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Free radical-induced lipid peroxidation has been implicated in a number of human diseases including atherosclerosis, cancer, and neurodegenerative diseases. F2-Isoprostanes (IsoPs) are isomers of prostaglandin PGF2α that are generated in vivo from the free radical-initiated peroxidation of arachidonic acid independent of cyclooxygenase enzymes. Since the discovery of the IsoPs in the early 1990s, a large body of evidence has been accumulated to indicate that quantification of these F2-IsoPs represents the most reliable biomarker to assess oxidative stress in vivo. A variety of analytical approaches have been developed for the quantification of these novel compounds; these methods include mass spectrometry (MS) detection coupled to gas chromatography (GC) or liquid chromatography (LC) separation, and detection using immunological approaches. This article summarizes our current methodology to quantify F2-IsoPs in biological fluids and tissues using GC-MS. This method includes solid phase extraction (SPE), thin layer chromatography (TLC) purification, chemical derivatization, and MS detection using negative ion chemical ionization (NICI) coupled with GC. The protocol described herein has been optimized and validated to provide the best sensitivity and selectivity for quantification of F2-IsoPs from a variety of biological sources.
Free radical-induced autooxidation of polyunsaturated fatty acids (PUFAs) has been linked to numerous human disorders, including atherosclerosis, cancer, neurodegenerative diseases, and aging [1–5]. Isoprostanes (IsoPs) are a series of prostaglandin (PG)-like compounds that are formed non-enzymatically in vivo via the peroxidation of arachidonic acid by a free radical-initiated mechanism [6–8]. An important aspect of the formation of IsoPs is that they are generated in situ from arachidonic acid esterified in phospholipids and are subsequently released by phospholipases, such as platelet-activating factor (PAF) acetylhydrolases . On the other hand, the PGs are generated from the oxidation of free arachidonic acid by cyclooxygenases (COXs) . Over the past two decades, quantification of F2-IsoPs, isomers of PGF2α, has emerged as one of the most accurate approaches to assess oxidant injury in vivo [11, 12]. In fact, in the Biomarkers of Oxidative Stress Study (BOSS) sponsored by the Nation Institute of Health (NIH), F2-IsoPs were shown to be the most accurate biomarker to assess in vivo oxidant stress status when compared against other well-known biomarkers . In addition to F2-IsoPs, D2/E2-IsoPs which contain E/D prostane rings analogous to COX-derived PGD2 and PGE2, and isomers of thromboxane, termed isothromboxanes (IsoTxs), are also generated in significant amounts in vivo [14, 15]. Some IsoPs exert potent biological activity by acting as ligands for either plasma membrane bound PG receptors or for nuclear receptors and thus are likely also mediators of oxidant injury [16–18].
F2-IsoPs are stable and robust molecules that are detectable in all human tissues and biological fluids analyzed, including plasma, urine, bronchoalveolar lavage (BAL) fluid, cerebrospinal fluid (CSF), and bile. The quantification of F2-IsoPs in urine and plasma is most convenient and least invasive. Several methods have been developed to analyze F2-IsoPs; these include chromatographic separation involving solid-phase extraction (SPE) or affinity chromatography with or without thin layer chromatography (TLC) followed by final determination by gas chromatography - mass spectrometry (GC–MS), liquid chromatography (LC)-MS, or enzyme immunoassay[19–24]. GC - negative ion chemical ionization mass spectrometric (GC/NICI-MS) employing stable isotope dilution is the preferred method for the quantification of F2-IsoPs and several alternative GC-MS assays have been developed by different investigators including FitzGerald and colleagues . For quantification purpose, we measure 15-F2t-IsoP (also known as 8-Iso PGF2α), and other F2-IsoPs that co-elute with this compound. The advantages of GC-MS include the high resolution of GC separation on fused silica capillary columns and the specificity and sensitivity of MS, which yield quantitative results in the low picogram range [26, 27].
A substantial body of evidence indicates that measurement of F2-IsoPs in body fluids provides a reliable approach to assess lipid peroxidation in vivo. Based on available data, quantification of these compounds in either plasma or urine is representative of their endogenous production and thus gives a highly precise and accurate index of in vivo oxidant stress [24, 28, 29]. Normal levels of F2-IsoPs in healthy humans have been defined [28–30]. Defining these levels is particularly important in that it allows for an assessment of the effects of diseases on endogenous oxidant tone and allows for the determination of the extent to which various therapeutic interventions affect levels of oxidant stress. Elevations of IsoPs in human body fluids and tissues have been found in diverse array of human disorders, including atherosclerosis, diabetes, obesity, cigarette smoking, neurodegenerative diseases, and many others . Further, treatments for some of these conditions, including antioxidant supplementation, antidiabetic treatments, cessation of smoking, and even weight loss, have been shown to decrease production of F2-IsoPs .
As has been mentioned, F2-IsoPs are formed non-enzymatically as a result of the free radical-mediated peroxidation of arachidonic acid. The mechanism outlined in Fig. 1 is based on the generation of bicyclic endoperoxide intermediates . Arachidonic acid initially undergoes hydrogen atom abstraction by a free radical at the three bisallylic positions of C7, C10, or C13 to yield a delocalized pentadienyl carbon-centered radical. Subsequently, there is an insertion of molecular oxygen to yield peroxyl radicals. These peroxyl radicals undergo further 5-exo cyclization, followed by the addition of another molecule of oxygen to yield the bicyclic endoperoxide (PGG2-like) . These intermediates are then reduced to form F2-IsoPs. Based on this mechanism, four F2-IsoP regioisomers are generated. These are named the 5-, 8-, 12-, and 15-series F2-IsoPs, based on the location of the side chain hydroxyl group in the final product [35, 36]. Each series contains sixteen possible diastereomers and a total of sixty four stereoisomers of F2-IsoPs can be generated.
The F2-IsoPs are initially formed esterified to phospholipids after the reduction of the bicyclic endoperoxides and the GC-MS can only analyze the derivatives of free F2-IsoPs. A derivatization protocol has been developed to make the samples suitable for GC-MS analysis (Fig. 2). Both esterified and free F2-IsoPs can be measured. To analyze the esterified F2-IsoPs in tissue samples, the F2-IsoPs are first released under basic condition. After addition of deuterated internal standard, solid phase extraction is carried out using both C18 and silica Sep-Pak to remove the contaminants. Pentafluorobenzyl (PFB) moiety is then introduced to the molecule to enhance the sensitivity of detection using electron capture chemical ionization technique . The last step is to cap the hydroxyl groups by trimethylsilyl (TMS) derivatization. Analysis of the derivatives of F2-IsoPs and the internal standard is carried out using selective ion monitoring (SIM) techniques; the ions monitored are m/z 569 for F2-IsoPs and m/z 573 for the internal standard respectively. The detailed procedures for the purification and derivatization steps will be described in the Protocol section.
Arachidonic acid in biological samples is susceptible to free radical oxidation and thus prevention of the ex vivo oxidation is crucial in the processing and storage of the samples especially plasma and tissue samples that contain abundant arachidonic acid. For the best results, samples should be flash frozen in liquid nitrogen immediately upon collection and not thawed until analysis. BHT and PPh3 are added to the Folch solution to prevent ex vivo oxidation during the extraction of lipids.
Levels of F2-IsoPs in a variety of biological sources can be measured as an accurate index of in vivo oxidant stress status. Although free or esterified F2-IsoPs (bound) can be quantified in plasma, we found no advantage in measuring total (free + bound) rather than free F2-IsoPs. Thus the present method measures only free F2-IsoPs. For urine sample, 24-h urine collection represents a reliable probe for the oxidant stress and only free F2-IsoPs is measured. For the tissue samples, the majority of the F2-IsoPs are esterified on phospholipids and basic hydrolysis is necessary to release these compounds after extraction of the total lipids from the tissue homogenates.
2–3 ml of plasma is added in 7 ml of pH 3 water or 0.25 ml of urine is added in 10 ml of pH 3 water and acidified to pH 3 with 1N HCl.
A representative chromatogram obtained from the analysis of F2-IsoPs in mouse liver is shown in Figure 3. For quantification purposes, we compare the height of the peak containing the derivatized 15-F2t-IsoP (m/z 569, peak labeled with * in Fig. 3) with the height of the deuterated internal standard peak (m/z 573). Levels of F2-IsoPs in plasma are reported in picograms or nanograms per milliliter, whereas levels in tissues are reported in nanograms per gram of tissue. Levels of F2-IsoPs in urine are normalized to creatinine clearance and reported as nanograms per milligrams of creatinine. It should be noted that the peak denoted with “+” in Figure 3 may also contain the COX product PGF2α when free F2-IsoPs are analyzed in biological fluids, such as plasma, CSF or urine. However, caution should be exercised regarding the enzymatic origin of this peak; our studies show that majority of this peak in human urine is actually derived from the free radical pathway . Furthermore, this peak is also present when the esterified F2-IsoPs are measured and it can only be derived from the free radical pathway in this case.
GC-MS method offers excellent sensitivity and selectivity for F2-IsoPs. However, the labor intensive purification and derivatization steps that are necessary for the GC-MS analysis make it difficult to improve the throughput of the assay. Secondly, experienced and trained personnel are required to set up and operate the rather expensive instrument. In addition the GC-MS method, a number of LC-MS methods for F2-IsoPs analysis have been developed [39–41]. As has been mentioned, F2-IsoPs consist a mixture of four series of regioisomers and each has sixteen diastereomers. These different stereoisomers may be formed and metabolized differently; thus the levels of these individual stereoisomers can be altered under various pathophysiological conditions [38, 40]. GC-MS methods quantify all possible F2-IsoPs stereoisomers while LC-MS methods permit to separate and identify selected regioisomers and diastereomers of F2-IsoPs. The other advantage of LC-MS methods is that the sample preparation for analysis is simpler than that for GC-MS because no derivatization of the molecule is required. In addition to analyze the free F2-IsoPs, we recently also developed LC-MS method to analyze the intact esterified F2-IsoPs on different phospholipid head groups . However, a concern with these LC-MS assays relates to the limits of detection in biological fluids that are often higher than those employing GC-MS .
Enzyme immunoassay has also been developed to quantify IsoPs using antibodies which generated against some selected isomers of F2-IsoPs, such as 8-iso-15(R)-PGF2α . Although mass spectrometric methods of F2-IsoP quantification are considered the best methods for analysis, immunoassays have expanded research in this area due to their low cost and relative ease of use [28, 29]. However, multistep sample purification is still necessary, and questions remain as to the specificity of the antibody. Studies comparing GC-MS and enzyme immunoassay assays of the same samples have demonstrated that the values from the assays are correlated but not identical [44, 45].
The measurement of free F2-IsoPs in urine can be confounded by the potential contribution of local F2-IsoP production in the kidney, although the extent to which this occurs is unclear. In light of this issue, we have identified the primary urinary metabolite of 15-F2t-IsoP to be 2,3-dinor-5,6-dihydro-15-F2t-IsoP, and we have developed a highly sensitive and accurate mass spectrometric assay to quantify this molecule . Thus, the quantification of 2, 3-dinor-5,6-dihydro-15-F2t-IsoP might represent a truly noninvasive, time-integrated measurement of systemic oxidation status that can be applied to living subjects.
This work is supported by NIH grants DK48831, GM15431, and ES31125. This manuscript is dedicated to the memory of Dr. Jason Morrow.
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