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Oxidative stress-induced lipid peroxidation (LPO) leads to the formation of cytotoxic and genotoxic 2-alkenals. LPO products such as 4-hydroxy-2(E)-nonenal (HNE) and 4-oxo-2(E)-nonenal (ONE) have been the subject of many studies due to their association with the development of cardiovascular and neurodegenerative diseases, as well as cancer. LPO products are excreted in the urine after conjugation with glutathione (GSH) and subsequent metabolism to mercapturic acid (MA) conjugates. Urinary LPO-MA metabolites are stable end-product metabolites and have gained interest as non-invasive in vivo biomarkers of oxidative stress. This protocol describes a method for the quantitative analysis of LPO-MA metabolites in urine using isotope-dilution liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). Included are protocols for preparation of labeled LPO-MA conjugates from unlabeled LPO products and deuterium labeled MA.
Lipid peroxidation (LPO) products are degradation products of polyunsaturated fatty acids, formed under conditions of oxidative stress. Reactive LPO products, such as 4-hydroxy-2(E)-nonenal (HNE) and 4-oxo-2(E)-nonenal (ONE), exert cytotoxic and genotoxic effects and may contribute to the development and progression of age related and chronic inflammatory diseases. HNE levels have been shown to increase with disease progression. Conjugation with glutathione (GSH), followed by further metabolism to mercapturic acid (MA) conjugates, can mitigate the deleterious effects of these LPO products by facilitating their excretion from the body. The increase of LPO products under conditions of oxidative stress suggests utility for their mercapturic acid conjugates as biomarkers of oxidative stress in vivo. This protocol describes a method for the quantitative analysis of LPO-MA metabolites in urine using isotope-dilution liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). Also included in the unit are synthetic preparations of individual LPO products, their LPO-MA conjugates, and deuterium labeled MA (MAd3) for the preparation of LPO-MAd3 internal standards (Support Protocols 1 to 5). In addition, details are also provided for the extraction of LPO-MA conjugates from urine (Basic Protocol), constructing a calibration curve (Support Protocol 6), and quantification of urinary creatinine (Support Protocol 7).
4-Hydroxy-2(E)-nonenal (HNE) is a well established lipid peroxidation (LPO) product that has been shown to contribute to the development and progression of age related diseases such as Alzheimer’s and atherosclerosis, in addition to being cytotoxic and genotoxic. HNE is formed from the linoleic acid breakdown product, 4-hydroperoxy-2(E)-nonenal (HPNE) via reduction. 4-Oxo-2(E)-nonenal (ONE) is another HPNE metabolite, formed by a loss of H2O. HNE and ONE can both undergo phase I metabolism, resulting in the formation of oxidation products: 4-hydroxy-2(E)-nonenoic acid (HNA) and 4-oxo-2(E)-nonenoic acid (ONA), and reduction products: 1,4-dihydroxy-2(E)-nonene (DHN) and 4-oxo-2(E)-nonenol (ONO; Figure 17.14.1). These LPO products are also subject to phase II metabolism, undergoing conjugation with glutathione (GSH) via Michael-type addition mediated by glutathione-S-transferase (GST; Figure 17.14.2). In the liver and kidneys, the GSH adducts are subject to enzymatic cleavage of the glutamyl and glycinyl residues, followed by N-acetylation of the remaining cysteine residue, to form N-acetyl cysteine metabolites, also referred to as mercapturic acid (MA) conjugates. The resulting LPO-MA conjugates can be quantified by isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods using deuterium-labeled internal standards.
This protocol describes the synthesis of the ONA standard for the analysis of LPO-MA conjugates. It is adapted from the method of Annangudi et al. (2005).
This protocol describes the synthesis of the HNA standard for the analysis of LPO-MA conjugates (Kuiper et al., 2008).
This protocol describes the synthesis of the ONO standard for the analysis of LPO-MA conjugates (Kuiper et al., 2008).
This protocol describes the synthesis of the MAd3 standard for the preparation of the internal standard LPO-MAd3 conjugates. It is adapted from the method of Slatter et al. (1991).
This protocol describes the preparation of a calibration curve for the quantitative analysis of the LPO-MA conjugates (Kuiper et al., 2010).
When spot urine is used for quantification of LPO product metabolites, it is recommended to correct the calculated analyte concentrations for variability in urinary output based on the level of creatinine in the urine. This protocol describes the quantification of creatinine for use in Step 21 of the Basic Protocol.
HNE-MAd3: Take 100 μl of the 100-μM solution (Support Protocol 5) and add 900 μl of 2:8 mobile phase A/mobile phase B to make a 10-μM solution. Store up to 6 months at −20°C.
ONO-MAd3: Take 100 μl of the 100-μM solution (Support Protocol 5) and add 900 μl of 2:8 mobile phase A/mobile phase B to make a 10-μM solution. Store up to 6 months at −20 °C.
DHN-MAd3: Use the 100-μM solution prepared in Support Protocol 5. Store up to 6 months at −20 °C.
Mobile phase solution A: Add 200 μl of formic acid (0.1% v/v) to 200 ml of water and mix. Make fresh daily.
Mobile phase solution B: Add 200 μl of formic acid (0.1% v/v) to 200 ml of acetonitrile and mix. Make fresh daily.
Dissolve 145 μl of H3PO4 (85 wt % in H2O) and 2.73 g NaH2PO4·2H2O in 200 ml of water and using a pH meter, adjust the pH to 3 with H3PO4 while stirring. Store up to 1 year at 4 °C.
Dissolve 0.21 g NaH2PO·2H2O and 3.32 g Na2HPO·2H2O in 200 ml of water and using a pH meter, adjust the pH to 8 with 1 M NaOH while stirring. Store up to 1 year at 4°C.
Under conditions of oxidative stress, reactive oxygen species (ROS) can react with polyunsaturated fatty acids (PUFAs), causing them to degrade and form electrophilic LPO products, such as HNE and ONE (Fig. 17.14.1). These reactive aldehydes have the ability to react with proteins, peptides, and DNA, causing covalent modifications of these biomolecules and altered cellular function (Benedetti et al., 1980; Esterbauer et al., 1991; LoPachin et al., 2009). This has led to LPO products being associated with the development and progression of age related diseases such as cardiovascular diseases (Rahman et al., 2002; Spiteller, 2007; Facchinetti et al., 2007), cancer (Barbin, 1998), and neurodegenerative diseases (Butterfield and Sultana, 2007; Picklo and Montine, 2007; Lovell and Markesbery, 2007). HNE and ONE react primarily with cysteine, histidine, and lysine via Michael-type additions (Doorn and Petersen, 2002; LoPachin et al., 2009) or in the case of lysine, Schiff base formation (Sayre et al., 1993). It has also been demonstrated that HNE can crosslink proteins by undergoing both Michael-type addition and Schiff base formation (Cohn et al., 1996). ONE has been shown to covalently modify DNA through formation of adducts with 2′-deoxyguanosine, 2′-deoxyadenosine, and 2′-deoxycytidine (Rindgen et al., 1999, 2000; Lee et al., 2000; Pollack et al., 2003), while HNE can form adducts with 2′-deoxyguanosine (Winter et al., 1986). Consequently, there is considerable interest in mitigating the effects of LPO products.
Multiple enzymatic pathways are involved in the metabolism of HNE and ONE. Phase I metabolism of HNE is a prominent metabolic pathway. Cytochromes P450 (Amunom et al., 2007) and aldehyde dehydrogenase (Mitchell and Petersen, 1987) can catalyze the oxidation of HNE to HNA, while aldo-keto reductase (Srivastava et al., 2000) has been shown to reduce HNE to DHN. Upon conjugation via phase II metabolism, HNA adducts can spontaneously undergo intramolecular condensation to form the corresponding γ-lactones (Alary et al., 1995). HNA lactone has also been reported to arise from the oxidation of HNE-GSH in the hemi-acetal form (Boon et al., 1999). ONE can be metabolized in similar fashion, forming ONA from aldehyde dehydrogenase-mediated oxidation (Doorn et al., 2006) or the HNE isomer, ONO, via aldo-keto reductase- (Doorn et al., 2003; Jian et al., 2005) mediated reduction. Carbonyl reductase has been demonstrated to reduce ONE at the C-4 position as well, resulting in HNE formation (Doorn et al., 2004). With the exception of DHN, these phase I metabolites of HNE and ONE retain their α, β-unsaturated carbonyl moiety, and are therefore able to form Michael-type conjugates with GSH. Phase II metabolism of HNE and ONE via Michael-type addition with GSH is mediated by GSTs. It has been demonstrated that GST 8-8 has a high specific activity for conjugation of GSH with HNE (Jensson et al., 1986), suggesting a major biological function of GSTs for the conjugation and deactivation of reactive electrophilic products. It has further been demonstrated that γ-glutamyl transferase (γ-GT) metabolizes HNE-GSH to HNE-cysteine glycine (CG; Enoiu et al., 2002). Metabolism by cysteinyl glycinase and N-acetyl transferase in the liver and kidneys results in HNE-MA conjugates which are excreted in the urine. This pathway for HNE removal can initially deplete GSH. HNE also plays a role in the recovery of depleted GSH levels by inducing the expression of glutamate cysteine ligase, the rate limiting enzyme in GSH synthesis (Iles and Liu, 2005).
Early detection of HNE and other reactive aldehydes was carried out by 2,4-dinitrophenylhydrazine (DNPH) derivatization (Esterbauer and Zollner, 1989; Esterbauer et al., 1991). The hydrazone derivatives have characteristic absorbance maxima at 360 to 390 nm and are easily detected by TLC or HPLC. Peak identification using this method was carried out by comparison to synthetic standards. The main drawback of DNPH derivatization is the lack of specificity, since DNPH will react with all carbonyls. Another derivatization method for HPLC analysis is the reaction of aldehydes with 1,3-cyclohexanedione to form fluorescent decahydroacridine derivatives (Esterbauer and Zollner, 1989). Direct HPLC determination of HNE at 220 nm has also been reported (Lang et al., 1985). These HPLC methods rely on retention time comparison with synthetic standards. Chromatography coupled with mass spectrometry provides a higher confidence level for structure identity and greater analytical selectivity and sensitivity. GC-MS methods for HNE analysis require derivatization, e.g., with pentafluorobenzyl-hydroxylamine followed by silylation (Van Kuijk et al., 1986). An enzyme immunoassay to quantitatively assess levels of DHN-MA was recently developed and validated by Guéraud et al. (2006). LC-MS/MS analyses of (S)-carbidopa derivatized HNE were employed to differentiate between the R- and S- enantiomers (Honzatko et al., 2007). HNE-GSH and HNA were also analyzed using this method. Isotope-dilution LC-MS3 methodology was demonstrated to be useful for the quantitative analysis of DHN-MA and could possibly be utilized to quantify other LPO-MA conjugates (Alary et al., 1998; Rathahao et al., 2005; Blair, 2010). LC-MS methods employing selected ion monitoring have also been used to analyze HNE-GSH, HNE, and ONO in mouse liver tissues (Warnke et al., 2008), although separate sample preparation methods were necessary for the conjugated and free metabolites. This protocol describes a quantitative LC-MS/MS method for the analysis of LPO-MA conjugates in urine. We have used this method for analysis of LPO-MA conjugates in an animal model of acute oxidative stress and in a smoking cessation study (Kuiper et al., 2008, 2010).
Oxidative stress-induced LPO leads to the formation of cytotoxic and genotoxic 2-alkenals, HNE and ONE, as well as their metabolites (Benedetti et al., 1980; Esterbauer et al., 1991). These LPO products have also been shown to contribute to the development and progression of age-related diseases such as Alzheimer’s and atherosclerosis (Spiteller, 2007; Butterfield and Sultana, 2007; Picklo and Montine, 2007; Lovell and Markesbery, 2007; Poli et al., 2008). Therefore, HNE and other LPO products show promise as biomarkers of oxidative stress and as diagnostic tools for assessing disease risk and/or development. In some cases, however, even these secondary products of oxidative stress such as HNE and ONE may not be suitable biomarkers due to high reactivity. By contrast, HNE-MA, ONE-MA, and their metabolites are stable end-product metabolites and more suitable to assess oxidative stress in vivo.
Existing methods for assessing in vivo oxidative stress are not without drawbacks. The thiobarbituric acid reaction, a colorimetric assay that is widely used to analyze levels of malondialdehyde lacks specificity (Knight et al., 1988). F2α-isoprostane analysis is currently considered the most reliable way to assess oxidative stress in vivo; however, F2α-isoprostanes are formed only from arachidonic acid degradation. Due to its specificity, F2α-isoprostane analysis may not provide a global assessment of oxidative stress (Guichardant and Lagarde, 2009). This protocol discusses appropriate analytical methods for the identification and quantitation of HNE-MA, ONE-MA, and their metabolites as in vivo biomarkers of oxidative stress.
Prior to analysis of LPO-MA conjugates, there are several important issues to consider. This protocol discusses the quantitative analysis of HNE-MA, ONO-MA, and DHN-MA. HNA-MA, HNAL-MA, and ONA-MA can all be detected in human urine as well, but have not been quantitatively analyzed. The endogenous amounts of ONA-MA, estimated at < 10 nM, were too small to quantify by isotope-dilution LC-MS/MS with satisfactory precision and accuracy. HNA-MA and HNAL-MA presented a different challenge. Due to the spontaneous conversion of HNA-MA into HNAL-MA under aqueous conditions, we have thus far been unable to obtain homogenous synthetic standards of either material for use in our calibration curves.
It should be noted that while we have previously demonstrated the presence of ONE-MA in rat urine (Kuiper et al., 2008), it was not detectable in our human urine samples. Its absence in human urine is likely due to preferential phase I metabolism of ONE or ONE-GSH resulting in metabolites ONO-MA and ONA-MA.
Note also that ONE-MA and HNAL-MA are isobaric and co-elute under the conditions described. Separation and analysis of these metabolites can be carried out by LC-MS/MS using a method described by Kuiper et al. 2008.
When preparing the urine samples for analysis, be sure to acidify. The LPO-MA conjugates will remain in the water layer if the sample is not acidic.
HNE-MA, ONO-MA, and DHN-MA are found in humans at low mg/g creatinine concentrations (Alary et al., 1998; Kuiper et al., 2010) and have been found in rats at the low end of the human range (Alary et al., 1998; Rathahao et al., 2005; Guéraud et al., 2006; Mally et al., 2007). The concentrations will vary between subjects based on type and level of oxidative stress. Typical human levels are 0.17 to 12.19 mg/g creatinine (7.4 to 225 nM) HNE-MA, 0.05 to 2.26 mg/g creatinine (1.7 to 177 nM) ONO-MA, and 0.22 to 17.90 mg/g creatinine (6.6 to 316 nM) DHN-MA. The limits of quantitation are 5 nM for HNE-MA, 0.5 nM for ONO-MA, and 10 nM for DHN-MA. When the samples undergo a volume concentration step as in this protocol, the limit of quantitation will change accordingly. A 2× sample volume concentration would cause the limits of quantitation to become 2.5 nM, 0.25 nM, and 5 nM for HNE-MA, ONO-MA, and DHN-MA respectively.
The synthesis of each LPO product takes a different amount of time. ONA preparation takes ~ 7 hr, HNA requires ~2 hr, ONO preparation takes ~17 hr, and MAd3 takes ~ 3 days. These products can all be prepared ahead of time and stored at −20°C for up to 6 months. The synthesis of LPO-MA standards and LPO-MAd3 internal standards takes ~ 4 or 5 hr. Urine preparation for 20 samples takes ~ 3 or 4 hr. LC-MS/MS analysis takes 26 min per sample. When calculating analysis time, sample blanks, standards, and calibration curve samples need to be counted too. The standards can be frozen prior to use and the urine samples can be stored for about 1 week at −20°C if necessary, allowing each step to be carried out on different days.
The research described in this protocol was supported by the National Institutes of Health (Grants R01HL081721, S10RR022589, and P30ES000210), the Medical Research Foundation of Oregon, and an OSU Center for Healthy Aging Research Fellowship.