Coulometric detection in high-performance liquid chromatographic analysis of cholesteryl ester hydroperoxides

Free Radical Biology& Medicine, Vol. 20, No. 3, pp. 365-371, 1996 Copyright© 1996ElsevierScienceInc. Printed in the USA. All rights reserved 0891-5849/96 $15.00 + .00 ELSEVIER

SSDI 0891-5849(96)02062-4

Original Contribution COULOMETRIC DETECTION IN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF CHOLESTERYL ESTER HYDROPEROXIDES HIROFUMI ARAI,* JUNJI TERAO, t DULCINEIA S. P. ABDALLA,* TETSUYA SUZUKI,* and K o z o TAKAMA* *Department of Marine Bioresources Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido, Japan; *National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki, Japan; and *Faculty of Pharmaceutical Sciences, University of SaO Paulo, Sa6 Paulo, Brazil (Received 16 May 1995; Revised 31 July 1995; Accepted 6 September 1995)

Abstract A highly sensitive and simple method for the determination of cholesteryl ester hydroperoxides (ChEOOH) was developed using high-performance liquid chromatography (HPLC) with coulometric electrochemical detection. The lowest detectable level by this technique was 2 pmol for cholesteryl linoleate hydroperoxides at the signal-to-noise ratio of 3. This method was applied to the determination of ChE-OOH presumably present in human plasma. Although ChE-OOH could not be detected, the ChE-OOH level in the fluid was estimated to be less than 27 nM. It was found that the extraction efficiency of an internal standard, cholesteryl nervonate, was decreased by lowering its amount spiked to the plasma. The concentration of ChE-OOH in human plasma and plasma lipoprotein, which were peroxidized with a radical initiator in vitro, could be determined by use of this standard. HPLC-coulometric technique is, therefore, useful to measure the peroxidation of plasma lipids in vitro. K e y w o r d s ~ o u l o m e t r i c electrochemical detection, HPLC, Human plasma, Cholesteryl ester hydroperoxide, Free radicals

glycerol hydroperoxides in oxidized vegetable oils. 5 However, it was found that the sensitivity by the amperometric detection with glassy carbon electrode was not enough to detect lipid hydroperoxides (LOOH) present in human plasma. 6 L O O H is likely to be present at extremely low level in human plasma because this biological fluid possesses a well-organized antioxidant defense system. 7'8 Sensitive methods for the determination of L O O H were then developed using high-performance liquid chromatography (HPLC)-chemiluminescence 9'~° or HPLC-fluorescence ~j'12 detections. The concentrations of phosphatidylcholine hydroperoxides (PC-OOH) and cholesteryl ester hydroperoxides (ChE-OOH) in human plasma or plasma lipoproteins were determined by these techniques) 3-~6 Recently, Girotti and co-workers ]7'Is improved the sensitivity of amperometric detection for L O O H by replacing glassy carbon electrode with mercury drop electrode. Coulometric detection is an alternative electrochemical detection system in H P L C analysis, and this technique has been utilized for the sensitive determination

INTRODUCTION Lipid peroxidation in biological tissues and fluids is recognized as one of the oxidative events leading to degenerative diseases.] In particular, much attention has been paid to the relationship between the lipoprotein oxidation in human plasma and the atherosclerosis. 2 Therefore, sensitive methods should be developed to determine the level o f peroxidized lipids in human plasma. W e previously applied a high-performance liquid chromatographic technique for the determination of lipid peroxidation products and achieved the specific detection of phospholipid hydroperoxides, primary products of phospholipid peroxidation, using amperometric electrochemical detection with glassy carbon electrode. 3 This analytical technique was subsequently employed to estimate the fatty acid hydroperoxide-reducing ability o f human plasma 4 and to measure triacylAddress correspondence to: Junji Terao, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan. 365

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of reducing compounds in biological samples such as ascorbic acid and a-tocopherol. ]9'2° We tried to apply this methodology for the determination of LOOH. In vitro studies suggest that ChE-OOH is a reliable index of the lipid peroxidation level in human plasma. 7 Therefore, this article describes the method for ChE-OOH determination using coulometry in HPLC. MATERIALS AND METHODS

Materials Cholesteryl linoleate (cholesteryl 9Z,12Z-octadecadienoate), cholesteryl nervonate (cholesteryl 15Z-tetracosenoate), sodium dextran sulfate (average molecular weight of 5 × 105), and soybean lipoxygenase (Type I-B, 2.06 × 105 units/mg protein) were purchased from Sigma Co. (St. Louis, MO). Methylene blue was obtained from Kanto Chemical Co. (Tokyo, Japan). 2,2'Azobis(2-amidinopropane) dihydrochloride (AAPH) was supplied from Wako Pure Chemical Co. (Osaka, Japan). All other chemicals and solvents were of reagent grade.

Preparation of cholesteryl ester hydroperoxide standards Cholesteryl linoleate hydroperoxides (ChL-OOH) were prepared by lipoxygenase reaction as follows. Cholesteryl linoleate (30 #tool) in chloroform/methanol (1:1, v/v) solution with 460 #mol of sodium deoxycholate was placed into a centrifuge tube and was evaporated under nitrogen gas, then in vacuo. The thin lipid film was dispersed in 28 ml borate buffer (200 mM, pH 9.0) with a vortex mixer for 1 rain followed by ultrasonic irradiation with a Heat Systems Sonifier (Model W-380; Farmingdale, NY) for 1 min. Then 2.06 × 10 6 units of soybean lipoxygenase in 2 ml borate buffer (200 mM, pH 9.0) was added to the cholesteryl linoleate emulsion. The reaction mixture was incubated at 25°C in the dark for 60 min with continuous shaking and O2 bubbling. The lipid fraction was extracted from the reaction mixture and purified by silica gel colunm chromatography in the following manner. The lipid fraction in 0.5 ml of nhexane was charged on a silica gel column (Wakogel FC-40, 10 × 30 mm, Wako Pure Chemical Co., Osaka, Japan) and fractionated by successive elution with 10 ml of n-hexane, 20 ml of n-hexane/chloroform (2:1, v/v), and 20 ml of chloroform. A ChL-OOH fraction eluted by chloroform was evaporated and the ChL-OOH were redissolved in chloroform/methanol (1:1, v/v). Cholesteryl nervonate hydroperoxides (ChN-OOH) were prepared by photosensitized oxidation using methylene blue 2~ and purified by the method described above. The hydroperoxide contents of ChL-OOH and ChN-OOH

standards were determined by iodometric assay. 22 In this method, the absorbance of triiodide produced by the reaction of hydroperoxides with iodide was measured at 353 nm.

High-performance liquid chromatographic analysis of ChE-OOH ChE-OOH were determined by reversed-phase high-performance liquid chromatography (HPLC) with spectrophotometric and electrochemical detection. HPLC was run on a column of TSKgel Octyl-80Ts (150 x 4.6 mm i.d.) with a guard column of TSKguardgel Octyl-80Ts (15 X 3.2 mm i.d.) (Tosoh, Tokyo, Japan) by using a Tosoh CCPS pump. The columns were kept at 40°C and eluted with methanol/water/ acetic acid (97:3:0.1, v/v/v) containing 50 mM lithium acetate at a flow rate of 1.0 ml/min. The premixed mobile phase, which was passed through a membrane filter (0.2 #m, Fuji Photo Film, Tokyo, Japan) and degassed under reduced pressure before use, was continuously purged with helium gas in the analysis. A Rheodyne 7125 injector (100 #1, Cotati, CA) was used to inject the sample solution onto the column. The eluent was monitored by a SPD-6AV spectrophotometfie detector at 235 nm (Shimadzu, Kyoto, Japan) and a Coulomet CEC-021 coulometric detector (Mitsubishi Petrochemical, Tokyo, Japan) connected in series. The working electrode of the coulometric detector is made of porous carbon and two electrodes positioned in series in the cell. The first (channel 1) and second (channel 2) electrode potentials of the coulometric detector were set at - 1 0 mV and - 5 0 mV vs. Ag/AgCI, respectively (range of 50 nA). The electrochemical signals of the second electrode (channel 2) were monitored in the coulometric detection. The functions of the coulometric detector was compared to that of an ICA-3062 amperometric detector (TOA Electronics, Tokyo, Japan). The glassy carbon electrode was used as working electrode of the amperometric detector and the electrode potential was set at - 1 0 0 mV vs. Hg/Hg2C12 (range of 102.4 nA). The electrodes of both electrochemical detectors were equilibrated by running of the mobile phase for 30 rain before analysis. The data were processed with a 807-IT integrator (JASCO, Tokyo, Japan).

Preparation of human plasma and the fraction of low-density lipoprotein plus very low-density lipoprotein Blood was drawn from three healthy male volunteers, aged from 26 to 44, using sodium heparin as an anticoagulant. Plasma was separated by centrifugation

Coulometry of lipid hydroperoxides at 1,500 × g for 10 min at 4°C. The fraction of lowdensity lipoprotein (LDL) plus very low-density lipoprotein (VLDL) was isolated from the fresh plasma by the precipitation method with dextran sulfate-Mg 2+ as follows. 16'23 An aliquot (2.0 ml) of plasma was mixed with 100/zl of a magnesium chloride solution (2.0 M) and 100/zl of a dextran sulfate solution (20 mg/ml), allowed to stand for 20 min in an ice bath. The mixture was centrifuged at 1,000 × g for 20 rain at 4°C. The supernatant was removed and the precipitate (LDL + V L D L fraction) was suspended in 2 ml of phosphatebuffered saline (10 m M phosphate-125 mM sodium chloride, pH 7.4) (PBS) containing 0.5 m M diethylenetriaminepentaacetic acid (DTPA) with a vortex mixer for 1 min followed by ultrasonic irradiation for 1 min. The protein content of the L D L + V L D L suspension was determined by the method of Bradford. 24

Analysis of cholesteryl ester hydroperoxides in human plasma Extraction procedure from human plasma was followed by the method of Yamamoto et al. 14 with slight modification. An aliquot of plasma (4.0 ml) was mixed with 4.16 nmol of C h N - O O H in 40/~1 of chloroform/ methanol (1:1, v/v) and 36 ml of methanol containing 2.5 m M 3,5-di-tert-butyl-4-hydroxytoluene (BHT). C h N - O O H was used as an internal standard. The mixture was sonicated for 1 min, and then 40 ml of nhexane was added and mixed vigorously for 1 min. After centrifugation at 1500 x g for 5 min at 4°C, the upper layer was transferred to another test tube. The extraction was carried out again by the addition of 40 ml of n-hexane to the lower layer by the same procedure. The n-hexane fractions were combined, and the solvent was removed using rotary evaporator at room temperature. The reduced pressure was restored to atmospheric pressure by the purge of nitrogen gas. The residue was immediately dissolved in 100 #1 of chloroform/methanol (1:1, v/v), and a 40 #1 portion was subject to the HPLC analysis for ChE-OOH.

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and 2 m M in the fraction of LDL + VLDL, respectively. The final concentration of DTPA was 0.5 mM in the two reaction mixtures. Both reaction mixtures were incubated with continuous shaking at 37°C in the dark. At regular intervals, the aliquots (100/zl) were withdrawn and mixed with 2.7 ml of methanol containing 2.5 mM BHT, 200 #1 of PBS containing 0.5 m M DTPA, and 3.12 nmol of ChN-OOH in 30 #1 of chloroform/methanol (1:1, v/v). The mixture was sonicated for 1 min, and then 3.0 ml of n-hexane was added and mixed vigorously for 1 rain. ChE-OOH fraction was extracted from the mixture twice with 3.0 ml of n-hexane in the same manner described in the procedure of the analysis of ChE-OOH in human plasma. The extract was dissolved in 40/zl of chloroform/methanol (1:1, v/v), and a 20 #1 portion was injected into the HPLC system. RESULTS

HPLC analysis of cholesteryl linoleate hydroperoxides Figure 1 shows the typical chromatograrns of ChLOOH standard monitored by the spectrophotometric detector (235 nm) (A) and the coulometric detector (B). In both chromatograms, ChL-OOH appeared as a prominent

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Free radical oxidation of human plasma and isolated lipoprotein fractions Peroxyl radical-mediated lipid peroxidation of fresh human plasma and its components, L D L + VLDL, were induced by A A P H as follows. To an aliquot (900 #l) of fresh human plasma, 100/zl of 200 m M AAPH in PBS containing 5 m M DTPA was added. To an aliquot of LDL + V L D L suspension (2.0 mg protein/ 900/zl), 100/~1 of 20 mM A A P H in PBS containing 0.5 mM DTPA was added. The final concentrations of A A P H in the reaction mixtures were 20 mM in plasma

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Fig. 1. Chromatograms of cholesteryl linoleate hydroperoxides (ChL-OOH) obtained by spectrophotometricdetection (A) and coulometfic detection (B) with dual electrode system. The amount of ChL-OOH applied on the column was 28.5 pmol. The wavelength of spectrophotometric detector was adjusted at 235 nm. Applied potential of first and second electrodes in dual electrode detection system were -10 mV and -50 mV vs. Ag/AgCI, respectively.The electrochemical responses were monitored by the second electrode.

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peak at a retention time of ca. 10 rain. On the other hand, the cholesteryl linoleate hydroxides (ChLIOH) prepared by the reduction of ChL-OOH with sodium borohydridetreatment gave no peak on the chromatogram by the coulometric detector, although the spectrophotometfic detection yielded a prominent peak at a retention time almost the same as that of ChL-OOH (data not shown). A linear relationship was observed between the amounts of ChL-OOH and the peak areas in the chromatograms obtained by the coulometric detection in the range from 4 to 142 pmol (correlation coefficient = 0.999) (Fig. 2). Its linearity was observed up to 635 pmol of ChL-OOH (data not shown). The lowest detectable level of ChLOOH was estimated to be 2 pmol for the coulometric detection and 14 pmol for the amperometric detection at a signal-to-noise ratio of 3. The electrode potential was set at - 5 0 mV vs. Ag/AgCl for the coulometric detection and - 100 mV vs. Hg/Hg2C12 for the amperometric detection, respectively, because higher applied voltages at reduction mode in both detection systems yielded the unstable electric currents in the HPLC analysis. The noise level in the second electrode of the coulometric detector was decreased by the application of the first electrode potential ( - 1 0 mV vs. Ag/AgC1), as shown in Fig. 3. The residual currents of the first (channel 1) and the second electrodes (channel 2) of the coulometric detector in the equilibrium state of the HPLC analysis were - 5 0 nA and - 1 4 7 nA, respectively.

Application of coulometry to human plasma ChE-OOH The calibration curve of ChL-OOH was used to determine the amount of ChE-OOH in human plasma because

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Fig. 3. Chromatogramsof ChL-OOHobtained by coulometricdetection with dual electrode system (A) and single electrode system (B). The analytical conditions in dual electrode detection system were the same as those described in the legend in Fig. 1. Applied potential of first and second electrodes in single electrode detection system were -50 mV and 0 mV vs. Ag/AgCI,respectively. The electrochemical responses in single electrode system were monitored by the first electrode. The amount of ChL-OOHapplied on the column was 28.5 pmol.

cholesteryl linoleate is the principal cholesteryl ester species in plasma lipidsY Figure 4 shows the typical chromatograms of human plasma lipids (top trace) and ChNOOH standard (bottom trace) obtained by the coulometric detection. ChN-OOH, the internal standard, appeared at the retention time of 19 min in both chromatograms. However, the peak corresponding to ChL-OOH was not found in the chromatogram of human plasma lipids from the volunteers. The lowest detectable level of ChL-OOH in the chromatogram of human plasma lipids was estimated to be 14 pmol because unidentified minor peaks at 5 - 1 5 min interfered the detection of lower level of ChL-OOH. Furthermore, the recovery of ChN-OOH from the plasma was found to be 13 ___ 2% (n = 3) by this procedure. Thus, the content of ChE-OOH in human plasma was estimated to be less than 27 nM (= 14 pmol/ 13%/100/4 ml plasma) from the lowest detectable level of ChE-OOH and the recovery of ChN-OOH. Figure 5 shows the relationship between the concentration of ChN-OOH spiked to human plasma and its recovery from human plasma. The recoveries of ChN-OOH increased with the concentration of ChN-OOH spiked to human plasma, and the recovery was reached to 99% at the concentration of ChN-OOH of 13.5 nmol/ml plasma. Figure 6 shows the typical chromatograms of the extracts from human plasma (A) and LDL + VLDL fraction (B)

Coulometry of lipid hydroperoxides

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after the addition of 20 mM and 2 mM AAPH, respectively. The prominent peaks detected at the retention time of 10 min and 19 min in the chromatograms obtained by the coulometric detection were identified as ChEOOH and ChN-OOH (internal standard), respectively, from the correspondence of retention times to those of the standards. On the other hand, ChE-OOH could not be determined exactly by the spectrophotometric detection because the large peaks interfered the peak of ChE-OOH in the chromatograms. The concentration of ChE-OOH in each reaction mixture was calculated by the internal standard method based on the correlation of peak area ratios of ChL-OOH to ChN-OOH with molar ratios of ChL-OOH to ChN-OOH (correlation coefficient = 0.998). ChE-OOH accumulated after a lag period of 30 min and 90 min in the AAPH-induced oxidation of the human plasma and the fraction of LDL + VLDL, respectively, as shown in Fig. 7.

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lometric detector. In theory, the electrolytic efficiency of coulometric detector is near to 100%, whereas that of amperometric detector is less than 10%. Our results indicate that the coulometric detection with porous carbon electrode is superior to the amperometric detection with glassy carbon electrode in terms of the sensitivity for ChL-OOH. In our previous reports, higher voltage ( - 3 0 0 mV) was required for the working electrode to obtain the high sensitivity in the amperometric detection for the LOOH. 3-5 It needed more than 3 h for the pretreatment with the eluting solvent to stabilize the base line. However, the coulometric detection technique does not require the high electrode potential to detect the picomole level of ChE-OOH, resulting in saving the analytical time. The interference by the noises in the coulometric detection was at least partly resolved by using dual electrode system. Yamamoto et al. ~° and Akasaka et al. ~2 devised HPLC for the determination of ChE-OOH using fluorescence or chemiluminescence detection system, respectively. However, these techniques are not direct measurements of hydroperoxyl group. In addition, they require special devices for the postcolumn reaction. We previously showed that the sensitivity of HPLC-amperometric electrochemical detection with glassy carbon electrode was relatively low in the determination of PC-OOH (--200 pmol) 3 and fatty acid hydroperoxides ( ~ 2 0 0 pmol). 4 However, HPLC-electrochemical detection is a direct and simple method for the detection of hydroperoxyl group. Here, we succeeded in developing highly sensitive technique for ChE-OOH using HPLC-electrochemical detection systems. In particular, the sensitivity obtained by coulometric detection system ( ~ 2 pmol) seems to be comparable to that 120.0 A

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DISCUSSION

There are two types of electrochemical detector in HPLC system, that is, amperometric detector and cou-

Fig. 5. The relationship between the concentration of internal standard (ChN-OOH) in human plasma and the recoveries of ChN-OOH from human plasma.

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that typical lipid extraction method using chloroform and methanol 27 was hardly applicable to coulometric detection of ChE-OOH because large interfering peaks appeared in the chromatogram (data not shown here). Interestingly, the recovery of ChN-OOH from plasma was influenced by the concentration of ChN-OOH spiked to plasma (Fig. 5). The disappearance of ChNOOH added to the plasma at low concentration may be derived from its decomposition or reduction during the extraction. This result implies that the extraction efficiency of endogenous ChE-OOH in plasma is altered by its concentration. A method using an internal standard that possesses hydroperoxyl group such as ChN-OOH may be necessary for the determination of ChE-OOH in human plasma. This technique is instructive for the specific detection of ChE-OOH in the measurement of oxidizability of plasma and plasma lipoprotein fraction (Fig. 7).

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Time (min) Fig. 6. Chromatograms of the extracts (ChE-OOH) of human plasma (A) and low-density lipoprotein (LDL) plus very low-density lipoprotein (VLDL) fraction (B) after peroxidation induced by 2,2'azobis(2-amidinopropane) dihydrochloride (AAPH) for 180 min and 150 min, respectively. Top and bottom trace in each panel represents coulometric detection and spectrophotometric detection at 235 nm, respectively. The reaction system in the oxidation of human plasma consisted of 20 mM AAPH and 0.5 mM diethylenetriaminepentaacetic acid (DTPA) in plasma/phosphate buffered saline (PBS, pH 7.4) (9:1, v/v). The reaction system in the oxidation of human LDL + VLDL fraction consisted of human LDL + VLDL fraction (2.0 mg protein), 2 mM AAPH, and 0.5 mM DTPA in PBS. ChN-OOH were used as the internal standard.

of fluorescence or chemiluminescence detection. The amount of ChE-OOH is expected to be suitable index of lipid peroxidation in human plasma because ChEOOH are most stable among the LOOH produced in human plasma. 7 Therefore, we applied this HPLC-coulometric electrochemical detection method to the determination of ChE-OOH in human plasma. Although we could not detect ChE-OOH in human plasma by this technique, we estimated that the concentration of ChEOOH in human plasma is less than 27 nM. This level of ChE-OOH in human plasma is not different from that determined by HPLC-chemiluminescence (3.4 nM, j4 4.2 nM 26) or HPLC-fluorescence (27.5 nM) ~ detection. In our study, ChN-OOH were used as the internal standard for the determination of ChE-OOH because of the structural similarity with ChL-OOH. We adopted hexane-methanol method as the extraction procedure for ChE-OOH from plasma. It was found

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Coulometry of lipid hydroperoxides In c o n c l u s i o n , c o u l o m e t r i c e l e c t r o c h e m i c a l d e t e c tion m e t h o d is s i m p l e and c o n v e n i e n t f o r the a n a l y s i s o f L O O H in b i o l o g i c a l s a m p l e s .

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Acknowledgements - - We thank T. Nishii for technical assistance

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REFERENCES

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AAPH-- 2,2' -Azobis(2-amidinopropane) chloride BHT--

dihydro-

3,5-di-tert-butyl-4-hydroxytoluene

C h E - O O H - - c h o l e s t e r y l ester h y d r o p e r o x i d e s ChL-OOH--cholesteryl linoleate hydroperoxides ChN-OOH--cholesteryl nervonate hydroperoxides DTPA -- diethylenetriaminepentaacetic acid HPLC--high-performance liquid chromatography LOOH--lipid hydroperoxides LDL--low-density lipoprotein P B S - - p h o s p h a t e - b u f f e r e d saline PC-OOH--phosphatidylcholine hydroperoxides VLDL--very low-density lipoprotein