The Amadori product 2 was prepared as reported (17). Nornicotine (250 mg, 1.69 mmol) and glucose (258 mg, 1.43 mmol) were dissolved in dry methanol (3.25 ml) and heated to reflux for 1 h. Malic acid (19 mg, 0.14 mmol) was then added, and the reaction was refluxed for an additional 2 h. The mixture was then concentrated under reduced pressure, and water (3 ml) was added to the residue. This solution was passed through a column of Amberlyte IR-120(plus) strongly acidic ion exchange resin and the column washed with deionized water (65 ml). The desired crude compound was then eluted with 2 M NH₄OH (125 ml), and the combined fractions concentrated under reduced pressure to remove any residual NH₃. The resulting aqueous solution was lyophilized to give a white powder, which was further purified on silica by using ethanol/water (30:70) as the eluent. (167 mg, 32%).

Nornicotine (10 mM) was incubated in a solution of glucose (200 mM) in PBS (200 mM, pH 7.4). The reaction was protected from light and heated to 37°C. At given time intervals, aliquots of the reaction (100 μl) were taken and analyzed by electrospray ionization MS for the presence of the Amadori product (m/z = 310). Additionally, aliquots (20 μl) were removed and diluted to a final volume of 1 ml with phosphate buffer. Aliquots of these diluted solutions (20 μl) were then removed and injected onto the HPLC system described above (HPLC conditions: isocratic mobile phase consisting of water with 0.1% trifluoroacetic acid. Solvent flow rate of 1 mlmin⁻¹ and detection at 254 nm). Independent confirmation of the Amadori product in these reactions was accomplished by coinjection with a known synthetically pure standard (retention time = 4.90 min).

Glucose (200 mM) was added to PBS (200 mM; pH 7.4) to obtain a glucose-enriched buffer. The protein in question [RNase A, BSA, or human serum albumin (HSA)] was then dissolved in this buffer in an microcentrifuge tube to attain a final concentration of protein of 10 mg/ml (final reaction volume = 1 ml). Immediately after the addition of protein, nornicotine was added to the reaction (0.77 μM), and the solution was filtered through a 0.2 μM syringe filter. The reactions were incubated at 37°C in the dark, and aliquots were removed at given time intervals, diluted 1:1,000 with PBS, and analyzed by ELISA.

Monoclonal antibody NIC6C12 does not react significantly with HSA, BSA, or RNase A at 1:1,000 dilution. Serum samples were obtained from subjects after informed consent was given in accordance with a protocol approved by the Institution Review Board at The Scripps Research Institute. Although some reactivity was observed with certain plasma proteins (see Results and Discussion), this did not interfere with the specific detection of glycated proteins in serum samples. Western blot analysis was performed by using 10% NuPage polyacrylamide gels (Invitrogen) according to published procedures (18). Blots were developed by using either the colorimetric reagent TMB (3,3′5,5′-tetramethylbenzidine; Pierce), or SuperSignal West Pico chemoluminescent horseradish peroxidase substrate (Pierce).

1D ¹H NMR spectra were acquired by using a standard pulse sequence with water suppression (512 scans) on a Bruker DRX 600 spectrometer at 298 K.

Immunoassays for the detection of glycation products have attracted significant attention (22). Although previous immunoassays have proven effective for the detection of AGE's both in vitro and in vivo, the inherent heterogeneity of both the antigen and polyclonal immune response significantly complicates understanding the chemical nature of in vivo glycation (23). To provide evidence that 2 can glycate proteins, a rapid and sensitive high-throughput immunoassay was developed to facilitate specific detection of nornicotine-derived protein glycation products. With this goal in mind, we chose to use the nornicotine alkaloid nucleus as the primary antigenic determinant. Our reasoning was based on our knowledge that the glucose-derived crosslinking region may undergo a variety of chemical transformations over time, whereas the 3-pyrrolidine-2-yl-pyridine nucleus is expected to be conserved. Thus, antibodies were raised against hapten 3 (Fig. ​(Fig.3),3), which prominently features the 3-pyrrolidine-2-yl-pyridine nucleus in enantiopure form (24). Our initial efforts focused on the detection of glycation of the model protein, RNase A (25). Nornicotine, glucose, and RNase A were incubated in buffer and aliquots of the mixture screened at given time points for binding to our panel of antibodies. Under our reaction conditions (see Materials and Methods), detectable glycation was first observed by ELISA after 7 days. Specific glycation resulting from nornicotine was detectable by using mAb NIC6C12, causing us to postulate that the recognized epitope of this antibody resides with the nornicotine alkaloid nucleus. As confirmation of this hypothesis, molecules that contain this nucleus such as nicotine and nornicotine, as well as 2 and nornicotine glycated RNase A, were found to compete for binding by ELISA (Table 1). To extend our studies to other proteins, we examined the glycation of HSA and BSA. Nornicotine-based glycation of these proteins occurred readily and again, only glycation in the presence of nornicotine and glucose could be detected by using mAb NIC6C12 (Fig. ​(Fig.44A). This assay is very sensitive, as glycation can be detected with as little as one covalently attached nornicotine molecule per protein. Even after extended incubations (>7 months), the nornicotine moiety remains covalently bound to the protein, implying that, similar to other reported AGE structures, the nornicotine-derived AGE also forms irreversibly. This result strongly suggests that the nornicotine AGE could exist for extended periods of time when attached to a suitable protein leading to increased antigenicity (25) and/or irreversible loss of protein function (26).

Previously, ¹H NMR has been exploited to directly observe conformationally flexible regions on protein surfaces as increased flexibility leads to less line broadening because of longer T₂ relaxation times (27). Although we could not directly detect covalently bound nornicotine moieties on the surface of BSA by NMR, we were able to develop a qualitative test for nornicotine-based glycation using 1D ¹H NMR. Proteins with glucose-based glycation displayed sharply defined peaks different from the native protein, whereas proteins that had been incubated with nornicotine and glucose displayed no analogous sharp peaks, possibly indicating a loss of conformational flexibility on nornicotine-based glycation (data not shown).

To determine whether nornicotine-derived glycation occurred preferentially in smokers, a set of plasma samples from both smoker and nonsmokers was analyzed by ELISA. The plasma of smokers was found to contain statistically higher amounts of nornicotine-modified proteins than the plasma of nonsmokers (P = 0.015, n = 10, unpaired Student's t test). Because mAb NIC6C12 displays cross-reactivity with certain high molecular weight species in plasma in some, but not all, nonsmokers (Fig. ​(Fig.44B, lanes 2–7), these proteins cannot be considered markers of smoking-related protein modifications. Our assumption was that this observed reactivity was caused by either recognition of NIC6C12 by serum proteins that bind antibody constant regions (e.g., C1q) or other immunoglobulins present in the serum. However, heat treatment of the serum before ELISA or treatment with immobilized protein G had little effect on the strength of the observed signal. Furthermore, assays using either the F(ab′)₂, Fab, or scFv fragments of NIC6C12 showed no significant reduction of this polyreactivity. Despite this complication, the lower molecular weight nornicotine-glycated proteins occur only in the serum of smokers (Fig. ​(Fig.44B, lanes 9, 10, and 13). As noted previously, nornicotine has been found in minor amounts both in tobacco and as a nicotine metabolite (see above). In light of these findings, it is evident that its concentration and extended half-life in vivo are sufficient to cause aberrant protein modification.