-phase HPLC
(20). The present report improves this methodology to
achieve resolution of regioisomers of linoleic, linolenic, arachidonic,
and docosahexaenoic acid FAOOHs and FAOHs. The method is applied to the
analysis of FAOOHs and FAOHs generated by in vitro oxidation of human
plasma by metal-catalyzed oxidation with the Cu2+
ion and hydrogen peroxide. Cu2+ has been shown to
react with any LOOH already present in a sample to cleave the LOOH:
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bulk quantities of FAOOHs for stability and recovery studies were synthesized using type 1 soybean lipoxidase to generate FAOOHs from pure linoleic, linolenic, arachidonic, and docosahexaenoic acid (20). Bulk FAOHs were synthesized by reduction of FAOOHs with sodium borohydride in ice-cold methanol followed by chloroform re-extraction. The isomeric identity of bulk FAOOH and FAOH preparations was confirmed against retention time and absorbance spectra of FAOOHs and FAOHs of known regioisomeric composition: 9S-hydroperoxy-10Z,12E-octadecadienoic acid (9-HpODE), 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE), 9S-10Z,12E-hydroxy-octadecadienoic acid (9-HODE), 13S-hydroxy-9Z,11E-octadecadienoic acid (13-HODE), 9S-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid (13-HpOTE), 9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid, 13S-hydroxy-9Z,11E,15Z-octadecatrienoic acid, 15-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HpETE), 12-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HpETE), 5-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE), 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HETE), 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE), and 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE). These compounds as well as an internal standard of (±)5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid-methyl ester (5-HETE-ME) and the R-enantiomer of 13-HODE (13R-hydroxy-9Z,11E-octadecadienoic acid) were obtained as pure solutions in ethanol and used as received (Caymen Chemical).
instrumentation
The equipment used was as follows: a UV-visible recording
spectrophotometer (Model 160 U) and HPLC system were from
Shimadzu. The HPLC system consisted of an LC-7A solvent delivery
system, an LPM-600 low-pressure mixing proportioning valve, a SIL-9A
automatic sample injector, an SPD/M6A UV/VIS photodiode array, and an
online IBM personal computer with Shimadzu Class-VP chromatograph data
processing software. The mobile phase reservoirs were continuously
sparged from a helium degassing system (Kontes Glassware). The
reversed-phase analytical HPLC column was a Supelcosil LC-18 (4.6
mm x 25.0 cm; 5.0-µm particle size; 100Å pore size), with a
Supelcoguard LC-18 guard column (4.6 mm x 2.0 cm; 5-µm particle
size; 100Å pore size; Supelco).
sample preparation
Hexane-isopropanol (HIP) total lipid extracts (21) were
prepared by adding 1.0 mL of isopropanol containing 0.1 g/L
butylated hydroxytoluene to 0.5 mL of EDTA plasma. After 2 mL
of hexane was added, the vial was perfused with nitrogen, capped,
vortex-mixed for 1 min, and centrifuged for 3 min at 3000g;
the upper hexane phase was collected by aspiration. The extraction was
repeated three times, and the hexane layers were pooled and evaporated
to dryness under nitrogen. To analyze the total lipid extraction
efficiency, the aqueous layer was re-extracted three times with
chloroform-methanol (2:1, by volume). Total lipids were analyzed on
adsorption thin-layer chromatography (TLC) plates (20 x 20 cm)
precoated with 0.25 mm of silica gel G without fluorescent indicator
(EM Laboratories); the plates were developed with hexane-diethyl
ether-acetic acid (80:20:1, by volume). Alkaline hydrolysis of total
dried lipid extracts was performed by dissolving the extracts in 0.95
mL of degassed, absolute ethanol. NaOH (50 µL of a10 mol/L solution)
was added, and the sample was perfused with nitrogen, capped, heated at
60 °C for 20 min, and neutralized with 30 µL of glacial acetic
acid. 5-HETE-ME (100 µL of a 1.0 µmol/L solution) was added as
internal standard. After the ethanol was evaporated under nitrogen, the
sample was dissolved in 1.0 mL of water and extracted twice with 2.0 mL
of n-heptane. The upper phase was collected, pooled, and
evaporated under nitrogen, and the residue resuspended in 250 µL of
ethanol. Completeness of the alkaline hydrolysis was assessed by TLC as
described above. Immediately before HPLC injection, 250 µL of water
was added to the samples to ensure efficient mass transfer to the
stationary phase.
hplc analysis
Sample (150 µL) was injected into the HPLC system, eluted
isocratically with a mobile phase containing 1 g/L acetic
acid-acetonitrile-tetrahydrofuran (52:30:18, by volume) over 60 min and
monitored at 200–300 nm by the photodiode array. It should be noted
that a photodiode array is not necessary for this methodology; a simple
UV detector could be used. Integration of peak areas was performed at
236 nm with an 8-nm bandwidth. This wavelength and bandwidth were
chosen to encompass the wavelength of maximum absorbance
(
max) of the octadecadienoic acids at 234 nm
and the eicosatetraenoic acids at 237 nm. Quantification was based on
an external calibration curve using ethanolic calibrators
prepared on a Shimadzu 160 UV scanning spectrophotometer applying the
max and absorptivity coefficients provided by
the manufacturer. Serial dilutions were made in ethanol-water (50:50,
by volume), and calibration curves were generated by triplicate
injections of each calibrator. Sample concentrations were interpolated
from calibration curves and corrected for recovery of 5-HETE-ME
internal standard.
FAOH recovery experiments were performed by adding known amounts of calibrators into human plasma. FAOOH recovery experiments were performed by adding calibrators into isopropyl alcohol-precipitated plasma. Reproducibility experiments were carried out on a pool of normal human EDTA plasma as a low-concentration control. A separate high-concentration pool was oxidized with 100 µmol/L Cu2+ ion for 12 h at 37 °C and then stabilized by addition of 250 µmol/L EDTA followed by portioning and freezing at -80 °C. Intraassay reproducibility was estimated by 20 replicates of each control pool in one batch. Interassay reproducibility was estimated by duplicate, daily analysis of control pools over a period of 20 days.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chloroform-methanol extracts of the remaining aqueous layer after HIP
extraction showed no detectable spots on TLC, indicating complete
extraction of total lipids. Fig. 1
shows the results of TLC analysis of lipid extracts after
alkaline hydrolysis. The lipid esters were completely hydrolyzed in 20
min in NaOH at a concentration of 0.5 mol/L.
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Thomas and Jackson (16) reported the conversion of small
amounts of FAOOHs to FAOHs during alkaline hydrolysis of lipid esters,
and the reaction of FAOOHs with strong alkali has been described in
detail by Gardener et al. (22). To account for possible
artifacts in our analysis, we studied the effect of alkaline hydrolysis
on the stability of FAOOH calibrators. A mixture of FAOOH calibrators
in ethanol (13-HpODE, 13-HpOTE, 15-HpETE, and HpDHE) was split into two
equal parts. One was analyzed directly by HPLC, whereas the other was
saponified as described above, neutralized, and then analyzed. Fig. 2
shows a comparison of these two chromatograms. Alkaline
hydrolysis produces an 8–12% loss of each FAOOH, which was recovered
as the corresponding FAOH. Three-dimensional diode array analysis
showed no change in the absorbance spectra of the FAOOHs or the FAOHs
after alkaline hydrolysis.
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Autooxidation of PUFAs during processing or analysis of biological
samples is a concern with all lipid peroxidation methodologies. To
assess possible lipid peroxidation during sample processing, we
prepared a mixture containing 12.5 mg each of linoleic, arachidonic,
linolenic, and docosahexaenoic acids in 1.0 mL of ethanol. 5-HETE-ME
was added to the solution. The solution was then split, and half was
diluted 1:2 in water and immediately subjected to HPLC using a mobile
phase of 1 g/L acetic acid-acetonitrile-tetrahydrofuran (41:41:18, by
volume). The photodiode array monitored the PUFAs at 215 nm and the
LOOHs and LOHs at 236 nm. The second split was evaporated to dryness
under nitrogen to remove the ethanol, redissolved in 1.5 mL of 0.1
g/L butylated hydroxytoluene in isopropyl alcohol-water (2:1,
by volume), processed as described for plasma, and analyzed under the
same HPLC conditions. Fig. 3
shows a comparison of these two chromatograms. This mobile
phase allowed the simultaneous determination of native PUFAs, FAOOHs,
and FAOHs within 60 min, although there was considerable loss of
resolution of the FAOOH and FAOH regioisomers. We noted that there were
substantial amounts of LOOHs and LOHs in pure commercial preparations
of PUFAs. Approximately 90% of the PUFAs, FAOOHs, and FAOHs were
recovered after sample processing, and there was no significant
increase in any FAOOH or FAOH relative to the corresponding PUFA. The
most notable difference was the presence of a sizeable peak at 17.5
min, which corresponded to 5-HETE and was attributable to alkaline
hydrolysis of the 5-HETE-ME internal standard during processing.
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To estimate the possible complexity of chromatograms generated from
biological samples and to assess the ability of the method to respond
to a known mechanism of induced oxidative damage, plasma was exposed to
CuSO4-H2O2.
Normal human plasma was obtained, and baseline FAOOH and FAOH
concentrations were determined. The specimens were incubated with 100
µmol/L CuSO4 and 0.3 mL/L
H2O2 at 37 °C for 2
h, and aliquots removed at 30, 60, and 120 min for FAOOH/FAOH analysis.
Fig. 4
shows the increase of plasma FAOOH and FAOH concentrations as a
function of time. The linoleic acid oxidation products 13-HODE and
9-HODE were the only quantifiable compounds in normal plasma and
constituted the major peaks in all analyses. 9-HpODE was detected at
baseline but was below the limit of quantification. 9-HpODE was the
first increased FAOOH and was quantifiable at 30 min,
13(S)HpODE was quantifiable by 60 min as were the
arachidonic acid products 15(S)HpETE, 12(S)HETE,
and 5(S)HETE. These products continued to rise in proportion
to each other as oxidation continued.
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After systematic changes in the mobile phase, the maximum resolution of
all calibrators with acceptable elution time was achieved using an
isocratic mobile phase consisting of 1 g/L acetic
acid-acetonitrile-tetrahydrofuran (52:30:18, by volume). A chromatogram
of all of the commercial calibrators is shown in Fig. 5
. Of the 15 FAOOH and FAOH isomers that we synthesized or
purchased, 13 peaks were resolved by the HPLC conditions described
above. Among the calibrators, co-elution occurred between 12-HETE and
HpDHE (peak 11) and between 12-HpETE and 5-HETE (peak 12). 15-HpETE and
15-HETE (peaks 6 and 10), which were not fully resolved from the down
slope of 9-HpODE and 9-HODE, respectively, were integrated by a manual
peak splitting function of the chromatography software.
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The method performance characteristics for all analytes detected in the
high- and low-concentration plasma pools are shown in Table 1
. The minimum detectable quantity (MDQ) was defined as a
peak signal greater that 3 SD above the baseline noise. For all
linoleic acid- and linolenic acid-derived FAOOHs and FAOHs, which gave
sharp peaks and were well resolved, the MDQ was 5–7 pmol on column.
The MDQs of the arachidonic acid-derived FAOOHs and FAOHs were lower
because they absorb more strongly at 236 nm ( = 27 000) than
the linoleates or linolenates ( = 23 000). The MDQs of
15-HpETE and 15-HETE were 5.1 and 5.2 pmol in pure solution but were
three times higher in a mixture with 9-HpODE and 9-HODE, which if
present in higher concentrations, mask these peaks. Each analyte
produced a linear response between the MDQ and 250 pmol, where the peak
width began to overlap adjacent peaks. Linear regression of all
calibration curves produced correlation coefficients (r)
>0.99.
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The CVs were <5% for 9- and 13-HODE, which were the only peaks detected in the low-concentration control pool. The high-concentration pool was inherently more complex, and the CVs for all peaks were between 8.4% and 12%, with most of the variability arising from the extraction process because replicate injections of a single extract showed CVs <1%. The moderate reproducibility is also likely to be a function of the reduction of FAOOHs to FAOHs during alkaline hydrolysis, which was evident in the low recovery of the FAOOH compounds (87–93%).
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Discussion |
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Once isolated, plasma total lipids were completely extracted by the HIP extraction system. We have compared HIP extraction with chloroform-methanol and ether-ethanol methodologies. Ether-ethanol was found to be unsuitable because of the epoxides that form in ether and catalyze in vitro peroxidation of the sample. Chloroform-methanol-extracted lipids are partitioned into the lower phase, are difficult to aspirate, and contain considerable water after drying. HIP extraction is favorable because no water is present after drying and the degree of toxicity of the solvents is much lower (19). The addition of butylated hydroxytoluene and the removal of dissolved oxygen from the solvents by sonication under reduced pressure and helium sparging prevents any detectable autooxidation of lipids during processing.
Total esterified PUFAs were completely hydrolyzed in 20 min by the alkaline hydrolysis procedure described, which produces an 8–12% conversion of FAOOHs to the corresponding FAOHs. This reduction of FAOOH to FAOH is higher than that reported by Thomas and Jackson (16), who described a 6–8% conversion. This reduction becomes less significant at lower concentrations of base, but as the base concentration is decreased, the amount of time required to completely hydrolyze the sample increases, leading to the potential for autooxidation.
The HPLC technique described can identify up to 13 distinct regioisomers of FAOOHs and FAOHs, constituting the most detailed HPLC separation of these compounds reported to date. The method has a low detection limit, detecting as little as 5 pmol, and is linear up to 250 pmol. Based on the procedure described, this converts to a linear range of 0.033–1.6 µmol/L, which encompasses the range of concentrations reported in normal human plasma by Wilson et al. (12). Analysis of the method performance characteristics indicated acceptable imprecision, with CVs of 7–12%, and recoveries >90%. It should be noted that FAOOH recovery experiments require that the FAOOH solution be added to the sample only after the sample is precipitated with isopropyl alcohol. Direct addition of FAOOH to intact plasma leads to a reduction of 25–50% of the FAOOH to the corresponding FAOH, depending on the time between addition of the FAOOH solution and alcohol precipitation. This phenomenon is most likely attributable to the activity of endogenous peroxidases such as glutathione peroxidase. Precipitation of the sample before the addition of exogenous FAOOH inactivates these enzymes and prevents the reduction of the FAOOH.
The in vitro oxidation of plasma demonstrated small but detectable FAOH concentrations at baseline and measurable increases in FAOOH and FAOH concentrations with time. The linoleic acid oxidation products 13-HODE and 9-HODE constituted the major peaks in all analyses. 9-HpODE was the only FAOOH visible at baseline but was below the MDQ. 9-HpODE and 13-HpODE were quantifiable by 60 min of oxidation. Teng and Smith (18) reported the inability to separate the cis-trans, trans-trans regioisomeric HODEs (13ZE, 13EE, 9EZ, and 9EE), the cis-trans HpODES (9EZ and 13ZE), and trans-trans HpODEs (9EE and 13EE) under similar chromatographic conditions. We have clearly demonstrated the separation of the four cis-trans regioisomers 9-EZ-HODE, 13-ZE-HODE, 9-EZ-HpODE, and 13-ZE-HpODE, with each calibrator giving a single resolve peak. This improvement may be attributable to the longer analytical column (25 cm), slightly higher tetrahydrofuran concentration, fivefold lower acetic acid concentration, or a combination of all these factors. However, because of the lack of suitable calibrators, we were unable to confirm the presence or absence of all trans (EE) regioisomers in Cu2+-H2O2-oxidized plasma. This is an acknowledged limitation of the method.
The arachidonic acid products 15-HpETE, 12-HETE, and 5-HETE were detectable by 60 min of oxidation. These products continued to rise in proportion to each other as oxidation continued. Nonenzymatic oxidation of arachidonic acid can also give rise to 8-, 9-, and 11-hydroperoxy and -hydroxy regioisomers in addition to the ones discussed here. Based on the elution pattern of 15-, 12-, and 5-HpETE and -HETE, we observed that the retention time of the particular isomer shortened as the oxygenation site moved farther from the carboxylic acid end of the acyl chain. This was also true for the linoleic and linolenic FAOOHs and FAOHs. The farther the negative charges of the hydroperoxy or hydroxy group are from the negative charge of the carboxylic acid group, the more independent they become, giving an increasingly polar character to the isomer and shortening the retention time in reversed-phase systems. We hypothesize that the regioisomers not characterized here, 11-, 9-, and 8-HETE and -HpETE, would elute in this order between the 12- and 5-regioisomers, where there is a moderate stretch of baseline. However, no peaks were detected in this region on analysis of Cu2+-H2O2-oxidized plasma, and these isomers were either not present or present in amounts below the detection limit of the method.
There were no peroxidation products detected that were attributable to linolenic or docosahexaenoic acid. These findings result from the small percentage that these PUFAs constitute of total plasma PUFAs. After 2 h of oxidation, FAOOH and FAOH isomers of linoleic and arachidonic acids account for <1% of the corresponding unoxidized PUFAs, which make up >85% of total plasma PUFAs. Unoxidized linolenic and docosahexaenoic acids constitute <5% of the total PUFAs, and their oxidation products are well below the MDQ of this method. These findings are in agreement with both Wilson et al. (12) and Spitellar (6), who reported that C20 and C18 fatty acids, especially linoleic acid, are the major precursors to plasma FAOOHs and FAOHs.
In conclusion, the HPLC methodology described here is sensitive and specific for the primary products of lipid peroxidation and facilitates the use of these products as markers of free radical-mediated lipid damage in the peripheral circulation.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: PUFA, polyunsaturated fatty acid; LOOH, lipid hydroperoxide; LOH, lipid hydroxy derivative; MDA, malondialdehyde; UV, ultraviolet; FAOOH, fatty acid hydroperoxide; FAOH, hydroxy fatty acid; 9-HpODE, 9S-hydroperoxy-10Z,12E-octadecadienoic acid; 13-HpODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid; 9-HODE, 9S-10Z,12E-hydroxy-octadecadienoic acid; 13-HODE, 13S-hydroxy-9Z,11E-octadecadienoic acid; 13-HpOTE, 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid; 15-HpETE, 15-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid; 12-HpETE, 12-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 5-HpETE, 5-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 15-HETE, 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid; 12-HETE, 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; 5-HETE, 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic; 5-HETE-ME, (±)5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid-methyl ester; 13(R)HODE, 13R-hydroxy-9Z,11E-octadecadienoic acid; HDHE, hydroxy-docosahexaenoic acid; HpDHE, hydroperoxy-docosahexaenoic acid; HIP, hexane/isopropanol (3:2, by volume); TLC, thin layer chromatography; and MDQ, minimum detectable quantity. 
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-tocopherol (VIT E), triolein
(TRIG), linoleic and arachidonic acids
(FFA), cholesterol (CHOL), phosphatidyl
choline (PC), and monoglycerides (MG).
