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

Article abstract of DOI:10.1039/c8ob00164b

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.

Full text of DOI:10.1039/c8ob00164b

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In vivo and in vitro identification of Z-BOX C - a new bilirubin oxi-
dation end product
Marcel Ritter,*a Sandesh Neupane, a Raphael A. Seidel a,b Christoph Steinbecka and
Georg Pohnerta,c
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
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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. It was
detected in pathological human bile samples using liquid
chromatography / mass spectrometry underlining its potential
relevance for liver dysfunction and cerebral vasospasms.
Heme, the prosthetic group of hemoproteins, is degraded at
the scale of around 375 mg per day in humans via two
different pathways.1 The enzymatic degradation involves the
catalysis by heme oxygenases to result in the formation of
iron, carbon monoxide and biliverdin, which is further
reduced, conjugated with glucuronic acid and excreted as
bilirubin diglucuronide into the bile.2 In a second pathway,
heme, biliverdin and bilirubin undergo nonenzymatic
degradation via reactive oxygen species (ROS). Especially
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 hitherto unknown biological function occur in
human bile in a total concentration of around 60 µM8.
The PDPs 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 in nanomolar and in human bile in up to
micromolar concentrations and affect the function and
integrity of the liver.12,5 The so far identified BOXes reflect only
a small fraction of oxidative products of bilirubin and PDPs.
This motivated our search for further degradation products in
patient samples as well as in a model system or bilirubin
oxidation. The mechanism of non-enzymatic heme
degradation is still partially unclear and many degradation
products remain unidentified.
In analogy to the cleavage of pyrrole rings of PDPs observed in
the generation of Z-BOX A/B9, we introduce an additional
pathway to give the novel BOX C, the monopyrrole carrying a
methyl and a propionic acid side chain (Scheme 1).
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To generate sufficient amounts of this new product, an in vitro
degradation of bilirubin with 10% hydrogen peroxide for 24 h
was performed based on Ritter et al. and Kranc et al..8,9 With e
help of ultra-high performance liquid chromatography coupled
to high resolution mass spectrometry (UHPLC-HR-MS)
a
promising candidate for the proposed BOX C molecule (Figure
S2) with a suggested sum formula of C10H13O4N2 (225.08691 u,
[M+H]+, Figure S4) was found. To separate Z-BOX A and B from
the reaction mixture a chloroform extraction was performed
according to 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
(Figure S1). To elucidate the structure, NMR experiments (1H;
13C; 1H,13C-HSQC and 1H,13C-HMBC) were performed,
confirming the proposed structure (Table 1, Figure 1, S6-10).
The Z-configuration of the double bond was concluded based
on DFT calculations of the E- and the Z-isomer with prediction
of the 13C-NMR shifts (Table S1 and S2). Further
characterization of the molecule via IR and UV spectroscopy
(Figure S10, 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 condition since
ca. 50% of the initial amount could be recovered after a four
week treatment with 1% hydrogen peroxide at neutral pH-
value.
Figure 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).
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 (Figure
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 derive from both propionic
acid substituted rings of the bilirubin molecule. Z-BOX A and B,
are only formed from the respective Z-PDP A or 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.
Table 1 NMR data of Z-BOX C in dimethyl sulfoxide-d6.
#
1
C/H
C
δH (ppm) δC (ppm)
HMBC
This indicates
a substantial influence of the electron
174.0
distribution in the educts (Figure 2 lower panels).
2
CH2
CH2
C
2.39
2.50
32.4
19.3
1, 3, 4
A possible mechanism for the formation of Z-BOX C from PDPs
is given in Figure 3. It involves the tautomerization of the OH-
groups from the PDPs followed by oxidative cleavage of the
intermediate lactame. Indication for the formation of the
3
1, 2, 4, 5, 8
4
133.3
168.4
5
C
6
NH
C
9.64
4, 7, 8
7
147.8
142.0
9.8
8
C
9
CH3
CH
C
1.97
5.54
4, 7, 8
10
11
12
97.9
7, 8, 11
170.9
NH2 7.22 / 7.66
Figure 3 Proposed mechanism of the oxidative degradation of all four PDPs to Z-BOX C.
Figure 1 HMBC coupling of Z-BOX C.
2 | J. Name., 2012, 00, 1-3
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proposed second product 2-methyl-3-oxo-pent-4-enoic acid acknowledged. Bile samples were provided by Falk Rauchfuß
and its isomer were obtained in LC/ HR-MS (supporting Figure and Utz Settmacher (Department of General, Visceral and
3).
Vascular Surgery, Jena University Hospital). Nico Ueberschaar
The occurrence of Z-BOX C in humans could be proven in an is acknowledged for helpful discussion during the preparation
initial study. Therefore we quantified Z-BOX C with a detection of this work.
limit of 25 nM in human bile samples obtained from patients
under
cholecystectomy
via
UHPLC-HR-MS.
Initial
Notes and references
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 storing 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 measurement.
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 PDPs formation. Accordingly, we conclude that
the degradation process is still ongoing in patient samples
even after work-up with organic solvents. This calls for strict
control of sampling, storage and analytic procedures in the
clinical monitoring of HDPs.
Bile samples were collected after approval by the ethics-committee
of Friedrich Schiller University Jena (4406-04/15).
1
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Westerhausen, S. Nietzsche, C. Sponholz, F. Cuperus, S. M.
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9
W. H. Schaefer, T. M. Harris and F. P. Guengerich,
Biochemistry, 1985, 24, 3254–63.
Compared to the four Z-PDPs with overall concentrations of
around 60 µM8 and Z-BOX A/B (0.5 µM)5 the concentrations of
Z-BOX C in human bile were with around 0.1 µM rather low.
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.
M. Ritter, R. A. Seidel, P. Bellstedt, B. Schneider, M. Bauer,
H. Görls and G. Pohnert, Org. Lett., 2016, 18, 4432–4435.
K. R. Kranc, G. J. Pyne, L. Tao, T. D. Claridge, D. A. Harris, T.
A. Cadoux-Hudson, J. J. Turnbull, C. J. Schofield and J. F.
Clark, Eur. J. Biochem., 2000, 267, 7094–101.
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Metab., 2002, 22, 472–478.
10
11
A. Joerk, R. A. Seidel, S. G. Walter, A. Wiegand, M. Kahnes,
M. Klopfleisch, K. Kirmse, G. Pohnert, M. Westerhausen, O.
W. Witte and K. Holthoff, J. Am. Heart Assoc., 2014, 3, 1–
13.
Conclusions
12
R. A. Seidel, M. Kahnes, M. Bauer and G. Pohnert, J.
Chromatogr. B, 2015, 974, 83–9.
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. 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
vasoconstrictions and liver dysfunction.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgement
Financial support by the German Research Foundation (DFG)
within the framework of the Research Group FOR1738 is
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O O
O
O
O
reactive oxy-
gen species
NH HN
NH
HN
H2
NH2
+
+
NH HN
NH2
N
NH
O
O
O
OH
HO
HO
O
O
O
Bilirubin
Z-BOX A
Z-BOX B
Z-BOX C
A new bilirubin degradation end product was isolated and characterized.