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Published in final edited form as: Toxicol Lett. 2019 Apr 23;311:11–16. doi: 10.1016/j.toxlet.2019.04.020

Detection and Quantification of 4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB) from Smoker Albumin and its Potential as a Surrogate Biomarker of Tobacco-Specific Nitrosamines Exposure and Bioactivation

Yi Wang a, Sreekanth Chanickal Narayanapillai a, Qi Hu a, Naomi Fujioka b, Chengguo Xing a,†
PMCID: PMC6551521  NIHMSID: NIHMS1528374  PMID: 31026483

Abstract

4-(Methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), two tobacco specific nitrosamine carcinogens, can form adducts with DNA and proteins via pyridyloxobutylation upon phase I enzyme-mediated bioactivation. Such DNA modifications have been proposed as the root cause to initiate carcinogenesis. Upon hydrolysis, both DNA and protein modifications would release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). The released HPB, being tobacco carcinogen specific, has the potential to serve as a surrogate biomarker for both tobacco exposure and carcinogen bioactivation. Because of its easy access, blood is a great source of such investigations with the potential in epidemiological application. HPB quantification from haemoglobin (Hb), however, has been demonstrated with limited success. To further explore this potentially paradigm-shift opportunity, we reported, for the first time, the detection and quantification of HPB from albumin (Alb) adducts formed by the tobacco-specific nitrosamines in mice and in human smokers. The time-course quantitative analysis of HPB from mouse Alb upon NNK exposure suggests that such an Alb adduct is stable. The amounts of HPB from Alb adducts in smoker plasma averaged 1.82 ± 0.19 pg/mg Alb (0.42 to 3.11 pg/mg Alb), which was 36 times the value in nonsmokers (0.05 ± 0.01 pg/mg Alb). Importantly, HPB level from Alb correlated positively with the level of human tobacco exposure estimated by urinary total nicotine equivalent (TNE) (R2=0.6170). For comparison, HPB level from Alb was 16.5 times that of Hb (0.12 ± 0.02 pg/mg Hb) in the plasma and red blood cell (RBC) samples of the same smokers. In addition, there was no significant correlation between HPB levels from Hb and TNE (R2=0.0719). These data overall suggest that HPB from Alb adducts can serve as a surrogate biomarker to monitor the level of tobacco exposure and carcinogenic nitrosamine bioactivation.

Keywords: protein biomarker, smoker, NNK, NNN, HPB, tobacco

1. Introduction

Tobacco use is one leading risk factor for cancers, particularly lung cancer (Siegel et al., 2016). Two tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and N-nitrosonornicotine (NNN, 2), induce lung and esophagus tumorigenesis respectively in various species (Hecht, 2003). Both chemicals have been categorized as group 1 carcinogens to humans by International Agency for Research on Cancer (IARC) (Organization and Cancer, 2007). Mechanistically, NNK and NNN can be bioactivated by phase I enzymes via α-hydroxylation to generate an electrophilic diazonium cation (3), which can react with nucleophiles on DNA or proteins to form adducts (Hecht, 1998, 2003). Quantitative monitoring of such DNA and protein adducts is expected to be useful for cancer risk prediction (Hecht, 2017; Hecht et al., 2016). Hydrolysis of such adducts from DNA or proteins will release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 4) (Scheme 1), which is tobacco specific and may be quantified to estimate NNK and NNN exposure/bioactivation.

Scheme 1.

Scheme 1.

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Formation of protein and DNA adducts by reactive metabolites of NNK and NNN, and hydrolysis to release HPB.

Although DNA adducts provide an additional indication on mutagenesis, protein adducts have several potential advantages as surrogate biomarkers. First of all, some DNA adducts are subjected to enzyme-mediated repairs (Turesky and Le Marchand, 2011). Their potential as surrogates may be compromised by the heterogeneity of the repair capacity among individuals. Protein adducts on the other hand are not known yet to be repaired until protein degradation. Second, the availability of human tissues is usually limited for DNA-based analysis, which makes it challenging to quantify trace-amount DNA adducts from the limited human tissues even with the most sensitive instruments (Turesky and Le Marchand, 2011). This is particularly problematic for epidemiological and longitudinal investigations due to the additional challenges associated with tissue sample acquisition. In contrast, human blood samples are easier to access with milligrams of albumin (Alb) and hemoglobin (Hb) recovered from 1 mL human blood (Tornqvist et al., 2002). Lastly, the half-life of Alb is around 30 days and the lifetime of red blood cells is around 120 days in human (Peters, 1985). Thus, the level of stable carcinogen adducts on Alb and Hb, such as HPB, may reflect long-term carcinogen exposure/bioactivation, potentially more predictive of cancer risk. HPB adducts on Hb have been explored by Carmella et al (Carmella and Hecht, 1987; Carmella et al., 1990a; Carmella et al., 1990b). Disappointly, the abundance differences between smokers and non-smokers were only ~ 2.7 folds; HPB was not detectable in 11 out of 40 smoker samples tested while it was not detected in only 4 out of 21 nonsmoker samples (Carmella and Hecht, 1987; Carmella et al., 1990a; Carmella et al., 1990b). These results overall suggest that HPB from Hb is not a promising biomarker candidate for NNK/NNN exposure and bioactivation.

Adduct formation of heterocyclic aromatic amines with Alb and Hb demonstrates that the adduct abundance on Alb and Hb may be carcinogen dependent (Sabbioni and Turesky, 2016; Turesky and Le Marchand, 2011). The objective of this study is test whether HPB from Alb can serve as a more promising surrogate biomarker. We detected HPB from A/J mouse Alb upon NNK exposure and characterized its time-course abundance via ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) coupled with high-resolution accurate-mass Orbitrap. Importnatly, we successfully detected and quanitifed HPB from Alb in all human smokers evaluated (n = 21) and its level was 36 times of that in non-smokers (n = 15). We also compared the levels of Alb HPB with Hb HPB from the same participants (n = 21). The results overall support the potential of Alb HPB as a surrogate biomarker for both tobacco exposure and tobacco specific nitrosamine carcinogen bioactivation.

2. Materials and methods

Caution: NNK is a human carcinogen and must be handled in well ventilated hoods with appropriate clothing.

2.1. Chemicals and Materials

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB) and 4-hydroxy-1-(3-pyridyl)-1-[2H4]-butanone ([2H4]HPB) were purchased from Toronto Research Chemical (Toronto, ON, Canada). Nicotine, [2H3]-nicotine, cotinine, [2H3]-cotinine, trans-3’-hydroxycotinine and trans-3’-[2H3]-hydroxycotinine were from Sigma-Aldrich (St. Louis, MO). LC/MS grade of water, formic acid, methanol and acetonitrile were from Fisher Scientific (Fair Lawn, NJ). Pierce BCA Protein Assay Kit was from Thermo Fisher (Rockford, IL). Pierce Albumin Depletion Kit was from Thermo Fisher (Rockford, IL). Amicon Ultra centrifugal filter units (10,000 molecular weight cuto) were from Millipore (Billerica, MA). StrataX reverse phase cartridges (33 μm, 30 mg/1 mL) were from Phenomenex (Torrance, CA). Other chemicals were from Sigma-Aldrich unless stated.

2.2. A/J mouse study

All mice were housed, tested, and cared for in accordance with the 2011 National Institutes of Health Guide for the Care and Use of Laboratory Animals, and handled according to the animal welfare protocols approved by Institutional Animal Care and Use Committee at the University of Florida. Five-week-old female A/J mice were purchased from Jackson Lab. After one week of acclimatization, mice were randomized into different groups (N= 3 except for the 0.5-h time point with 5 mice). Mice were dosed with NNK (100 mg/kg bodyweight) in 200 μL sterilized saline via i.p. injection, and control mice were given 200 μL saline. This dose of NNK has been widely used in the A/J mouse model to characterize lung tumor formation and its associated DNA damage (Hecht, 1998). Mouse sera were collected at 0.5, 8, 24 and 96 hours after NNK administration, snap-frozen in liquid nitrogen and stored at −80 ºC.

2.3. Human Samples

Plasma, red blood cells, and matched urine samples from 21 human smokers were obtained from a clinical trial conducted at the University of Minnesota. Those samples were stored at −80 °C within 4 hours after collection. The study was approved by the IRB at the University of Minnesota and all subjects provided informed, written consent. Plasma and urine samples from the same nonsmoker individuals were purchased from Bioreclamation IVT (Bioreclamation Inc., Baltimore, MD). Demographic information of smokers and nonsmokers were summarized in Table S3.

2.4. Purification of Alb and Hb

Free HPB in human plasma or mouse sera, produced from the hydrolysis of NNK and N-nitrosonornicotine (NNN) reactive intermediate (Jalas et al., 2003), was removed by ethyl acetate extraction. Briefly, mouse sera (10 μL) or human plasma (150 μL) was extracted with ethyl acetate (1 mL). Mouse sera was mixed with 10 mM potassium phosphate buffer (pH 7.4) (100 μL) before ethyl acetate extraction. The aqueous solution was then subjected to dialysis [450 μL of 10 mM potassium phosphate buffer (pH 7.4), three times] using centrifugal filters (10k cutoff). Alb was purified from the desalted solution using the Pierce Albumin Depletion Kit following the manufacture protocol (Wang et al., 2015). Alb eluted from the purification kit (10 mM potassium phosphate buffer (pH 7.4) with 1.5 M KCl) was desalted again as described above. Isolation and enrichment of Hb from red blood cells was performed as previously described (Pathak et al., 2016). Enriched Hb was subjected to a buffer exchange. Alb concentration was determined by using the BCA assay. HbO2 concentration was estimated by UV spectrophotometer at 542 nm (Pathak et al., 2016).

2.5. Hydrolysis of Alb and Hb to release HPB

Alb or Hb solution (3–7 mg in 100–150 μL), with the addition of [2H4]HPB (25 pg), was mixed with KOH (final concentration 0.1 N), and agitated on a Thermo Mixer (700 rpm) at 37 °C for 6 h. The mixture was neutralized with HCl (0.1 N), mixed with cold C2H5OH (1 mL) to precipitate proteins and centrifuged at 13000 g for 20 min. The supernatant was concentrated to ~100 μL via speed vacuum. The solution was diluted with H2O (1 mL) and applied to StrataX preconditioned with C2H5OH (1 mL) and H2O (2 mL). The cartridge was washed with 10% C2H5OH (2 mL) followed with H2O (2 mL), and eluted with C2H5OH (1 mL). The eluent was speed vacuumed to dryness and the residue was dissolved in 25 μL 10 mM ammonium acetate in H2O for UHPLC-MS/MS analysis.

2.6. UHPLC-MS/MS analyses of HPB from Alb-HPB and Hb-HPB adducts

Mass spectral data of HPB and [2H4]HPB were acquired by direct infusion with a Q Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer (Thermo Fisher, San Jose, CA) and a Heated Electrospray Ionization (HESI-II) source (Thermo Fisher, San Jose, CA). The flow rate of direct infusion was set at 10 μL/min. Instrument tuning parameters were as follows: sheath gas, 5; auxiliary gas, 0; auxiliary gas temperature, 50 °C; capillary temperature, 300 °C; spray voltage, 4 kV; 1 μscan; maximum injection time, 200 and 1000 ms for full MS and MSn respectively; HCD, 20 for MS2. Resolution was set as 70,000 and 17,500 at m/z 200 for full MS and MSn respectively. The isolation width was set at m/z 1.5 for MS2 scan modes. All analyses were conducted in the positive ionization mode.

HPB from Alb and Hb was assayed by targeted UHPLC-MS2 consisting of a Dionex Ultimate 3000 RS and a Q Exactive Hybrid Quadrupole Orbitrap Mass Spectrometer. The Orbitrap was configured as Parallel Reaction Monitoring (PRM) mode for data acquisition. HPB samples (20 μL) were resolved through a Waters ACQUITY HSS T3 C18 column (150 × 2.1 mm, 1.8 μm particle size, 100 Å) with a 20-min linear gradient from 99% A (2 mM ammonium acetate in 10% CH3CN) to 80% B (2 mM ammonium acetate in CH3CN with 10% H2O) at a flow rate of 150 μL/min. Chromeleon 7.2 Chromatography Data System was used for the UHPLC management. The parameters for HESI-II were set as follows: sheath gas, 35; auxiliary gas, 10; auxiliary gas temperature, 200 °C; capillary temperature, 300 °C; spray voltage, 4 kV; 1 μscan; maximum injection time, 1000 ms for MS2; HCD, 20. Resolution was set as 17,500 at m/z 200 for MS2. The isolation width was set at m/z 1 for MSn scan mode. AGC (automated gain control) was set at 50,000 for Orbitrap (FT) MS2. The Orbitrap was routinely calibrated in the positive ion mode using Pierce LTQ Velos ESI Positive Ion Calibration Solution (2 μg/mL caffeine, 1 μg/mL MRFA, 0.001% Ultramark 1621 and 0.00005% n-butylamine).

2.7. Comparison of acid and base hydrolysis of Alb-HPB adducts

To explore the hydrolysis efficacy of acid and base treatments, Alb from the sera of 0.5-h NNK-treated mice was used. Purified Alb was subjected to hydrolysis with HCl (0.1 N) or KOH (0.1 N) at 37 °C for 6 h. Three biological replicates were performed for each treatments following the procedures detailed above.

2.8. Demonstration of the efficient removal of free HPB

To ensure our sample preparation procedures efficiently removed free unbound HPB, nonsmoker plasma (150 μL) was mixed with HPB standards (120 ng), followed by ethyl acetate extraction, albumin purification, and UHPLC-MS/MS quantification for HPB as described above.

2.9. Measurement of urinary total nicotine equivalent (TNE) and creatinine

TNE in the urine samples of human nonsmokers and smokers was measured as previously described (Murphy et al., 2014) to confirm their smoking status. Creatinine was measured using a modified Jaffe reaction method (Slot, 1965). The level of TNE was expressed as ng/mg creatinine.

2.10. Calibration Curve

An eight-point calibration curve was constructed by adding [2H4]HPB (5 pg/mg protein) and varying ratios of HPB to [2H4]HPB (0, 0.05, 0.1, 0.25, 0.5, 2.5, 5, 10 pg HPB/mg protein) into pooled non-smoker plasma samples. The amounts of [2H4]HPB internal standard and the range of HPB were selected based on a previous study on the level of Hb-HPB adducts in smokers (Carmella et al., 1990a). Limit of detection (LOD) and limit of quantification (LOQ) were estimated by 3.3σ/s and 10σ/s, respectively (σ is the standard deviation of the slope (s) of the calibration curve) (Food and Administration, 2001).

2.11. Method validation

Method validation followed the FDA guideline (Food and Administration, 2001). Performance of this method was evaluated for precision, accuracy, within-day and between-day reproducibility by six measurement of the addition of known amounts of HPB with four different concentrations to the control matrices. Accuracy was expressed as the percentage of the mean of measured values to theoretical values.

2.12. Statistical analyses

GraphPad Prism (v. 7.00) was used for statistical analyses. Two-tailed unpaired t test was performed for the analysis of the Alb-HPB adducts between smokers and non-smokers. Two-tailed paired t test was performed for the analysis of Alb-HPB and Hb-HPB adducts in smokers. P < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Method validation

The method was validated using pooled plasma from human nonsmokers. HPB standard was added at four different levels, ranging from lower limit of quantification (LLOQ) to high levels. Limit of detection (LOD) was estimated to be 11.3 fg/mg Alb (~5 HPB per 109 Alb molecules) (Fig. S1). The sensitivity of this method was comparable to those that measure protein adducts formed with heterocyclic aromatic amines (Bellamri et al., 2018; Wang et al., 2017). The accuracy ranged between 93.0–110.0%. The intraday and interday variations were 2.5–11.2% and 2.4–12.6%, respectively (Table S1). As illustrated in scheme 1, direct hydrolysis of the diazonium intermediate (3) from NNK and NNN intermediate also forms HPB (4), which needs to be completely removed before quantification of Alb-bound HPB. To demonstrate this, non-smoker plasma (150 μL) was added with HPB standards (120 ng). UHPLC-MS/MS analyses showed that solvent extraction and albumin purification removed >99.9% of the free HPB (Fig. S4). Furthermore, the potential influence from residual free HPB was negligible relative to HPB from Alb in smokers (Fig. S4 and Fig. 3). These results overall demonstrate the feasibility of our analytical method and sample processing method.

Fig. 3.

Fig. 3.

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(Left panel) reconstructed ion chromatograms of HPB in human Alb and Hb upon basic hydrolysis. Scales of the ion signals were normalized to the response of HPB from Alb of the smoker. Mass tolerance, ± 5 ppm. (Right panel) Correlation of Alb-HPB and Hb-HPB with urinary TNE.

3.2. Mass spectrometric characterization of HPB-Alb adducts in mice

We first tested whether Alb-HPB adducts could be detected in A/J mice upon nitrosamine exposure using NNK as the model carcinogen. The dose of NNK used herein has been widely employed to characterize NNK-induced DNA damage and tumor burden in the A/J mouse lung carcinogenesis model (Hecht, 1998). Briefly, six-week old female A/J mice were given a single dose of NNK (100 mg/kg bodyweight) via i.p. injection with sera collected at different time points (0.5, 8, 24 and 96 hours after NNK administration). Mice without NNK treatment served as the control. Using the 0.5-h serum sample, HPB recovered from Alb upon base hydrolysis was about 4 times as that from acid hydrolysis (Table S2), consistent with a previous report of a higher recovery of Hb-HPB adducts with base treatment (Carmella and Hecht, 1987). Base hydrolysis was used in the rest of the studies. Alb-HPB adducts were detected in all NNK-treated mice (Fig. 1A), ranging from 747 ± 258 pg/mg Alb at 0.5 h to 113 ± 24 pg/mg Alb at 96 h (Fig. 1A). Reconstructed ion chromatograms of HPB from mouse sera were shown in Fig. S2. A simple first-order exponential decay fitting of Alb-HPB adduct estimated its half-life (t1/2) to be 19.6 h (Fig. 1B), comparable to the half-life of mouse Alb (Dixon et al., 1953). This suggests that Alb-HPB adducts are likely stable. If such adducts are stable in humans, HPB quantity may reflect the exposure and activation of NNK over a period of time, which may have more predictive power of carcinogenesis risk.

Fig. 1.

Fig. 1.

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HPB from of A/J mouse Alb upon NNK treatment. (A) The amount of HPB in different treatment groups (n = 3 per group). (B) A first-order exponential decay fitting of Alb-HPB adducts.

3.3. Mass spectrometric characterization of HPB-Alb adducts in human nonsmokers and smokers

We next quantified HPB from Alb in the plasma from twenty-one smokers and fifteen non-smokers (demographic information in Table S1). The smoker plasma samples were collected from a clinical trial (NCT02500472) while the non-smoker plasma were purchased from Bioreclamation IVT. The smoking status of these subjects was verified by quantifying their urinary TNE, which is the sum of nicotine-N-oxide, free and glucuronides of nicotine, cotinine and 3-hydroxycotinine that provides an estimate of tobacco exposure/use (Murphy et al., 2014). The urinary TNE values in smokers ranged between 40 – 425 nmol/mg creatinine, and the average TNE value (152.5 ± 18.3 nmol/mg creatinine) was 92 times the level in nonsmokers (1.7 ± 0.4 nmol/mg creatinine) (Fig. S3), which corroborates their smoking and non-smoking status.

HPB from Alb-HPB was successfully detected in all plasma samples from smokers with the concentrations ranging from 0.42 to 3.11 pg/mg Alb, which were 12 – 91 times above LOQ (Fig. 2). The significant variations of HPB levels among the smokers may indicate different carcinogenic risk from NNK and NNN. The mean value (1.82 ± 0.19 pg/mg Alb) was 36 times the mean value in nonsmokers (0.05 ± 0.01 pg/mg Alb) (Fig. 2), indicating its high dependence of tobacco use. Even the lowest level of HPB in the smoker plasma was 3.3 times of the highest among the non-smokers. The levels of HPB from the non-smoker plasma, on the other hand, were mostly around or slightly above LOQ, again supporting its specificity to tobacco use/exposure. Figure 3 shows the representative reconstructed ion chromatograms of HPB from human nonsmoker and smoker plasma. Mass spectra of HPB and [2H4]HPB recovered from the human sample had excellent agreement with the spectra of their pure standards (Fig. S5). Since urinary TNE has been used to assess the amount of tobacco exposure (Murphy et al., 2014), we explored the relationship between urinary TNE and Alb-HPB adduct (Fig. 3), which demonstrated a positive linear correlation with R2=0.6170. These data overall suggest that Alb-HPB has the potential as a molecular dosimetry to assess human exposure to tobacco smoke. Alb-HPB also captures NNK and NNN bioactivation, which cannot be reflected by either urinary TNE or urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (Yuan et al., 2014). Alb-HPB therefore may be a better surrogate biomarker for cancer risk prediction relative to TNE and NNAL.

Fig. 2.

Fig. 2.

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HPB from Alb (nonsmokers and smokers) and Hb (smokers). HPB of Alb and Hb from smokers were analyzed with two tailed paired t-test. Two-tailed unpaired t-test for HPB from Alb between smokers and non-smokers. ****, p<0.0001.

3.4. Comparison of HPB-Alb and HPB-Hb adducts in human smokers

We next compared the biomarker potential of HPB from Alb and Hb by quantifying the amount of HPB recovered from proteins adducts formed with Alb and Hb in 21 smokers. The mean value of HPB from Alb was 16.5 times that from Hb (0.12 ± 0.02 pg/mg Hb) (Fig. 2). There was also minimal linear correlation between urinary TNE vs. Hb-HPB with R2=0.0719 (Fig. 3). In addition, the level of HPB on Hb from three sample were around LOQ. These data overall suggest that HPB from Alb would be a more promising biomarker than HPB from Hb.

4. Conclusions

In summary, we detected and quantified HPB from Alb-HPB adducts derived from NNK and NNN in human smoker and nonsmoker plasma (150 μL) by targeted UHPLC-MS/MS. HPB from Alb has the potential as a practical surrogate biomarker to assess human exposure and bioactivation of NNK and NNN for cancer risk prediction. First, the abundance differences of Alb-HPB between smokers and non-smokers are much more pronounced than that of Hb-HPB and there is a positive linear correlation of Alb-HPB adducts with urinary TNE. Second, its abundance was estimated to be about 8 HPB adducts per 107 Alb molecules (1.8 pg/mg Alb) in smokers, which is >100 times higher than the reported levels of DNA adducts in human (Hecht, 2017; Xiao et al., 2016). Limited amount of plasma is sufficient for its quantification. Thirdly, Alb-HPB adducts, based on the A/J mouse time-course data, are stable. The stable protein adducts may contribute to its high cumulative abundance and allow the assessment of long-term exposure which might have better diagnostic value for cancer risk prediction. Lastly and more importantly, it is much easier to acquire human blood samples for Alb-HPB analysis than human tissues for DNA adducts.

Supplementary Material

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2

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Acknowledgements

We would like to thank Jordan Paladino and Vickie Nguyen for the help of sample collection and preparation. We would also thank the Department of Medicinal Chemistry at University of Florida for the Orbitrap mass spectrometry usage.

Funding Sources

The work was part-funded by R01CA193286 (CX), University of Minnesota Masonic Cancer pilot program (NF and CX), University of Florida Medicinal Chemistry Mass Spectrometry Support (CX), College of Pharmacy Frank Duckworth Endowment (CX), University of Florida Health Cancer Center Startup fund (CX), and Florida Cancer Health Disparities-Focused NCI SPORE (CX).

Footnotes

Conflicts of interest

The authors declare no competing financial interest.

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Associated Data

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Supplementary Materials

1
NIHMS1528374-supplement-1.docx (552.7KB, docx)
2
NIHMS1528374-supplement-2.docx (509.2KB, docx)

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