UV light absorption parameters of the pathobiologically implicated bilirubin oxidation products, MVM, BOX A, and BOX B

The formation of the bilirubin oxidation products (BOXes), BOX A ([4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide]) and BOX B (3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide), as well as MVM (4-methyl-3-vinylmaleimide) were synthesized by oxidation of bilirubin with H2O2 without and with FeCl3, respectively. Compound identity was confirmed with NMR and mass spectrometry (MS; less than 1 ppm, tandem MS up to MS4). UV absorption profiles, including λmax, and extinction coefficient (ε; estimated using NMR) for BOX A, BOX B, and MVM in H2O, 15% CH3CN plus 10 mM CF3CO2H, CH3CN, CHCl3, CH2Cl2, and 0.9% NaCl were determined. At longer wavelengths, λmax's for 1) BOX A were little affected by the solvent, ranging from 295–297 nm; 2) BOX B, less polar solvent yielded λmax's of lower wavelength, with values ranging from 308–313 nm, and 3) MVM, less polar solvent yielded λmax's of higher wavelength, with values ranging from 318–327 nm. Estimated ε’s for BOX A and BOX B were approximately 5- to 10-fold greater than for MVM.


Subject area
Chemistry More specific subject area Bilirubin oxidation products detection Type of data Table, figure How data was acquired NMR, mass spectroscopy, UV spectrometry, HPLC Data format Raw, analyzed Experimental factors Oxidation of bilirubin, extraction with chloroform Experimental features Bilirubin oxidation products BOX A, BOX B, and MVM were synthesized by the oxidation of bilirubin, purified by HPLC and UV absorption profiles and extinction coefficients determined Data source location Cincinnati, OH USA Data accessibility The data are accessible within the article.

Value of the data
First report (to our knowledge) of UV absorption profile, including λ max , of MVM in solvents relevant to detection in biologic/pathobiologic samples.

Comparison of UV absorption profiles of MVM with BOX A and BOX B.
First report (to our knowledge) of BOX B extinction coefficient (ε; estimated using NMR), along with comparison to BOX A and MVM estimated ε's in different solvents, along with MS at less than 1 ppm and tandem MS up to MS 4 .
Novel methodology to increase MVM yield through FeCl 3 inclusion in oxidation reaction mixture. Data will potentially assist in the detection and determination of these BOXes in pathobiologies associated with elevated bilirubin.

Data
The bilirubin oxidation products (BOXes), MVM (4-methyl-3-vinylmaleimide), along with BOX A ([4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide]) and BOX B (3-methyl-5-oxo-4vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide), have been implicated in the deleterious effects associated with subarachnoid hemorrhage (SAH; [1][2][3][4][5]). The detection method utilized to determine the presence of these compounds is UV absorption associated with reversed phase-HPLC [1]. However, reports (to our knowledge) of the UV absorption profile and/or λ max of MVM have not been reported for the solvent utilized in their detection (H 2 O/CH 3 CN), but are limited to CH 3 OH [6,7]. Also, reports of these absorption characteristics are limited (to our knowledge) for BOX A to H 2 O and CH 3 CN, and for BOX B to H 2 O [1,8]. Further, extinction coefficients (ε) for MVM and BOX A are limited (to our knowledge) to CH 3 OH and CH 3 CN, respectively [6,7,9], and are lacking for BOX B. Thus, it is anticipated that the present data will assist in the detection and quantitative determination of BOXes levels in biologic samples from SAH, as well as in other pathobiologies associated with elevated bilirubin.

Synthesis
Bilirubin solubilization was performed at room temperature in an aluminum foil wrapped vessel due to the reported light sensitivity of BOX A, BOX B, and MVM [1,8,10]. One or more 50 mg portions of bilirubin were incubated in 25 ml 0.2 M NaOH(aq) with occasional vortexing over 24-72 h [1,10]. The dark red bilirubin solution was then buffered by addition of 7.5 ml of 0.5 M Tris base before neutralization with 0.4 ml of 12.3 M HCl(aq) to pH 7.0. Overtitration of the dark red solution to lower pH resulted in a green solution. The neutralized (pH 7) buffered bilirubin solution was immediately used for oxidation with H 2 O 2 . With prolonged storage, bilirubin precipitated from this supersaturated solution.
As performed under dim ambient light and in an unlit fume hood (and with dim ambient light) the neutral buffered solution (now in 0.1 M TrisHCl, pH 7.0, 0.15 M NaCl) was oxidized for 24 h with 8% H 2 O 2 (final concentration). For MVM synthesis, 0.5 M FeCl 3 was added (novel procedure) to the bilirubin solution prior to H 2 O 2 and the oxidation allowed to proceed for 10 min. Each aqueous reaction mixture (about 45 ml per 50 mg bilirubin) was extracted twice with 6 ml CHCl 3 or CH 2 Cl 2 (recoveries of BOX A, BOX B, and MVM were similar with CHCl 3 and CH 2 Cl 2 ) and the combined organic phase extracted once with 1 ml water, evaporated to~2 ml at o50°C and atmospheric pressure, transferred to microfuge tubes, and evaporated to near dryness. Additional~2 ml aliquots of extract were repeatedly added, each followed by evaporation to near dryness. The final addition of washed extract was evaporated to dryness and reconstituted in 1 ml 1% CH 3 CN(aq) for purification by reversed phase (RP)-HPLC.

Purification by RP-HPLC
RP-HPLC (0.1 cm light path; Shimadzu LC-10AT, Shimadzu Scientific Instruments, Columbia, MD) was used for both purification and analysis of the bilirubin oxidation products. The column was eluted (1 ml/min) with a continuous gradient of 0.5% CH 3 CN/min (2% to 18% CH 3 CN) over 32 min, followed by steeper gradients and higher CH 3 CN concentration for washing the system between runs. Eluates were monitored from 210-350 nm using a diode array spectrophotometer and flow cell and were collected in aluminum foil wrapped test tubes. RP-HPLC of the combined products of bilirubin-H 2 O 2 reaction mixtures with and without Fe 3 þ yielded three peaks with retention times at 26.0, 28.7, and 31.2 min, respectively (Fig. 2). These retention times corresponded to eluting CH 3 CN concentrations of 12.8, 14.4, and 15.6% (v/v), respectively. UV absorption at other retention times was not detected at 297, 310, and 327 nm, i.e., at the longer wavelength λ max 's of the compounds with 26.0, 28.7, and 31.2 retention times, respectively, as well as at 223 nm (Figs. 1 and 2; Table 1), indicative of a purified preparation. This relative order of retention time of MVM, BOX A, and BOX B differs from that which a laboratory previously reported, which was BOX A, BOX B, and then MVM [1,5]. While this difference in relative order of retention time may be due to differences in column properties, it should also be considered that the present inclusion of CF 3 CO 2 H in the solvent resulted in ion pairing with BOX A and BOX B (retention times of the ion pairs would be increased as compared to the non-paired species).
From the bilirubin-H 2 O 2 oxidation in the absence of Fe 3 þ , the ratio of MVM:BOX A:BOX B (BOX A set at absorption unity) formed at their respective λ max 's ( Figs. 1 and 2, Table 1) was 0.10 7 0.03:1.0:0.95 7 0.05, respectively (mean 7 SE; n ¼ 5). Several minor peaks were also observed (determined prior to further RP-HPLC purification). Incubation at times shorter or longer than 24 h did not result in additional MVM formation. Yields after purification of BOX A and BOX B were~1% each, based on starting material and measured by UV spectroscopy (calculated with ε's as described below; Table 1).
From the bilirubin-H 2 O 2 oxidation in the presence of Fe 3 þ , the ratio of MVM:BOX A:BOX B (MVM set at absorption unity) formed at their respective λ max 's ( Figs. 1 and 2, Table 1) was 1.0:0.05 7 0.01:0.04 7 0.01, respectively (mean 7 SE; n ¼ 5). Several minor peaks were also observed (determined prior to further RP-HPLC purification). Incubation for 1, 5, 30, 45, and 60 min did not increase BOX A and BOX B formation while MVM formation was reduced. The reaction yielded~5% MVM, based on starting material and measured by UV spectroscopy (calculated with ε; Table 1). Increased MVM formation with Fe 3 þ inclusion in the bilirubin-H 2 O 2 reaction mixture is consistent with the dependency of MVM formation following H 2 O 2 oxidation of ferriprotoporphyrin IX on the chelated iron [11] as well as the oxidation of bilirubin by CrO 3 [6].
Present yields are generally consistent with earlier reports of o5% and 4% formation of BOX A, BOX B and MVM [1,10]. While one of these reports [1] also demonstrated significant MVM synthesis (MVM:BOX A:BOX B ¼ 2.8:1:0.9; determined at 320 nm and as presently calculated with BOX A set to unity), the increased MVM formation was possibly due to a somewhat greater H 2 O 2 concentration in the reaction mixture with bilirubin (13% H 2 O 2 ). On the other hand, highly variable amounts of MVM were formed by oxidation of bilirubin with~10% H 2 O 2 [10]. Hydrogen peroxide oxidation of biliverdin instead of bilirubin did not increase the yield of MVM.

Stability
After purification, BOX A, BOX B, and MVM samples shielded with aluminum foil from light were stable for at least 6 mo at −20°C and for 24 h at room temperature in 14.6% CH 3 CN (eluting solvent), as determined by RP-HPLC; i.e., no loss of compound or detection of additional absorption peaks through the UV absorption spectrum. Removal of the aluminum foil and exposure of BOX A, BOX B, and MVM (in 14.6% CH 3 CN in a clear polypropylene microfuge tube) to ambient light for 24 h decreased recovery by 10%, 15%, and 5%, respectively, and the appearance of peaks at 18.3 min and 20.8 min with a ratio 1.13:1, and with λ max 's of 288 and 296 nm, respectively.  Fig. 1 and Table 1).

UV absorption spectrometry
UV spectra were performed in a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA).

1 H-NMR
For compound identification and ε determinations, analytic samples of BOX A, BOX B, and MVM (CHCl 3 extraction) were loaded onto a C 18 separation cartridge (Sep-Pak), washed with 1 ml D 2 O and eluted with 1.5 ml, 80% CD 3 CN (in D 2 O). Samples were then evaporated to dryness under N 2 and reconstituted in 1 ml CD 3 CN. BOX A, BOX B, and MVM chemical shifts and coupling constants were determined on a DMX-500. Extinction coefficients (ε) at the respective λ max 's for BOX A, BOX B, and MVM were determined by titration in CH 3 OH (3.49 ppm singlet) and integration of signals relative to CH 3 OH under conditions of long recycle delay, and determination of UV absorption. 1 H-NMR spectroscopy yielded chemical shifts and coupling constants for BOX A, BOX, B, and MVM consistent with previous reports (Fig. 3; Table 2; [1,6,9]).

MS
Samples for MS were prepared by evaporation of compounds in aqueous CH 3 CN to dryness in an N 2 stream at 40°C, followed by reconstitution in 10% CH 3 Table 3.
Lyophylization was avoided due to apparent loss of compounds. Samples obtained from RP-HPLC were infused into a Thermo Scientific LTQ-FT™ hybrid MS consisting of a linear ion trap and a Fourier transform ion cyclotron resonance (FT-ICR) MS. The standard electrospray ionization (ESI) source was operated in a profile mode for both positive and negative ions as indicated (Fig. 4, Table 3). The only possible elemental composition at 2 ppm mass error, but also even at 5 ppm, for 0-10 nitrogen, 0-15 oxygen, 0-30 carbons, and 0-60 hydrogens are those of BOX A and BOX B (as assessed for the positive ion mode), and for MVM (as assessed in both the positive and negative ion mode; Fig. 4, Table 3; consistent with 1, 6,9,10). With MVM as the protonated molecular ion, the observed mass was m/z 138.05498 with a mass error of 180 ppb. For MVM, MS also suggested the apparent presence of the plastic antioxidant/stabilizer 1,10-bis(2,2,6,6-tetramethyl-4-piperidinyl-decanedioate), resulting from the (initial) carrying out of the FeCl 3 -bilirubin-H 2 O 2 oxidation in a polypropylene vessel ("2. Experimental design, materials and methods; 2.1. Synthesis"; subsequent oxidations were performed in glass containers).