A Specific, Accurate and Sensitive Measure of Total Plasma Malondialdehyde by HPLC. Hamdy F Moselhy1, Raymond G Reid2, Saeed Yousef1 and Susanne P Boyle2** 1
Faculty of Medicine and Health Sciences, University of the United Arab Emirates.
2
School of Pharmacy and Life Sciences, Robert Gordon University, Schoolhill, Aberdeen, AB10 1FR.
**
Corresponding author: Dr Susanne P Boyle, email:
[email protected], Tel. + 44 1224 262529, Fax. +44 1224 262555.
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Abbreviations DAD
Diode Array Detection
LC-DAD-MS
Liquid chromatography- diode array detector-mass spectrometry
LOD
Limit of Detection
LOQ
Limit of Quantitation
MDA
Malondialdehyde
MS
Mass Spectrometer
%RSD
% Relative Standard Deviation
Sd
Standard deviation
SIM
Selected Ion Monitoring
TBA
2 Thiobarbituric acid
TBARS
Thiobarbituric Acid Reactive Substances
TEP
Tetraethoxypropane
1
Abstract
Keywords: Plasma Malondialdehyde, TBARS, assay specificity, assay bias, lipid peroxidation.
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Malondialdehyde is one of the most commonly reported biomarkers of lipid peroxidation in clinical studies. The reaction of thiobarbituric acid with Malondialdehyde to yield a pink chromogen attributable to an MDA-TBA2 adduct is a common assay approach with products being quantified by UV-Vis assay as non specific thiobarbituric acid reactive substances (TBARS) or chromatographically as Malondialdehyde. The specificity of the TBARS assay was compared to both chromatographic assays for total plasma MDA. The levels of total plasma MDA were significantly lower than the plasma TBARS in each of the samples examined and interestingly the inter-individual variation apparent in the level of plasma MDA was not evident in the plasma TBARS assay. Each of the 4 online chromatographic detectors yielded a precise, sensitive and accurate determination of total plasma MDA and selected ion monitoring was the most accurate assay (101.3%, n=4). The online diode array detectors provided good assay specificity (peak purity index of 999), sensitivity, precision and accuracy. This research demonstrates the inaccuracy that is inherent in plasma TBARS assays which claim to quantify Malondialdehyde and it is proposed that the TBARS approach may limit the likelihood of detecting true differences in the level of lipid peroxidation in clinical studies.
Introduction Lipid peroxidation is one of the most commonly reported indices of oxidative stress and has been implicated as a contributing factor in a range of degenerative diseases including diabetes 1, cardiovascular disease, Parkinson’s disease, Alzheimer’s disease2 and other psychiatric disorders including Schizophrenia3. The process of lipid peroxidation results in a range of intermediates and end products including lipid hydroperoxides and aldyehydes including malondialdehyde. These aldehydes and lipid hydroperoxides form DNA adducts with may result in extensive single strand and double strand breaks4. Various intermediates and end products generated during the lipid peroxidation cascade have been assayed but the most commonly employed approach continues to be the thiobarbituric acid (TBA) test5.
Despite the widely acknowledged limitations of the TBARS test14 and in particular its lack of specificity it continues to be reported as a true measure of MDA in clinical disease 1,15,16. It is proposed that the poor assay specificity associated with TBARS assays may lead to an overestimation of the levels of MDA in human plasma and other biological tissues and fluids and this in turn may limit the likelihood of detecting true differences in the level of lipid peroxidation in clinical studies. This research investigated the absolute levels of plasma MDA employing two chromatographic assays (HPLC-DAD-Fluoro and LC-MS-DAD) and compared this with the level of TBARS determined by UV spectrophotometry. Within physiological systems malondialdehyde may exist in a free state or protein bound form and various studies have looked at the relative proportions of malondialdehyde in human plasma and report that between 83% - 92% of the MDA is protein bound17,18. Hydrolysis of the protein bound MDA fraction may be achieved by either an alkaline18,19 or acid treatment20 of the sample prior to the acid catalysed TBA reaction. An approximately two fold increase in plasma MDA levels determined by HPLC-UV was observed following alkaline hydrolysis of human plasma by Hong et al., (2000)18 and it was concluded that this additional sample pre-treatment was a useful step which enhanced assay reliability ensuring a more complete and uniform release of the MDA. In light of this and similar studies21 which report good assay precision when measuring total plasma MDA a similar alkaline hydrolysis method was employed herein. 3
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Whilst it is widely acknowledged that TBA reacts with a range of oxidised lipids, both saturated and unsaturated aldehydes6,7 sucrose and urea8 to form various chromogens referred to as TBA reactive substances (TBARS) it is the reaction of TBA with malondialdehyde to produce a pink pigment9 with an absorption maximum at 532 nm10 and mass ion at 323 amu11 which is a true indicator of lipid peroxidation. Historically the TBARS test has been assayed by UV spectrophotometry or fluorescence assays but the specificity of the TBA test, enabling the selective determination of malondialdehyde, may be achieved via chromatographic separation of the pink TBA2-MDA adduct12. The adoption of HPLC techniques improves both assay specificity and sensitivity of MDA determination and we have previously confirmed the validity of quantifying the TBA2-MDA adduct as a specific measure of lipid peroxidation in human biological fluids13.
Methods Materials Reagent grade sodium hydroxide, sulphuric acid, trichloroacetic acid and HPLC grade solvents were purchased from Fisher Scientific. 1,1,3,3 tetraethoxpropane and 2-thiobarbituric acid were purchased from Sigma Chemical Co. Acetic acid (99.5% pure) was purchased from Arcos Organics. HPLC grade water (H20) was used throughout. Ethical Approval
Preparation of MDA Calibration Line and Human Plasma Assay for Total MDA 1,1,3,3 tetraethoxypropane (TEP) was used as the MDA standard and under conditions of acid and heat condensed with 2-thiobarbituric acid to form a stable adduct (TBA2-MDA). An alkaline hydrolysis step was then employed21 enabling a measure of total plasma MDA to be determined by HPLC or LC-MS. Assays were performed in acid washed pyrex tubes to which was added 100 μl human plasma (or TEP std or H2O blank) and 25 μl of 3 M NaOH. The tubes were capped, vortexed and placed in a 60oC water bath for 30 minutes. Post alkaline hydrolysis 1 ml of 0.05 M sulphuric acid and 0.5 ml of 20% w/v TCA were added, the tubes vortexed and centrifuged (3000 rpm x 10 mins) and 1 ml of supernatant layer removed to clean tubes to which was added 0.5 ml of 0.355% w/v TBA. The tubes were vortexed and then heated to 90 oC for 40 minutes. Upon cooling the tubes were centrifuged at 3000 rpm for 10 minutes and an aliquot of the aqueous phase was analysed chromatographically for MDA or by UV spectrophotometry for determination of TBARS10. UV Spectrophotometric Determination of TBARS TBARS determinations were performed using a Hewlett Packard Diode Array spectrophotometer with spectral scans being collected and absorbances being measured at 532 nm10. HPLC and LC-MS Analysis HPLC separation was performed at room temperature using a Shimadzu Prominence liquid chromatography system (LC-20AD) with degasser, a SPD-M20A diode array detector (DAD) and a Shimadzu RF-10 AXL fluorescence detector. Analysis was achieved using a 5μm Hyperclone C18 column (150 x 4.6mm) and a gradient elution programme as summarised in Table I (Supplemental files). 4
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Participant recruitment for this collaborative research study was managed by clinicians in the University of the United Arab Emirates and the study protocol was approved by the Research Ethics Committee, Faculty of Medicine and Health Sciences, United Arab Emirates University. The study was conducted in accordance with Good Clinical Practice and all participants gave their informed consent prior to sampling. Blood was collected (10 ml) from each participant by venepuncture into EDTA vacutainers. The blood was centrifuged at 3500 g for 15 minutes, the plasma fractionated, aliquoted into cryovials and stored at -80oC until required for analysis. Pooled plasma samples were used for assay validation and then two individual volunteer samples used to test the application of the validated assay.
A constant flow rate of 1 ml/min was employed and the eluent monitored at 532 nm10 by DAD with spectral scans being collected over the range of 500 to 580 nm. The fluorescence detector used an excitation wavelength of 515nm and an emission wavelength of 553nm8. An Agilent Technologies 1200 series LC coupled to an Agilent 6130 Quadrupole LC-MS was utilised with a 5 μm Hyperclone C18 column (150 x 4.6mm) analytical column maintained at 40oC, a flow rate of 1 ml/minute and the gradient conditions summarised in Table I (Supplemental files). The MS detector was set to measure in the negative ion mode and scans were collected over a mass range of 200-450amu with single ion monitoring (SIM) at 323amu. All other settings for the MS were standard for the instrument. For chromatographic analyses, quantification was primarily by external standard calibration using TEP as standard. Daily calibration lines were prepared across the range of 0 - 24.3 μM MDA and regression lines of MDA concentration versus detector response (peak area) constructed. Determination of total TBARS used the assay conditions described above with calibration curves of TEP concentration versus UV absorption at 532 nm being constructed. Validation of the HPLC Assay
Statistical Analyses Student’s t tests were carried out to test for statistically significant differences between the various chromatographic detectors.
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Assay linearity and precision were determined in accordance with the ICH Guideline for Validation of Analytical Procedures (1994)22. Peak purity was evaluated by diode array detection22 and sensitivity of the chromatographic assays was determined in terms of Limits of Detection (LOD) and Limits of Quantitation (LOQ) derived from residuals method of Sanagi et al., (2009)23 using the standard deviation (sd) of the response and the slope. Assay accuracy was determined by a process of spiking of human plasma (n=6 replicates) and evaluated by HPLC and LC-DAD-MS analysis.
Results Validation of the TBARS and MDA Assays TBARS analysis of the TEP standards produced a pink chromogen which had a maximal absorption at 532 nm. The linearity of the TBARs assay was demonstrated over the range of 0-30 µM and the precision of assay response was acceptable with a mean slope of 0.00457 (% RSD = 6.79, n=3). A similar pink coloured pigment was observed following TBARS analysis of the pooled plasma but in this matrix the UV Vis spectral analysis showed a maximum absorption at 532 nm and a further absorption band at 490 nm (see Supplemental Figure I).
The linearity of MDA determination across the range of 0 - 24.3 μM was demonstrated on all 4 online chromatographic detectors as illustrated in Supplemental Table II and the precision of assay response was good (%RSD ≤ 8%, n=4) on each of the 4 detectors employed. Replicate analysis (n=8) of a pooled plasma sample demonstrated detector equivalence in terms of determination of total plasma MDA, see Table 1. The assay proved to be quite reproducible with % RSD typically 999) was demonstrated following analysis of both the calibration standard and the human plasma samples by HPLC-DAD indicating assay specificity and accuracy was demonstrable by this approach. The validated assay was then applied to individual human plasmas to further evaluate assay performance in terms of precision, specificity and general fitness for purpose. Replicates (n=6) of 6
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Analysis of the MDA standard by HPLC-DAD- Fluorescence and LC-MS-DAD yielded a single HPLC peak with a mean retention time in the range of 9.46-9.67 minutes and was highly reproducible (%RSD< 0.25, n=5) for each of the 4 online detectors. Spectral scans indicated a λmax of 532 nm and the predominant mass ion was at 323 amu; characteristics indicative of the MDA-TBA2 adduct, (Figure 1A). Similarly, replicate assay of the plasma samples and analysis by HPLC-DAD- Fluoro and LC-MS-DAD yielded a single HPLC peak with a mean retention time in the range of 9.49-9.70 minutes and was highly reproducible (%RSD< 0.03, n=10) for each of the 4 online detectors. Spectral scans indicated a λmax of 532 nm and the predominant mass ion was at 323 amu; characteristics indicative of the MDA-TBA2 adduct, (Figure 1B).
each plasma sample were processed through the total MDA assay and analysed by HPLC-DADfluorescence and LC-MS-DAD. The precision of assay response was good (≤ 8%, n=4) on each of the 4 detectors employed, the mean plasma MDA concentration was 4.50 and 5.98 µm in subjects 1 and 2 respectively (see Figure 2).
Comparison of TBARS and MDA Levels in Human Plasma The validated assays were then employed to undertake a comparison of the levels of TBARS and plasma MDA using test plasmas. The test plasmas included two pools of human plasma (pool 1 and pool 2) plus two further participant plasmas. Quantification of “MDA” at 532 nm indicated the mean concentration of TBARS in the pooled plasma ranged from 16.94 µM (n=6 assays) through to 24.80 µM in pool 2 (n=6 assays). The plasma TBARS level in subjects 1 and 2 was remarkably similar with a mean of 18.42 and 18.92 µM respectively as illustrated in Figure 3a.
A positive correlation between the plasma MDA and TBARS concentrations was observed r=0.709, (data not shown) n=24 plasma assays.
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The same set of plasma samples were simultaneously analysed by HPLC-DAD-Fluorescence and the level of plasma MDA quantified. The levels of plasma MDA were significantly lower than the plasma TBARS in all 4 of the test samples. Plasma MDA levels ranged from 13.79 µM in plasma pool 2 through to 5.78 µM in subject 1. Critically there was a 30% difference in the level of plasma MDA measured in subjects 1 and 2 (see Figure 3b) whereas previously an almost identical level of plamsa TBARS had been determined (see Figure 3a).
Discussion It has been reported that acid precipitation of lipoprotein fractions reduces soluble TBARS components and enables a specific determination of lipid peroxidation without the need for HPLC 24 however it is generally acknowledged that the specificity of MDA assay as a true indicator of lipid peroxidation in biological matrices13 is more consistently achieved when a chromatographic assay is employed12. This research investigated the absolute levels of plasma MDA employing two chromatographic assays (HPLC-DAD-Fluoro and LC-MS-DAD), compared this with the level of TBARS determined by UV spectrophotometry and systematically examined the equivalence and sensitivity of each of the LC detectors employed.
This observation concurs with that reported by Hong et al., (2000)18 and indicates that TBARS determination by UV spectrophotometry leads to an over estimation of the levels of plasma “MDA” and critically in this study the significant inter-individual differences observed in plasma MDA levels were not apparent in plasma TBARS determinations. This may be due to the poor assay selectivity of the TBARS assay leading to an assay bias. The source of the bias was evident from the UV-Vis spectrum analysis which illustrated two absorption bands (532 and 490 nm) indicating that in a human plasma matrix the TBARS method lacks specificity for MDA determination and essentially a chromatographic separation of the MDA-TBA2 adduct is required for a true measure of lipid peroxidation to be achieved. The interfering TBARs compounds in human plasma may be attributable to the reaction of TBA with dienals7 and/or the formation of 2:1 barbituric acid adducts with MDA11. Such interferences are likely to be common in many MDA assays but may be effectively resolved from the adduct of interest by application of the HPLC mobile phase conditions reported. Additionally, the current HPLC assay resulted in an improvement on previously reported assays for total MDA in terms of efficiency of sample preparation with no need for the sample extraction step of Grotto et al. (2007)21. The concentration of total plasma MDA was comparable to previously published data21. Comparable concentrations of MDA were demonstrated by all 4 online HPLC detectors and there was no evidence of signal suppression or enhancement on LC-MS suggesting that the acid precipitation and alkaline hydrolysis steps efficiently removed the phospholipid fraction. Whilst the LC-MS-DAD method with single ion monitoring offers a very specific and accurate measure (mean 101.3%, n=4) of the TBA2-MDA adduct it is recognised that the costs of LC-MS equipment may be prohibitive for some researchers and importantly the HPLC-DAD or HPLC-Fluorescence methods offer a rapid, selective determination of total plasma MDA which showed acceptable sensitivity given that the mean plasma MDA concentration was typically in the range of 5.78 through to 13.79 µM (see Figure 3b) and a satisfactory assay accuracy of 87.6% (n=4 replicates). 8
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A positive correlation between the plasma MDA and TBARS concentration was observed r=0.709, (data not shown) n=24 plasma assays. A significant difference (up to 3-fold ) in the concentration of total Malondialdehyde was noted when determined by HPLC or measured as TBA reactive substances in human plasma.
In summary, this research demonstrates conclusively the inaccuracy that is inherent in TBARS assays which claim to quantify Malondialdehyde in biological tissues and fluids and it is proposed that the continued reporting of such ambiguous data may limit the likelihood of detecting true differences in the level of lipid peroxidation in clinical studies.
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Interestingly, a similar observation of bias in the TBARS method has been reported by Tug et al., (2008)25 in a study of patients with chronic obstructive pulmonary disease in which they reported a 5-fold increase in levels of free MDA determined by TBARS compared to that determined by HPLC and similarly by Hong et al (2000)18 who reported an approximate 2-fold increase in total plasma MDA in a Taiwanese population. Furthermore, the elegant study of Breusing et al., (2010) 26 which investigated inter- and intra-laboratory performance of a range of lipid peroxidation assays in human plasma included a study of 8 laboratories which measured MDA using a range of derivatisation methods. It reported within day precisions of between 1- 30% MDA when analysed by HPLC assay and in that regard the current method compares favourably with a within day precision of ≤ 8% (n=4) being demonstrated. Whilst the study of Breusing et al., (2010)26 did not examine the performance of a spectrophotometric TBARs assay it did report on a spectrophotometric assay employing the 1-methyl-2-phenylindole method of Gerard-Monnier et al., (1998)27. This assay recorded the highest MDA plasma level and was approximately 2-fold greater than the median MDA level determined by HPLC suggesting once more an issue of assay bias when colorimetric assays are employed for plasma or serum MDA quantitation. Consequently a number of research groups18,25,27 have recommended HPLC as the preferred MDA assay for human plasma samples but notwithstanding these previous recommendations the field continues to support the publication of research articles reporting TBARS values as a specific measure of Malondialdehyde levels /lipid peroxidation 12,28,29,30.
Acknowledgments Elements of this research are related to an ongoing study in First Episode Psychosis which is partially funded by a grant from The Cunningham Trust, St Andrews, Scotland, UK.
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Figures and Tables
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Table 1: Determination of Total Plasma MDA Detector
Mean Plasma MDA (μM) (n=8)
HPLC-DAD
6.03
HPLC-FLUORO
5.54
LC-DAD
6.56
LC-MS-SIM
5.78
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Figure 1A: HPLC CHROMATOGRAM AND DAD UV VIS SPECTRUM OF PEAK AT 9.52 MINUTES FOLLOWING ANALYSIS OF A CALIBRATION STANDARD
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1,1,3,3 tetraethoxypropane (TEP) was used as the MDA standard and under conditions of acid and heat condensed with 2-thiobarbituric acid to form a stable adduct (TBA2-MDA). An alkaline hydrolysis step was then employed enabling a measure of total plasma MDA to be determined by HPLC employing the chromatographic conditions described previously. The diode array detector collected scans over the range of 500 to 800 nm and a monitoring wavelength of 532 nm was employed.
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Figure 1B: LC-MS-DAD CHROMATOGRAM and NEGATIVE ION ESI SPECTRUM OF PEAK AT 9.57 MINUTES FOLLOWING ANALYSIS OF A HUMAN PLASMA
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An Agilent Technologies 1200 series LC coupled to an Agilent 6130 Quadrupole LC-MS was utilised with a 5 μm Hyperclone C18 column (150 x 4.6mm) analytical column maintained at 40 oC, a flow rate of 1 ml/minute and the gradient conditions summarised in Table 1. The MS detector was set to measure in the negative ion mode and scans were collected over a mass range of 200-450amu with single ion monitoring (SIM) at 323amu. The diode array detector collected scans over the range of 500 to 800 nm and a monitoring wavelength of 532 nm was employed.
Figure 2: Total Plasma MDA Determined by HPLC-DAD-Fluorescence and LC-MS-DAD
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Data are the mean of quadruplicate assays ± sd. Samples were analysed by HPLC-DAD(UV)–Fluorescence(Fl) and by LC-DAD(UV-2)-MS(SIM)
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Figure 3a: Plasma TBARS Level Determined by UV Spectrophotometry
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Figure 3b: Plasma MDA Level Determined by HPLC-UV-FLUORO
TBARS determinations were performed using a Hewlett Packard Diode Array spectrophotometer with spectral scans being collected and absorbances being measured at 532 nm. Samples were analysed by reverse phase HPLC-DAD(UV)–Fluorescence (Fl) and by LC-DAD(UV-2)-MS(SIM). The data are the mean of replicate assays (n=6) ± sd.
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