Development of a Method for Quantification of Acrolein

Aug 18, 2005 - Xinli Liu,† Mark A. Lovell,†,‡ and Bert C. Lynn*,†,‡. Department of Chemistry, University of Kentucky, Lexington, Kentucky 40...
0 downloads 0 Views 156KB Size
Anal. Chem. 2005, 77, 5982-5989

Development of a Method for Quantification of Acrolein-Deoxyguanosine Adducts in DNA Using Isotope Dilution-Capillary LC/MS/MS and Its Application to Human Brain Tissue Xinli Liu,† Mark A. Lovell,†,‡ and Bert C. Lynn*,†,‡

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, and Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 40536-0230

Acrolein is a highly reactive r,β-unsaturated aldehyde and is known to react with DNA forming exocyclic acroleindeoxyguanosine adducts (Acro-dG). These aldehydeDNA lesions may play a role in mutagenesis, carcinogenesis, and neurodegenerative diseases. In the present work, we described the development and evaluation of a highly sensitive and selective capillary liquid chromatography nanoelectrospray isotope dilution tandem mass spectrometry method for quantitatively analyzing AcrodG in DNA hydrolysates. This was achieved by applying a stable isotope-labeled analogue Acro-dG-13C10,15N5 as an internal standard to the DNA to be analyzed and then hydrolyzing the DNA enzymatically to nucleosides. The acrolein-modified nucleosides were separated from normal nucleosides by capillary liquid chromatography and quantified by a high-capacity ion trap mass spectrometer in the MS/MS mode. The developed method achieved attomole-level sensitivity (limit of detection was 10 fg, 31 amol on column) for detection of pure Acro-dG adduct standards. The limit of quantification of Acro-dG adducts obtained in 10 µg of DNA hydrolysates was 1.5 fmol, which corresponded to 50 adducts/109 normal nucleosides. Application of this method to the analysis of AcrodG adducts in acrolein (10-fold)-treated calf thymus DNA showed ∼830 lesion/106 DNA nucleosides using as low as 50 ng of DNA. Application of this method to DNA samples (1-2 µg) isolated from brain tissues from Alzheimer’s disease subjects and age-matched controls demonstrated 2800-5100 Acro-dG adducts/109 DNA nucleosides. Statistically significant differences (P < 0.05) in levels of Acro-dG between AD subjects and controls were observed in DNA isolated from the hippocampus/ parahippocampal gyrus. Humans are exposed to a wide array of potentially harmful substances in both their work and home environments. Acrolein (2-propenal), the simplest R,β-unsaturated aldehyde (enal) is * To whom correspondence should be addressed. Phone: (859) 257-2300; ext. 287. Fax: (859) 257-2489. E-mail: [email protected]. † Department of Chemistry. ‡ Sanders-Brown Center on Aging.

5982 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

ubiquitously distributed in the environment.1 Acrolein can be generated by burning fats during cooking, is present in automobile exhaust, and is also found in tobacco smoke at relative high concentrations (25-140 µg/cigarette).2 Small amounts of acrolein may also be formed endogenously from polyunsaturated fatty acids during lipid peroxidation.3 In addition, there is increasing evidence that acrolein participates in many important pathological states such as cell apoptosis4 and mediates cell damage in various disease processes such as Alzheimer’s disease (AD)5,6 and cancer.7 Acrolein has been regarded as genotoxic and mutagenic in bacteria,8 cultured cells,9 and human cells.10 Acrolein is a strong electrophile, and it shows high reactivity with biologically importantly nucleophiles, including thiol and amino groups of cysteine, histidine, and lysine to form DNA-protein cross-links.11,12 Although several mechanisms are possible for the genotoxic and mutagenic effects of acrolein, the most probable mode of action is the formation of exocyclic adducts with DNA bases. Since the adducted bases do not form base pairs in the usual manner, these mutations result in misreplication of DNA.13,14 Like many other reactive bifunctional aldehydes, acrolein reacts with DNA bases including guanine,15-17 adenine,18,19 cytosine,20-23 and thymidine22,23 (1) Izard, C.; Libermann, C. Mutat. Res. 1978, 47, 115-138. (2) Biswal, S.; Maxwell, T.; Rangasamy, T.; Kehrer, J. P. Carcinogenesis 2003, 24, 1401-1406. (3) Uchida, K.; Kanematsu, M.; Morimitsu, Y.; Osawa, T.; Noguchi, N.; Niki, E. J. Biol. Chem. 1998, 273, 16058-16066. (4) Kern, J. C.; Kehrer, J. P. Chem.-Biol. Interact. 2002, 139, 79-95. (5) Lovell, M. A.; Xie, C.; Markesbery, W. R. Neurobiol. Aging 2001, 22, 187194. (6) Picklo, M. J.; Montine, T. J. Biochim. Biophys. Acta 2001, 1535, 145-152. (7) Cohen, S. M.; Garland, E. M.; St. John, M.; Okamura, T.; Smith, R. A. Cancer Res. 1992, 52, 3577-3581. (8) Marnett, L. J.; Hurd, H. K.; Hollstein, M. C.; Levin, D. E.; Esterbauer, H.; Ames, B. N. Mutat. Res. 1985, 148, 25-34. (9) Curren, R. D.; Yang, L. L.; Conklin, P. M.; Grafstrom, R. C.; Harris, C. C. Mutat. Res. Lett. 1988, 209, 17-22. (10) Yang, I.-Y.; Chan, G.; Miller, H.; Huang, Y.; Torres, M. C.; Johnson, F.; Moriya, M. Biochemistry 2002, 41, 13826-13832. (11) Yang, X.; Wu, X.; Choi, Y. E.; Kern, J. C.; Kehrer, J. P. Toxicology 2004, 204, 209-218. (12) Furuhata, A.; Ishii, T.; Kumazawa, S.; Yamada, T.; Nakayama, T.; Uchida, K. J. Biol. Chem. 2003, 278, 48658-48665. (13) Eder, E.; Hoffman, C.; Bastian, H.; Deininger, C.; Scheckenbach, S. Environ. Health Perspect. 1990, 88, 99-106. (14) Boerth, D. W.; Eder, E.; Hussain, S.; Hoffman, C. Chem. Res. Toxicol. 1998, 11, 284-294. (15) Galliani, G.; Pantarotto, C. Tetrahedron Lett. 1983, 24, 4491-4492. 10.1021/ac050624t CCC: $30.25

© 2005 American Chemical Society Published on Web 08/18/2005

Scheme 1. Formation of the Exocyclic Acro-dG Adducts of Acrolein with Deoxyguanosine

to form several cyclic adducts and DNA interchain cross-links.24-26 Among the exocyclic adducts, acrolein-deoxyguanosine (AcrodG) was the major adduct detected in DNA extracted from rodent and human tissues.27-29 The chemistry of formation of Acro-dG is illustrated in Scheme 1. Exocyclic adducts are formed through a pair of regioisomeric Michael additions with initial bond formation occurring at either the N2 or N-1 position of deoxyguanosine, followed by ring closure to form exocyclic hydroxylisomeric adducts, the R and S isomers of 3H-8-hydroxy-3-(β-D-2′deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purin-9-one (8OH-PdG) and the R and S isomers of 3H-6-hydroxy-3-(β-D-2′deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purin-9-one (6OH-PdG).16 The 8-OH-PdG adduct dominates over the 6-OH-PdG. The 8-OH-PdG adduct is biologically important in the formation of the ring-opened, acyclic N2 oxopropyls in duplex DNA, which are capable of forming DNA-DNA, DNA-peptide, and DNAprotein cross-links.26 The development of a fast, sensitive, and selective method for detection of DNA adducts in DNA is a crucial and a challenging (16) Chung, F. L.; Young, R.; Hecht, S. S. Cancer Res. 1984, 44, 990-995. (17) Cheng, G.; Shi, Y.; Sturla, S. J.; Jalas, J. R.; McIntee, E. J.; Villalta, P. W.; Wang, M.; Hecht, S. S. Chem. Res. Toxicol. 2003, 16, 145-152. (18) Smith, R. A.; Williamson, D. S.; Cerny, R. L.; Cohen, S. M. Cancer Res. 1990, 50, 3005-3012. (19) Kawai, Y.; Furuhata, A.; Toyokuni, S.; Aratani, Y.; Uchida, K. J. Biol. Chem. 2003, 278, 50346-50354. (20) Sodum, R. S.; Shapiro, R. Bioorg. Chem. 1988, 16, 272-282. (21) Smith, R. A.; Sysel, I. A.; Tibbels, T. S.; Cohen, S. M. Cancer Lett. (Shannon, Ireland) 1988, 40, 103-109. (22) Smith, R. A.; Williamson, D. S.; Cohen, S. M. Chem. Res. Toxicol. 1989, 2, 267-271. (23) Chenna, A.; Iden, C. R. Chem. Res. Toxicol. 1993, 6, 261-268. (24) Kozekov, I. D.; Nechev, L. V.; Sanchez, A.; Harris, C. M.; Lloyd, R. S.; Harris, T. M. Chem. Res. Toxicol. 2001, 14, 1482-1485. (25) Kim, H.-Y. H.; Voehler, M.; Harris, T. M.; Stone, M. P. J. Am. Chem. Soc. 2002, 124, 9324-9325. (26) Kozekov, I. D.; Nechev, L. V.; Moseley, M. S.; Harris, C. M.; Rizzo, C. J.; Stone, M. P.; Harris, T. M. J. Am. Chem. Soc. 2003, 125, 50-61. (27) Nath, R. G.; Chung, F.-L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 74917495. (28) Nath, R. G.; Ocando, J. E.; Chung, F.-L. Cancer Res. 1996, 56, 452-456. (29) Nath, R. G.; Ocando, J. E.; Guttenplan, J. B.; Chung, F.-L. Cancer Res. 1998, 58, 581-584.

task to improve understanding of the impact of DNA adducts in disease processes. Because levels of DNA adduction are typically in the range of 1 in 1 million or 1 in 1 billion normal nucleotides, highly sensitive techniques with high dynamic range are required for the analysis of small amounts of DNA (less than 100 µg) available from many human studies. The 32P-postlabeling technique has been widely used for the measurement of DNA adducts. This assay uses γ-32P-labeled adenosine triphosphate to incorporate a radioactive phosphorus group into nucleotides derived from enzymatic hydrolysis of DNA. Chung and co-workers reported that use of a sensitive 32P-postlabeling method combined with HPLC enables detection of Acro-dG in human oral tissue (1.36 ( 0.90 µmol/mol guanine in smokers versus 0.46 ( 0.26 µmol/ mol guanine in nonsmokers).28,29 Although highly sensitive, 32Ppostlabeling requires use of an energetic radioactive isotope and cannot provide structural information about adducts. In the bioanalytical arena, high-performance liquid chromatography coupled with ion trap tandem mass spectrometry (LC/ MS/MS) has been widely employed for the analysis of adducts in biological samples because of its excellent specificity, speed, sensitivity and the ability for the ion trap to manipulate ions to perform multiple-stages mass analysis. The advent of capillary LC with nanoelectrospray ionization mass spectrometry and its application to peptides30 has clearly demonstrated outstanding sensitivity (low-femotomole to attomole detection limits) and quantitative capabilities for charged molecules. Isotope dilution mass spectrometry (IDMS) is an established method for unequivocal detection and accurate quantification of analytes in complex mixtures. The quantification of an analyte is calculated from the ratio of the peak areas of analyte versus isotope internal standard that has the same chemical and physical properties as the analyte. Variation in sample recovery can be monitored and adjusted during each step of the assay; therefore, an accurate quantification is ensured. In the present study, we developed a highly sensitive capillary liquid chromatography nanoelectrospray isotope dilution tandem mass spectrometry (capLC-nanoESIIDMS/MS) method for quantitatively analyzing Acro-dG in DNA hydrolysates. The developed method has achieved attomole-level sensitivity (limit of detection was 10 fg, 31 amol on column) for detecting pure Acro-dG adducts. Several studies observed that endogenously generated acrolein levels increased in the brain of AD subjects compared with age-matched control subjects,5,31 which suggests that acrolein may play an important role in the pathophysiological process of AD. In this study, the sensitive and selective capLC-nanoESI-IDMS/MS method was applied to quantify Acro-dG adducts in the hippocampus/parahypocampal gyrus (location of a high degree of pathology) of postmortem brains from AD and age-matched control subjects. A statistically significant difference was observed (P ) 0.025). EXPERIMENTAL SECTION Caution: Acrolein is highly cytotoxic and mutagenic and should be handled using gloves in a well-ventilated hood. Chemicals and Materials. Acrolein was purchased from Aldrich (Milwaukee, WI). 2′-deoxyguanosine, deoxyribonuclease I (from bovine pancrease), phosphodiesterase I (from Crotalus (30) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (31) Lovell, M. A.; Ehmann, W. D.; Mattson, M. P.; Markesbery, W. R. Neurobiol. Aging 1997, 18, 457-461.

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

5983

adamanteus venom), phosphodiesterase II (from bovine spleen), and alkaline phosphatase were obtained from Sigma (St. Louis, MO). 2′-Deoxyguanosine-13C10,15N5-5′-triphosphate sodium salt (dGTP-13C10,15N5, U-13C10, 98%; U-15N5, 98%) was obtained from Isotec (Miamisburg, OH). Calf thymus DNA was purchased from Worthington Biochemical Co. (Lakewood, NJ). HPLC grade water, acetonitrile, and formic acid were purchased from Fisher Scientific (Fair Lawn, NJ). The 100 µm i.d. × 358 µm o.d. fused-silica capillary tubing (Polymicro Tech. Phoenix, AZ) was used in column fabrication. Bulk stationary-phase packing material, Macrosphere 300-Å bonded C18, 5 µm, was purchased from Alltech Associates, Inc. (Deerfield, IL), Luna C18, 5-µm and Polymer X 3-µm packing materials were obtained from Phenomenex (Torrance, CA). All other miscellaneous LC accessories, including zero dead volume unions and 2-µm stainless steel frits, nuts, ferrules, and PEEK (polyetheretherketone) tubing for column construction were obtained from Upchurch Scientific (Oak Harbor, WA). Packing of Capillary Column. Capillary columns used in this study were constructed with a 100 µm i.d. × 358 µm o.d. bare fused-silica column packed to a length of 15 cm with different reversed-phase materials including Microsphere C18-bonded (5µm, end capped), Luna C18-bonded (5-µm, end capped), and Polymer X (3-µm polystyrene-divinylbezene). The choice of these materials was dictated by the commercial availability of highquality matrix. All columns were manufactured in-house following our previously described slurry-packing procedures.32 CapLC-NanoESI-MS/MS. CapLC-nanoESI-MS/MS analysis was performed on an UltiMate Capillary/Nano LC System (LC Packing, Sunnyvale, CA) coupled to a Bruker Daltonics highcapacity ion trap (HCT) mass spectrometer (Billerica, MA) through a Bruker nanoelectrospray ion source. Mobile phase A was water (0.1% formic acid), and mobile phase B was acetonitrile (0.1% formic acid). The separation of normal nucleosides and modified Acro-dG was performed isocratically (95(A)/5(B), v/v). A 5-µL injection loop was manufactured in-house by measuring an appropriate length of fused-silica capillary (28.7 cm × 149 µm i.d.), and full loop injection was achieved by total injection of 6 µL via a six-port switching valve injector (Valco, Houston, TX) mounted on the LC packing. The pump output (150 µL/min) was split before the injection port to a flow rate of 500 nL/min. The column effluent enters the spray chamber through a tapered stainless steel emitter with 50-µm i.d. constructed according to the method of Ishihama et al.33 and directly electrosprayed into the Bruker HCT ion trap mass spectrometer in the positive mode for nanoESI-MS/MS analysis. Emitter position was adjusted under a microscope and positioned in front of the orifice at a distance of ∼1.5 mm. Spray voltage was set between -1400 and -1600 V to achieve a stable current. Heated nitrogen drying gas (3.0 L/min, 200 °C) was introduced in the spray chamber to aid in desolvation. No nebulizing gas was employed. Source voltages were adjusted such that the precursor ion generated maximized product ion signal from the tandem experiment. The mass spectrometer was configured for multiple reaction monitoring (MRM) scan mode composed of two alternating scan functions. The first scan function isolated the protonated Acro-dG ([M + H]+, m/z 324) with a 3 m/z wide window and fragmented this

precursor ion (MS/MS mode) into a unique product ion ([M 116 + H]+, m/z 208). The second scan function isolated the protonated Acro-dG-13C10,15N5 ([M + H]+, m/z 339) with a 3 m/z wide window and fragmented this precursor ion into a unique product ion ([M - 121 + H]+, m/z 218). Adduct Synthesis and HPLC Purification. Incubation of acrolein with 10 equiv of 2′-deoxyguanosine or 2′-deoxyguanosine13C ,15N -5′-triphosphate in phosphate buffer (25 mM, pH 7.4) at 10 5 37 °C for 7 days produced crude products of Acro-dG and AcrodGTP-13C10,15N5, respectively. The latter was redissolved in 1 mL of 4 mM Tris-HCl buffer (pH 8.0) and incubated with alkaline phosphatase (4 units/mg of sample) at 37 °C for 24 h to produce Acro-dG-13C10,15N5. The enzyme was removed by centrifugation at 6000g for 1 h using a Millipore centrifugal filter with a molecular mass cutoff of 5 kDa. The crude isotope-labeled and unlabeled adducts were dried under SpeedVac and redissolved in water for HPLC-UV purification using a Thermo Separation SpectraSYSTEM P4000 pump with a UV6000LP detector (Themo Separation products, San Jose, CA) at λ ) 260 nm and a Rheodyne injector on a PR Phenomenex Luna 5-µm 250 × 4.60 mm C18 column (Phenomenex, Torrance, CA) with an isocratic separation (water/ acetonitrile 93:7) at flow rate of 1 mL/min over 30 min. Composition and purity of collected fractions of each adduct were verified by full-scan MS (m/z range 100-650). Linearity and Calibration Curves. Calibration of the assay was performed in at least triplicate runs by addition of a fixed amount (20 pg/µL, 100 pg on column) of Acro-dG-13C10,15N5 internal standard with various quantities of an authentic standard solution of Acro-dG ranging from 0.1 to 150 pg (0.1, 2, 4, 8, 10, 15, 20, 25, 50, and 150 pg) of adducts on column (corresponds to 310 amol to 464 fmol). The precision of the method was determined by at least triplicate analyses of adducts at all concentrations. On the day of in vivo sampling, blanks were analyzed in order to eliminate false positives derived from potential carryover in the analytical system, digestion enzymes, solvents, and other workup apparatus. Care was taken to inject a dilute sample such that the syringe needle and sample loop were not contaminated before analysis of in vivo sample. A procedural blank was prepared by analyzing unmodified calf thymus DNA that had been carried through the entire workup procedure. A system blank was composed of an injection of 5% acetonitrile in water. In Vitro Generation of DNA Adducts and Enzymatic Cleavage of DNA. Acrolein-modified calf thymus DNA (ctDNA) was generated by reaction of ctDNA with acrolein. Briefly, ctDNA was weighed and dissolved in 5 mM Tris/10 mM MgCl2 pH 8.5 buffer to achieve a final concentration of 0.5 µg/µL stock, and the concentration was checked by UV absorbance (assuming A260 ) 1 at 50 µg/mL). A total of 500 µL of the above ctDNA solution (250 µg of ctDNA) was added to an Eppendorf tube containing 7.5 µmol of acrolein. (assuming that 100 µg of ctDNA contains ∼0.3 µmol of nucleotides,34 this amounts to a 10-fold excess of acrolein). The solution was incubated at 37 °C for 7 days and dried under SpeedVac. The residue was reconstituted in buffer. Control ctDNA and modified ctDNA samples were spiked with the isotopelabeled internal standard Acro-dG-13C10,15N5 (100 pg). The DNA was hydrolyzed enzymatically to nucleosides according to the

(32) Wenner, B. R.; Lovell, M. A.; Lynn, B. C. J. Proteome Res. 2004, 3, 97-103. (33) Ishihama, Y.; Katayama, H.; Asakawa, N.; Oda, Y. Rapid Commun. Mass Spectrom. 2002, 16, 913-918.

(34) Beland, F. A.; Doerge, D. R.; Churchwell, M. I.; Poirier, M. C.; Schoket, B.; Marques, M. M. Chem. Res. Toxicol. 1999, 12, 68-77.

5984

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

Figure 1. Chromatogram obtained by HPLC purification of reaction mixtures of acrolein and deoxyguanosine. Separation was achieved on a Phenomenex Luna 5-µm 250 × 4.60 mm C18 column with an isocratic separation (water/acetonitrile, 93:7, v/v) at flow rate of 1 mL/min over 30 min at 260 nm. Free deoxyguanosine and three major adduct peaks were observed, and each adduct fraction was collected. Peak 1 was identified as deoxyguanosine, Peaks 2 and 3 correspond to 6-OH-pdG, and peak 4 corresponds to 8-OH-pdG. Only peak 4 (8-OH-pdG) was used as standard for the calibration assay.

method described by Mecocci et al.35 with minor modification with deoxyribonuclease I (DNase I from bovine pancreas, 200 U/mg DNA), Crotalus adamanteus venom phosphodiesterase I (5′-exonuclease, 0.3 U/mg DNA), bovine spleen phosphodiesterase II (3′-exonuclease, 0.01 U/mg DNA), and alkaline phosphatase (10 U/mg DNA) in the presence of 4 mM Tris-HCl (pH 8.0) buffer containing 10 mM MgCl2. Samples were incubated for 6 h in a 37 °C water bath. The enzyme digests were desalted and purified by solid-phase extraction (SPE). Sample Cleanup Procedure for LC/MS. SPE columns of various inner diameter and length, including Oasis HLB (30 mg/ mL, Waters), PrepSep C18 (100 mg/mL, Fisher), C8 SPE (50 mg/ mL, Supelco), and graphitized carbon SPE (150 mg/4.0 mL, Alltech), were evaluated to determine maximal sample processing capability that provided maximized sample recovery. Solid-phase sample extraction was facilitated by using a vacuum manifold. Authentic standards were spiked in water and applied to the above SPE cartridge using the generic procedure as follows: (1) 1 mL of MeOH condition; (2) 1 mL of water condition; (3) load DNA digest aqueous solution (∼200 µL); (4) wash with 1 mL of water; (5) elute with 1 mL of MeOH. The methanol eluent was evaporated to dryness under SpeedVac and reconstituted in 95:5 (v/v) water/acetonitrile (the initial mobile-phase composition) prior to injection into the capLC-nanoESI-MS/MS system. Isolation of Human Brain DNA. Brain specimens were removed at autopsy from AD subjects and age-matched control subjects, immediately frozen in liquid nitrogen, and subsequently stored at -70 °C per University of Kentucky approved IRB protocols. Tissue was homogenized and nuclear DNA isolated using a previously described procedure.5 The concentration of DNA was determined at 260 nm (A260 ) 1 at 50 µg/mL).

Absorbance was also measured at 280 nm, with a mean 260/280 ratio less than 1, indicating a relatively pure DNA preparation with little or no contamination by proteins or RNA. These human DNA samples were subjected to the same processing procedures as the modified ctDNA samples. Statistical Analysis. Acro-dG adducts level are presented as means ( standard deviation (SEM). The stastistical significance of difference in means was evaluated by the unpaired Student’s t-test using SigmaPlot 8.0 software (Systat Software Inc. CA). Statistically significant differences were assumed for P < 0.05.

(35) Mecocci, P.; MacGarvey, U.; Kaufman, A. E.; Koontz, D.; Shoffner, J. M.; Wallace, D. C.; Beal, M. F. Ann. Neurol. 1993, 34, 609-616.

(36) Basu, A. K.; O’Hara, S. M.; Valladier, P.; Stone, K.; Mols, O.; Marnett, L. J. Chem. Res. Toxicol. 1988, 1, 53-59.

RESULTS AND DISCUSSION Standard Characterization. The ultimate goal of this study is to develop an analytical protocol to quantify Acro-dG adducts at physiologically relevant levels. Since authentic Acro-dG was not commercially available, it was prepared and purified from a reaction mixture of acrolein and deoxyguanosine. Figure 1 illustrates a typical HPLC-UV chromatogram obtained upon purification. Unreacted deoxyguanosine (peak 1) elutes first, followed by three major adduct peaks (labeled as 2, 3, and 4). Each adduct fraction was collected and proved to be structural isomers as all mass spectra showed a molecular weight of 323. Tandem spectra of structural isomers 2-4 were identical. An equilibrium existed between adducts 2 and 3, because when pure adduct 2 or 3 was injected onto the HPLC a mixture of adducts 2 and 3 was observed. Based on published observations, peaks 2 and 3 were assigned as mixtures of the two enantiomers of 6-OHPdG and peak 4 was racemic mixtures of 8-OH-PdG.13,16,17,36 The stereoselective formation of 8-OH-PdG has been demonstrated in vivo in rodents and humans;27-29 therefore, only 8-OH-PdG (peak 4) was used as a standard for the calibration assay.

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

5985

Figure 2. (A) Reconstructed ion chromatograms of m/z 218 (Acro-dG-13C10,15N5) and 208 (Acro-dG) recorded during the capLC-nanoESIIDMS/MS analysis. Solvents, water and acetonitrile (95/5, v/v, with 1% formic acid); flow rate, 500 nL/min. (B) Tandem mass spectrum of Acro-dG-13C10,15N5 (m/z 339 f 218). (C) Tandem mass spectrum of Acro-dG (m/z 324 f 208). Insets: structures and fragmentation pathways of Acro-dG-13C10,15N5 and Acro-dG. The stars indicate the labeled atoms in Acro-dG-13C10,15N5.

Stable isotope-labeled analogues of Acro-dG-13C10,15N5 were obtained from desphosphorylation of the Acro-dGTP-13C10,15N5 by alkaline phosphatase (∼95% yield, determined by UV absorbance). When purified by HPLC-UV, a similar chromatogram was obtained, consisting of the labeled adduct 6-OH-PdG-13C10,15N5 (R and S, two peaks) and a racemic mixture of 8-OH-PdG-13C10,15N5 (one peak) with HPLC retention times and UV spectra similar to those observed for unlabeled analogues. Collected fractions from several HPLC runs were combined and dried in a SpeedVac under vacuum. The isotopic purity and chemical composition of the collected Acro-dG-13C10,15N5 was determined by LC/MS full-scan (m/z 100-650) and LC/UV with a purity of >98%. This assay also revealed that the Acro-dG-13C10,15N5 internal standard was not contaminated with unlabeled adducts. The pure Acro-dG and Acro-dG-13C10,15N5 adducts (both R and S 8-OH-PdG forms) coeluted on the capLC-nanoESI-MS system. As expected, the mass spectrum of Acro-dG-13C10,15N5 consisted of the protonated molecular ion at m/z 339, which corresponded to the protonated molecular ion m/z 324 ions from unlabeled Acro-dG, a 15 amu mass shift. The major product ion observed in the tandem mass spectrum of Acro-dG-13C10,15N5 internal standard was m/z 218, which resulted from the loss of the deoxyribose (121 amu) and was similar to the product ions from Acro-dG ([M + H]+ at m/z 208) also due to the loss of the deoxyribose (116 amu) (Figure 2). Adduct Enrichment and Sample Cleanup. DNA samples were analyzed by adding Acro-dG-13C10,15N5 as an internal standard to the DNA to be analyzed and then hydrolyzing the DNA enzymatically to nucleosides. The acrolein-modified nucleosides were separated from normal nucleosides by capillary liquid 5986 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

chromatography. Before loading the DNA digest on the capillary column, nonvolatile salts and enzymes were removed to minimize matrix suppression effects commonly observed with electrospray and preconcentrate adduct sample. Currently, the most widely used biological sample preparation methodologies include SPE, liquid-liquid extraction, and protein precipitation.37 Liquid-liquid extraction was evaluated in this study, but poor recovery and lack of reproducibility were observed due to the polar nature of AcrodG, which could not be extracted with good recoveries regardless of the organic solvent selected. To find a more selective method to efficiently extract the relatively polar Acro-dG from the matrix and quantitatively elute from the cartridge, several SPE columns with different sorbent materials were evaluated. C18-bonded silica cartridges were initially selected due to their broad applicability for cleanup of biological fluids. Using the protocol described in the Experimental Section, ∼30% Acro-dG was recovered with the C18 sorbent. The C8 sorbent achieved similar recovery. Graphitized carbon showed higher efficiency for trapping Acro-dG (∼85%). However, use of the polymer-based Waters Oasis HLB sorbent (hydrophilicliphophilic balanced sorbent, macroporous copolymer consisting of lipophilic divinylbenzene and hydrophilic n-vinylpyrrolidone)38 increased recoveries of Acro-dG to 95% on the basis of mass spectrometric ion chromatogram peak area comparisons between samples spiked either before or after extraction. Moreover, superior reproducibility (relative standard deviation within 5%) was attained using the Oasis HLB sorbent compared to the traditional (37) Henion, J.; Brewer, E.; Rule, G. Anal. Chem. 1998, 70, 650A-656A. (38) Huck, C. W.; Bonn, G. K. J. Chromatogr., A 2000, 885, 51-72.

C18 silica packing. The high recovery and reproducibility of AcrodG obtained by the Oasis HLB sorbent was supported by the similar high extraction efficiency achieved from structurally similar polar nucleoside derivatives reported in the literature.39,40 Capillary Chromatography. In the present study, we focused our attention on the excellent sensitivity of capillary HPLC. It is well known that the use of small-inner diameter capillary columns and requisite low flow rates greatly improves the sensitivity of electrospray since the combination of mass spectrometric detection is concentration-sensitive.41,42 The concentration of equally abundant solutes in the LC mobile phase is proportional to the inverse square of the column inner diameter, which substantially determines the ESI-MS signal.43 Smith and co-workers44-46 successfully developed a high separation efficiency nanoscale LC/ MS system for ultrasensitive proteomic analysis using 75-µm-i.d. capillaries. Sensitivity improvements to the low-picogram (on column) range have been demonstrated by several groups using 300-µm-i.d. capillary columns.47,48 Vanhoutte et al. 49 reported that sensitivity improved by a factor of 3300 using nanoLC/MS compared to conventional LC/MS. In this study, we used a 100µm-i.d. LC column with an aim of increasing sensitivity without compromising loading capacity and operational robustness. Due to comparable polarity and chromatographic properties of Acro-dG adducts relative to intact nucleosides, we evaluated different types of HPLC packing materials for the separation of Acro-dG adducts from natural nucleosides, including Macrosphere C18-bonded 5-µm end-capped particles, Luna C18-bonded 5-µm, end-capped particles, and Polymer X 3-µm polystyrenedivinylbezene polymer particles, to achieve most efficient separatons between free dG and Acro-dG possible and optimal peak shape. The retention of the highly polar Acro-dG adducts was achieved on both the Macrosphere and Luna C18 packing material, but only using predominately aqueous mobile phases. However, after careful evaluation using a variety of mobile-phase conditions, we successfully optimized the Polymer X packing material to provide good chromatographic peak shapes and baseline separation of normal nucleosides from Acro-dG. However, the isomeric forms of Acro-dG adducts (6-OH-PdG and 8-OH-PdG) were not well resolved on the capillary column when compared with the prep-analytical column and a loss of separation efficiency was observed. This slight loss in separation efficiency was attributed to extra column dead volume in the system (5-µL (39) Poirier, J. M.; Robidou, P.; Jaillon, P. Ther. Drug. Monit. 2002, 24, 302309. (40) Scalia, S.; Simeoni, S.; Dalpiaz, A.; Villani, S. J. Pharm. Biomed. Anal. 2001, 24, 1131-1136. (41) Bruins, A. P. Mass Spectrom. Rev. 1991, 10, 53-77. (42) Hopfgartner, G.; Bean, K.; Henion, J.; Henry, R. J. Chromatogr., A 1993, 647, 51-61. (43) Chervet, J. P.; Ursem, M.; Salzmann, J. P. Anal. Chem. 1996, 68, 15071512. (44) Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235-4249. (45) Shen, Y.; Moore, R. J.; Zhao, R.; Blonder, J.; Auberry, D. L.; Masselon, C.; Pasa-Tolic, L.; Hixson, K. K.; Auberry, K. J.; Smith, R. D. Anal. Chem. 2003, 75, 3596-3605. (46) Smith, R. D.; Shen, Y.; Tang, K. Acc. Chem. Res. 2004, 37, 269-278. (47) Geromanos, S.; Freckleton, G.; Tempst, P. Anal. Chem. 2000, 72, 777790. (48) Gangl, E. T.; Turesky, R. J.; Vouros, P. Anal. Chem. 2001, 73, 2397-2404. (49) Vanhoutte, K.; Van Dongen, W.; Hoes, I.; Lemiere, F.; Esmans, E. L.; Van Onckelen, H.; Van den Eeckhout, E.; van Soest, R. E.; Hudson, A. J. Anal. Chem. 1997, 69, 3161-3168.

Figure 3. Calibration curve for the capLC-nanoESI-IDMS/MS analysis for Acro-dG at m/z 208 and Acro-dG-13C10,15N5 at m/z 218. Samples containing various amounts (0.1-150 pg) of Acro-dG were added to fixed amount of Acro-dG-13C10,15N5 (100 pg) and subjected to the assay procedures described in the Experimental Section (R2 ) 0.9987). Inset: enlargement of the calibration curve containing low amounts (0.1-10 pg) of Acro-dG. Each data point represents the mean of three determinations ( standard deviation.

sample loop and required transfer lines from HPLC to MS) at very low, nanoliter per minute flow rates. Sensitivity with CapLC-nanoESI and High-Capacity Ion Trap-MS/MS. Based on the chromatographic and mass spectrometric properties of the Acro-dG, quantification of Acro-dG adducts in enzymatic hydrolysates of ctDNA was performed by capLC-nanoESI-IDMS/MS in the MRM scan mode. The characteristic transitions from the [M + H]+ precursor ion to the unique m/z 218 product ion for Acro-dG-13C10,15N5 and corresponding [M + H]+ precursor ion to the unique m/z 208 product ions for Acro-dG were recorded. A calibration curve was obtained for the response of the mass spectrometer to known quantities of both Acro-dG (0.1-150 pg on column) and Acro-dG-13C10,15N5 (100 pg on column). As shown in Figure 3, integrated peak area ratios (peak area of the ion m/z 208 divided by peak area of the m/z 218) were plotted against the amount of authentic Acro-dG on column (0.1-150 pg). A linear response ratio versus picogram of Acro-dG on column was determined (y ) 0.0428x + 0.0218) using the method of least squares with a resulting correlation coefficient R2 of 0.9987 and the percent relative standard deviation ranging from 1 to 8%. Using Acro-dG standards, the lowest amount detected with quantitative linearity was 100 fg on column (310 amol, LOQ), the limit of detection (LOD, signal-to-noise ratio of ∼3) was observed to be 10 fg (31 amol) on column. The determination of the limit of quantification in the presence of complex ctDNA digest matrix was achieved by spiking the adduct standard in untreated ctDNA hydrolysate. The smallest amount of the Acro-dG adduct standard was 500 fg in 10 µg of DNA. When an equivalent of 10 µg of DNA was loaded on column (i.e., 1.5 fmol in 30 nmol of DNA), this corresponded to an adduct level of 50 adducts/109 normal nucleosides. Attomole sensitivity was achieved in this analysis for the following reasons. First, the sensitivity of capLC-nanoESI-IDMS/ Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

5987

Figure 4. Reconstructed ion chromatograms from the capillary LC-nanoESI-IDMS/MS analysis of a DNA sample from Alzheimer’s disease subject (A) internal standard, Acro-dG-13C10,15N5 and (B) Acro-dG. Table 1. Summary of Average Acro-dG Adducts in AD and Controlsa brain region H H

AD control

subjects

gender (F/M)

age (years)

PMI (h)

Braak score

Acro-dG per 109

P

n)8 n)5

4M/4F 3M/2F

77.9 ( 3.4 82.8 ( 7.1

3.1 ( 0.2 3.0 ( 0.2

6.0 ( 0.0 1.3 ( 1.2

5150 ( 640 2800 ( 460

0.025

a Values are expressed as mean ( SEM. Brain region (H) is the hippocampus/parahippocampal gyrus. PMI refers to postmortem interval. P value was calculated from unpaired Student’s t-test.

MS at low flow rate increases substantially as the result of improved “overall ion-transfer efficiency” (tip to detector).30 Second, the new geometry of the HCT enables higher ion storage capacity over conventional trap designs, leading to improved sensitivity in both full-scan MS and MS/MS modes and a broader dynamic range. Third, miniaturized LC columns with inner diameters of 100 µm provide increased sensitivity compared to conventional columns due to lower chromatographic dilution of the sample. Quantification of Acro-dG in ctDNA. The method was applied to the analysis of control ctDNA and acrolein-treated ctDNA. Acrolein-treated (10-fold acrolein) ctDNA (10 µg, with internal standard) was digested and prepared for analysis on an Oasis SPE column, and 50 ng of this sample was loaded on the capillary column. The concentration of Acro-dG was measured as 830 adducts in 106 normal nucleosides. The calculation is based on the equation “1 nmol of a lesion/mg of DNA ) 308 lesions/ 106 DNA bases”, which is derived from the percent levels of adenine, guanine, thymine, cytosine, and 5-methylcytosine nucleotides in mammalian DNA and their molecular weights. 50 Control ctDNA contained Acro-dG levels below the detection limit (S/N < 3) even though typical recoveries of the isotope-labeled internal standard were observed. The ability to manipulate and effectively utilize small in vivo DNA samples is an important and challenging aspect for analyses using capLC-nanoESI-IDMS/MS. The maximum sample loading for capillary LC-nanoESI-MS is limited by the column sample capacity, ESI process, and ruggedness of the method. Our study showed that when Acro-dG adduct levels were ∼1 in 106, 50 ng of DNA digest on column was sufficient for quantification. To quantify in vivo DNA samples with adduct levels as low as 100 in 109, a larger mass of DNA digest will be needed. For instance, Figure 4 shows an analysis of 1 µg of human nuclear DNA digest (50) Davidson, J. N. The biochemistry of nucleic acids; Chapman and Hall: New York, 1972.

5988

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

isolated from brain tissue from an AD where the Acro-dG content was calculated to be 3800 adducts in 109 normal nucleosides. The mass of DNA used in the method was significantly less than that used by 32P postlabeling methods that typically require 180 µg of ctDNA to quantify Acro-dG adducts.51 This level of sensitivity is an advantage of this method over conventional LC/MS methods that usually require more than 100 µg of DNA for quantification of DNA adducts. Quantification of Acro-dG in Human Brain Tissues. Gibson et al.52 demonstrated the presence of free acrolein in greater than 50% of AD neurofibrillary tangles and in dystrophic neurites surrounding senile plagues using immunostaining. Lovell et al. also demonstrated that free acrolein was significantly elevated in AD amygdala (2.5 ( 0.9 nmol/mg of protein) compared with controls (0.3 ( 0.05 nmol/mg of protein), and in hippocampus/parahippocampal gyrus in AD (5.0 ( 1.6 nmol/mg of protein) compared with control (0.7 ( 0.1 nmol/mg of protein) by using acrolein-cyclohexanedione derivatization HPLC analysis.5 To evaluate the acrolein-modified DNA adduct levels in AD and control subjects, a blind study for quantification of AdG-dG adducts in the hippocampus/parahippocampal gyrus of postmortem brains from AD and age-matched control subjects was carried out using this sensitive and selective capLC-NanoESI-IDMS/MS method. A total of 13 DNA samples from hippocampus/parahippocampal gyrus (AD and control, identities of the samples were unknown prior to the analysis). DNA sample were pure (UV A260 nm:A280 nm ) ∼1.8). Definitive diagnosis of AD was made at autopsy where the severity of the disease is assessed according to the Braak staging model, which evaluates the distribution pattern and density of neurofibrillary tangles and neuropil threads in the brain (Braak (51) Nath, R. G.; Chen, H. J. C.; Nishikawa, A.; Young-Sciame, R.; Chung, F. L. Carcinogenesis 1994, 15, 979-984. (52) Gibson, G. E.; Park, L. C.; Zhang, H.; Sorbi, S.; Calingasan, N. Y. Ann. N. Y. Acad. Sci. 1999, 893, 79-94.

as diet and smoking. Our study indicated statistically significant differences (P < 0.05) in Acro-dG adducts in the hippocampus/ parahippocampal gyrus of AD subjects compared with agematched control subjects. This result may have biological implications in the pathogenesis of Alzheimer’s disease because the hippocampus is a key region affected by the pathology of Alzheimer’s disease.

Figure 5. Acro-dG adducts/109 nucleosides observed in hippocampus/parahippocampal gyrus of AD and control subjects (P ) 0.025).

1-2 ) very little AD pathology, Braak 3-4 ) moderate AD pathology, Braak 5-6 ) advanced AD pathology).53 Representative DNA samples (10 µg of each) were hydrolyzed in the presence of an internal standard. Only 1 or 2 µg of the DNA digests was loaded on the capillary column and analyzed according to the method described earlier. The mean ( SEM ages, postmortem intervals (PMI), and Acro-dG adducts (adducts/109 nucleosides) between AD and control subjects in hippocampus/parahippocampal gyrus are presented in Table 1. For AD subjects, the range of the Acro-dG adducts (per 109 normal nucleosides) was from 3360 to 7990, with a mean ( SEM of 5150 ( 640. In control subjects, the levels of Acro-dG adducts (per 109 normal nucleosides) ranged from 1060 to 3720, with a mean ( SEM of 2800 ( 460. The elevated levels of Acro-dG observed in the AD subjects were significantly increased compared to controls (P ) 0.025) as illustrated in Figure 5. The detection and quantification of DNA adducts in human subjects is multivariate due to large subject-to-subject variability, exposure to complex mixtures, or other confounding factors such (53) (a) Braak, H.; Braak, E. Acta Neuropathol. 1991, 82, 239-259. (b) Braak, H.; Braak, E. Neurobiol. Aging 1997, 18, S85-88. (c) Braak, H.; Braak, E. In Neurodegenerative Diseases, Caine, D. B., Ed.; W. B. Saunders Co.: Philadelphia, 1994; pp 585-618.

CONCLUSIONS In postmitotic cells such as neurons, the presence of DNA adducts could affect binding of transcription factors, thus causing changes in critical protein expression. In light of widespread exposure to acrolein and its ability to react with DNA generating DNA adducts, we sought to develop a sensitive, selective method for the detection and quantification of Arco-dG adducts in DNA by using capLC-nanoESI-IDMS/MS. Current methods offer attomole sensitivity when measuring pure Acro-dG standard. This method could be applied in quantification of endogenous generated Acro-dG from DNA samples of human subjects using only 1-2 µg of DNA, an important advantage for studies of human samples. Improved analytical performance compared to the 32P postlabeling method was achieved by using 100-µm-i.d. slurrypacked RP-Polymer X capillary columns coupled with a highcapacity ion trap tandem mass spectrometer in the MRM mode, efficient sample cleanup obtained with a Waters Oasis HLB SPE, high quantification accuracy achieved by use of an isotopically labeled internal standard. Application of this method for the quantification of Acro-dG adducts in human brain nuclear DNA samples from AD and agematched controls showed sufficient sensitivity to observe ∼1000 Acro-dG adducts in 109 normal nucleosides. The method provides a means to correlate the incidence of the acrolein DNA adducts with certain disease processes. ACKNOWLEDGMENT This work was supported by grants from the National Institutes of Health (5P50-AG05114 and 5P01-AG05119) and the Abercrombie Foundation. X.L. acknowledges support from the Research Challenge Trust Funds Fellowship from University of Kentucky. Received for review April 12, 2005. Accepted July 6, 2005. AC050624T

Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

5989