In vivo and in vitro identification of Z-BOX C – a new bilirubin oxidation end product

Marcel Ritter a, Sandesh Neupane a, Raphael A. Seidel ab, Christoph Steinbeck a and Georg Pohnert *ac
aInstitute of Inorganic and Analytical Chemistry, Friedrich Schiller University, Lessingstrasse 8, D-07743 Jena, Germany. E-mail:
bDepartment of Anesthesiology and Intensive Care Medicine/Center for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, D-07747 Jena, Germany
cMax-Planck-Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, D-07745 Jena, Germany

Received 19th January 2018 , Accepted 1st March 2018

First published on 1st March 2018

A new bilirubin oxidation end product (BOX) was isolated and characterized. The formation of the so-called Z-BOX C proceeds from bilirubin via propentdyopents as intermediates. This BOX was detected in pathological human bile samples using liquid chromatography/mass spectrometry and has potential relevance for liver dysfunction and cerebral vasospasms.

Heme, the prosthetic group of hemoproteins, is degraded at a scale of around 375 mg per day in humans via two different pathways.1 The enzymatic degradation involves catalysis by heme oxygenase resulting in the formation of iron, carbon monoxide and biliverdin, which is further reduced to bilirubin, which is conjugated with glucuronic acid and excreted into bile.2 In the second pathway, heme, biliverdin and bilirubin undergo non-enzymatic degradation via reactive oxygen species (ROS). In particular, bilirubin is a potent endogenous antioxidant that reduces oxidative stress in vitro and in vivo.3,4 The non-enzymatic degradation leads to several mostly uncharacterized higher order heme degradation products (HDPs).5 As such, four dipyrrolic regioisomers of the propentdyopent-type (PDP) were found as prominent cleavage products of bilirubin (Scheme 1).6–8 These isomers differ from each other in the position of the methyl and vinyl groups (PDP A and B) and in the position of the OH-group, which is either at the methyl/vinyl pyrrole (1) or at the propionic acid pyrrole (2). In protic solvents and body fluids, the position of the OH-group can undergo a rearrangement resulting in an equilibrium between Z-PDP A1 and A2 as well as B1 and B2. These four PDPs with a hitherto unknown biological function occur in human bile at a total concentration of around 60 μM.8
image file: c8ob00164b-s1.tif
Scheme 1 Oxidative degradation of bilirubin (ROS, reactive oxygen species; PDP, propentdyopent; BOX, bilirubin oxidation end product).

The PDP isomers are intermediates to the monopyrrole bilirubin oxidation end products (BOXes),8,9 which are discussed as contributors of cerebral vasoconstriction, a severe complication of subarachnoid hemorrhage.10,11 BOXes occur in human serum at nanomolar and in human bile at up to micromolar concentrations and affect the function and integrity of the liver.5,12 The so far identified BOXes reflect only small fractions of oxidative products formed from bilirubin and PDPs. The mechanism of non-enzymatic heme degradation is still partially unclear and many degradation products remain unidentified. This motivated our search for further degradation products in patient samples as well as in a model system of bilirubin oxidation.

In analogy to the cleavage of pyrrole rings of PDPs observed in the generation of Z-BOX A/B,9 we introduce an additional pathway to give the novel BOX C, a monopyrrole carrying a methyl and a propionic acid side chain (Scheme 1).

To generate a sufficient amount of this new product, an in vitro degradation of bilirubin with 10% hydrogen peroxide for 24 h was performed based on the studies reported by Ritter et al. and Kranc et al.8,9 With the help of ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HR-MS), a promising candidate for the proposed BOX C molecule (Fig. S2) with the suggested sum formula C10H13O4N2 (225.08691 amu, [M + H]+, Fig. S4) was found. To separate Z-BOX A and B from the reaction mixture, a chloroform extraction was performed according to the study reported by Seidel et al.12 To isolate further compounds, the water phase of the chloroform extraction was subjected to solid phase extraction (SPE, hydrophilic lipophilic balanced). After washing with brine to remove excessive oxidant, a fraction containing the target compound was eluted with water. A reversed-phase HPLC method was established to isolate BOX C in a preparative scale from this water fraction (Fig. S1). To elucidate the structure, NMR experiments (1H; 13C; 1H,13C-HSQC and 1H,13C-HMBC) were performed, confirming the proposed structure (Table 1, Fig. 1 and S6–10). The Z-configuration of the double bond was concluded based on DFT calculations of the E- and the Z-isomers with a prediction of the 13C-NMR shifts (Tables S1 and S2). Further characterization of the molecule via IR and UV spectroscopy (Fig. S10 and S11) was carried out and complemented with stability studies via UHPLC-HR-MS analysis. We observed that Z-BOX C is stable for at least 4 weeks at pH values of 2.5, 8.0 and 12.5. Z-BOX C is also resistant to oxidative conditions since ca. 50% of the initial amount could be recovered after a four week treatment with 1% hydrogen peroxide at a neutral pH-value.

image file: c8ob00164b-f1.tif
Fig. 1 HMBC coupling of Z-BOX C.
Table 1 NMR data of Z-BOX C in dimethyl sulfoxide-d6
# C/H δ H (ppm) δ C (ppm) HMBC
1 C 174.0
2 CH2 2.39 32.4 1, 3, 4
3 CH2 2.50 19.3 1, 2, 4, 5, 8
4 C 133.3
5 C 168.4
6 NH 9.64 4, 5, 8
7 C 147.8
8 C 142.0
9 CH3 1.97 9.8 4, 7, 8
10 CH 5.54 97.9 7, 8, 11
11 C 170.9
12 NH2 7.22/7.66

To obtain further insights into the degradation mechanism, PDPs were oxidized with 1% hydrogen peroxide and the transformation was monitored via UHPLC-HR-MS. In fact, all PDPs of the A and B series were precursors of Z-BOX C (Fig. 2). Within two days under these oxidative conditions we could clearly detect an increasing amount of Z-BOX C from all Z-PDPs. This result shows that Z-BOX C can be derived from both propionic acid substituted rings of the bilirubin molecule. Z-BOX A and B are only formed from the respective Z-PDP A and B. Interestingly, the amount of Z-BOX C formed from PDPs of the A and B series differs. A preferred formation of Z-BOX C from PDP B1/2 is observed under otherwise identical conditions. This indicates a substantial influence of the electron distribution in the educts (Fig. 2, lower panels).

image file: c8ob00164b-f2.tif
Fig. 2 Oxidation of Z-PDP A1/2 and B1/2 to Z-BOX A, Z-BOX B and Z-BOX C. UHPLC-HR-MS of Z-BOX A/B (upper, from Ritter et al.8) and Z-BOX C mass trace (lower).

A possible mechanism for the formation of Z-BOX C from PDPs is given in Fig. 3. This involves the tautomerization of the OH-groups from the PDPs followed by the oxidative cleavage of the intermediate lactam. The indication for the formation of the proposed second product 2-methyl-3-oxo-pent-4-enoic acid and its isomer was observed by UHPLC-HR-MS (Fig. S3).

image file: c8ob00164b-f3.tif
Fig. 3 Proposed mechanism of the oxidative degradation of all four PDPs to Z-BOX C.

The occurrence of Z-BOX C in humans could be proven in an initial study. Therefore, we quantified Z-BOX C with a detection limit of 25 nM in human bile samples obtained from patients under cholecystectomy via UHPLC-HR-MS. Initial measurements after protein precipitation with acetonitrile revealed Z-BOX C in three out of twelve samples (Table S3). Surprisingly, we observed a substantial increase in Z-BOX C concentrations when re-measuring the samples after storage at room temperature (3-fold increase of Z-BOX C after 9 h; 7-fold after 33 h). To exclude that this effect is due to the denaturation step, we prepared samples from bile samples stored at room temperature directly before measurements. Z-BOX C concentrations in these samples ranged from 70 to 130 nM (Table S4). Even under these conditions we observed a significant increase in Z-BOX C concentrations in the three Z-BOX C containing samples (2.5-fold after 9 h; 5-fold after 32 h, Table S4). This trend was also confirmed for Z-BOX A and B as well as for PDP formation. Accordingly, we conclude that the degradation process is still ongoing in patient samples even after work-up with organic solvents. This calls for a strict control of sampling, storage and analytic procedures in the clinical monitoring of HDPs.

Compared to the four Z-PDPs at overall concentrations of around 60 μM (ref. 8) and Z-BOX A/B (0.5 μM),5 the concentration of Z-BOX C in human bile was rather low at around 0.1 μM. HDPs show a strong structural dependence of activities as evidenced for Z-BOX A and B.5 Therefore, despite overall lower concentrations, Z-BOX C activity will have to be monitored in further experiments to clarify its contribution to the observed overall activity in liver failure, SAH, or further hitherto unidentified diseases.


We investigated the oxidative transformation of the heme degradation product bilirubin. Thereby, we isolated and structurally elucidated Z-BOX C, a new monopyrrole bilirubin degradation end product. The formation of Z-BOX C occurs via PDPs as intermediates. We provide evidence for the occurrence of Z-BOX C in human bile and its fast formation even in isolated bile samples at room temperature. This adds a new potentially bioactive compound to the family of oxidative degradation heme products with consequences in the field of vasoconstriction and liver dysfunction.

Conflicts of interest

The authors declare no competing financial interest.


Financial support by the German Research Foundation (DFG) within the framework of the Research Group FOR1738 is acknowledged. Bile samples were provided by Falk Rauchfuß and Utz Settmacher (Department of General, Visceral and Vascular Surgery, Jena University Hospital). Nico Ueberschaar is acknowledged for helpful discussion during the preparation of this manuscript.

Notes and references

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Electronic supplementary information (ESI) available. See DOI: 10.1039/c8ob00164b
Bile samples were collected after approval by the ethics committee of Friedrich Schiller University Jena (4406-04/15).

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