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Photoisomerization Neutralizes Vasoconstrictive Activity of a Heme Degradation ProductClick to copy article linkArticle link copied!
- Raphael A. SeidelRaphael A. SeidelInstitute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingstraße 8, 07743 Jena, GermanyDepartment of Anesthesiology and Intensive Care Medicine/Center for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, 07747 Jena, GermanyDevie Medical, c/o Jena University Hospital, Bachstraße 18, 07743 Jena, GermanyMore by Raphael A. Seidel
- Marcel RitterMarcel RitterInstitute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingstraße 8, 07743 Jena, GermanyMore by Marcel Ritter
- Alexander JoerkAlexander JoerkHans Berger Department of Neurology, Jena University Hospital, Am Klinikum 1, 07747 Jena, GermanyResearch Program “Else Kröner-Forschungskolleg AntiAge”, Jena University Hospital, Am Klinikum 1, 07747 Jena, GermanyMore by Alexander Joerk
- Stefan KuschkeStefan KuschkeInstitute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingstraße 8, 07743 Jena, GermanyMore by Stefan Kuschke
- Niklas LangguthNiklas LangguthHans Berger Department of Neurology, Jena University Hospital, Am Klinikum 1, 07747 Jena, GermanyMore by Niklas Langguth
- Daniel SchulzeDaniel SchulzeInstitute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Humboldtstraße 8, 07743 Jena, GermanyMore by Daniel Schulze
- Helmar GörlsHelmar GörlsInstitute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Humboldtstraße 8, 07743 Jena, GermanyMore by Helmar Görls
- Michael BauerMichael BauerDepartment of Anesthesiology and Intensive Care Medicine/Center for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, 07747 Jena, GermanyMore by Michael Bauer
- Otto W. WitteOtto W. WitteHans Berger Department of Neurology, Jena University Hospital, Am Klinikum 1, 07747 Jena, GermanyResearch Program “Else Kröner-Forschungskolleg AntiAge”, Jena University Hospital, Am Klinikum 1, 07747 Jena, GermanyMore by Otto W. Witte
- Matthias WesterhausenMatthias WesterhausenInstitute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Humboldtstraße 8, 07743 Jena, GermanyMore by Matthias Westerhausen
- Knut HolthoffKnut HolthoffHans Berger Department of Neurology, Jena University Hospital, Am Klinikum 1, 07747 Jena, GermanyMore by Knut Holthoff
- Georg Pohnert*Georg Pohnert*Email: [email protected]. Phone: +49 3641 948170. Fax: +49 3641 948173.Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingstraße 8, 07743 Jena, GermanyMore by Georg Pohnert
Abstract
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Delayed cerebral ischemia (DCI) caused by cerebral vasospasm is the leading determinant of poor outcome and mortality in subarachnoid hemorrhage (SAH) patients, but current treatment options lack effective prevention and therapy. Two substance families of heme degradation products (HDPs), bilirubin oxidation end products (BOXes) and propentdyopents (PDPs), are elicitors of pathologic cerebral hypoperfusion after SAH. Z-configured HDPs can be photoconverted into the corresponding E-isomers. We hypothesize that photoconversion is a detoxification mechanism to prevent and treat DCI. We irradiated purified Z-BOXes and Z-PDPs with UV/Vis light and documented the Z–E photoconversion. E-BOX A slowly reisomerizes to the thermodynamically favored Z-configuration in protein-containing media. In contrast to vasoconstrictive Z-BOX A, E-BOX A does not cause vasoconstriction in cerebral arterioles in vitro and in vivo in wild-type mice. Our results enable a critical assessment of light-induced intrathecal photoconversion and suggest the use of phototherapy to prevent and cure HDP-mediated cerebral vasospasms.
Copyright © 2020 American Chemical Society
Introduction
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Subarachnoid hemorrhage (SAH) is a severe and life-threatening medical condition with an overall incidence of 9 per 100 000 per year (1) and occurs upon aneurysmal bleeding or traumatic cranial injury. The early location of the bleeding focus with aneurysm treatment and the interventional release of intracranial pressure, e.g., via external ventricular drainage (EVD), are mandatory to limit the volume of tissue damage. However, about 50% of the survivors of the initial hemorrhagic event develop a secondary ischemic period, which is caused by delayed arterial vasospasm in the perihematomal space. The vasospasm onset is most likely between 3 and 21 days post-SAH with a peak incidence between day 5 and day 7 after the bleeding event. (2) Importantly, the vasoconstrictions may occur in both the larger arteries preceding the brain parenchyma and the microvessels, (3) which lead to microthrombosis, (4) perfusion deficits, and delayed cerebral ischemia (DCI). (3) Since the measurement of a vasospastic blood flow acceleration via Doppler sonography is limited to larger vessels, it is mostly the clinical appearance that leads to the diagnosis of DCI, which is to be confirmed via contrast-enhanced MR or CT perfusion imaging. (5,6) The consequences of DCI include transient and persistent neurological deficits and an unfavorable outcome in general with an increased six-month mortality rate up to 60%. (1) Although DCI is in a clear causal relation to a preceding bleeding event, the molecular pathogenesis of delayed vasospasm represents a multifactorial network. It includes heme-mediated depletion of vasodilatory nitric oxide (NO), vasoconstrictive hemolysate compounds, humoral factors such as serotonin, oxidative stress-mediated inflammation, and an impaired neurovascular coupling. (7,8) The current treatment options for SAH-induced vasospasm and DCI focus on the application of vasodilative agents like nimodipine, the hemodynamic management, including hypertonia and euvolemia, or invasive interventions such as balloon angioplasty or intra-arterial spasmolysis. (9) However, the achieved effects of contemporary therapy are not sufficient because they neither provide causal prophylaxis to block the formation of mediators nor offer a therapy, which eliminates vasoconstrictive effectors.
Higher-order heme degradation products (HDPs) (Figure 1), such as the monopyrrolic bilirubin oxidation end products (BOXes) and propentdyopents (PDPs), arise upon nonenzymatic break-down of the tetrapyrrole heme, biliverdin, and bilirubin. (10−12) Inflammatory processes and further conditions with elevated concentrations of reactive oxygen species are likely to increase their production. (13,14)
Figure 1
Figure 1. Heme degradation products (HDPs), obtained upon nonenzymatic degradation of heme, biliverdin, or bilirubin, may occur in the Z- and E-configurations. Naturally occurring HDPs with Z-configuration (white background) cause vasoconstriction of cerebral arteries, whereas the effect of their isomers with E-configuration (red background) is to be investigated. BOX: bilirubin oxidation end product and PDP: propentdyopent.
Among these HDPs, the regioisomers Z-BOX A and Z-BOX B as well as the regioisomer pairs Z-PDP A1/2 and Z-PDP-B1/2 (Figure 1) were recently shown to cause vasoconstriction of cerebral arterioles in different in vivo and in vitro models. (15,16) These Z-BOXes and Z-PDPs were quantified in the cerebrospinal fluid of SAH patients, resulting in nanomolar concentrations in patients with SAH, whereas these compounds were hardly traceable in the control group without intracranial bleeding. (15) This suggests that the compound class of HDPs has a high potential to influence the development of vasospasms after SAH. Their mode of action includes the inhibition of BKCa potassium channels in vascular smooth muscle cells and contribution of astrocytes and the complete neurovascular unit. (15,16) Nevertheless, despite this evidence for the contribution of these heme-derived substance classes, a causal prophylaxis or therapy concerning these effectors of vasospasm is still missing.
In analogy to the open-chain tetrapyrrole precursors biliverdin and bilirubin, (17) these higher-order degradation products may exist in two configurations, i.e., with Z- and E-configurations at the exocyclic double bonds (Figure 1). It is striking that state-of-the-art analytical determination with high-performance liquid chromatography coupled to mass spectrometry only detected the more stable Z-BOXes and Z-PDPs in human specimens so far. (12,14,15)
The (neuro-)toxicity of bilirubin is linked to its chemical structure in the all-Z-configurations, exhibiting high lipophilicity and, therefore, poor excretability. The successful application of phototherapy converts the bulk part of Z-bilirubin to bilirubin with an E-configuration, which is far less toxic and has an increased water solubility and excretability. (17,18) We assume that also the detrimental effects of Z-configured HDPs, such as Z-BOXes and Z-PDPs, occurring after SAH may be treated via irradiation to generate the corresponding E-isomers with potentially less or even without toxicity and faster excretion.
In the initial description of BOXes, the use of UV-irradiation to reduce the amount of such vasoconstrictors was already mentioned. (10) However, the proposed photodegradation of the BOX A and BOX B isomers to methyl vinyl maleimide could not be confirmed so far. The first evidence of photoisomerization was obtained in the course of the total syntheses of Z-BOXes (19,20) and while investigating the stability of serum samples during preanalytical sample preparation. (21)
The aim of this work is to study the physicochemical and biological feasibility of a phototherapeutic treatment to prevent and/or cure cerebral HDP-mediated vasospasms after subarachnoid hemorrhage. We therefore studied the formation of E-HDPs via photoconversion of the naturally occurring Z-isomers, evaluated suitable irradiation conditions, clarified the chemical structure and tested the stability, established protocols for the isolation, and finally studied the vasoactive potency of the representative photoconversion product E-BOX A in comparison to the naturally formed Z-HDPs in mouse in vitro and in vivo.
Results
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Wavelength-Dependent Photoconversion of Z-Configured Heme Degradation Products BOXes and PDPs with UV/Vis Light
To find the optimal conditions for a targeted photoconversion of Z-configured BOXes and PDPs, we investigated the influence of the deployed wavelengths on E-isomer formation in aqueous solutions. The wavelength range was modified by using different K-edge glass filters in the light path, which led to a sharp cut-off transmission of light above the indicated wavelength (Supporting Information Figure S1). Especially with UV light, we observed a decrease in the total peak areas, which is due to the already reported, overlaying photodegradation. (21) The following considerations focus on the peak area ratio of the E- to Z-isomers and thus exclude the analysis of photodegradation.
Measurements of the peak area ratios of E-BOX A/B and Z-BOX A/B after a 10 min irradiation period of pure Z-BOXes yielded a 50:50 ratio of the E/Z-isomers when the irradiation wavelength cut-offs were between 275 and 345 nm (Figure 2). At a wavelength cut-off of 360 nm, the peak areas of E-BOXes were slightly lower with ∼30%, whereas above a wavelength cut-off of 485 nm, no photoconversion of Z-BOX A and Z-BOX B to the corresponding E-isomers was detected. When Z-PDPs were irradiated likewise, the peak area ratios of E-PDPs to Z-PDPs were below 40% E (Figure 2B). In contrast to BOXes, the ratio of E- to Z-isomers of PDPs is decreasing already at wavelength cut-offs between 275 and 345 nm. Irradiation wavelength above 360 nm did not cause photoconversion of Z-PDPs. To foster interpretation of the peak area/wavelength diagrams, we provide UV/Vis absorbance spectra (Supporting Information Figure S2). Taken together, a fast and substantial (>40%) conversion of Z-BOXes and Z-PDPs to their corresponding E-isomers is achieved with a wavelength of up to 345 nm. Irradiation with a wavelength cut-off of 350 nm is only sufficient for the photoconversion of Z- to E-BOXes.
Figure 2
Figure 2. Relative peak areas of the Z- and E-isomers of BOXes (A) and PDPs (B) in relation to the transmission cut-off wavelengths after irradiation of Z-isomers.
Preparative Isolation of E-BOX A
To characterize the physicochemical properties of E-BOX A and E-PDPs as well as to provide the compounds for individual biological testing, we studied their preparative formation and purification via analytical and semipreparative high performance liquid chromatography (HPLC). We established an irradiation procedure with a Xenon arc lamp and carried out the photoconversion from saturated BOXes solutions in 3 mL of acetonitrile. E-BOX A and E-BOX B were isolated after a 12 min irradiation period of synthetic Z-BOX A and Z-BOX B using nonacidified isocratic (17% acetonitrile/water) liquid chromatography on the octadecyl column material. Due to a several-minute difference in retention time (Figure 3), E-BOX A (11.3 min retention time) was easily separated from Z-BOX A (28.0 min). By this method, about 50% of E-BOX A and B was produced from the corresponding Z-isomers. It was not possible to achieve a higher conversion ratio in favor of the E-BOX isomers, as longer irradiation time led to photodegradation and, therefore, a decreased yield. After removal of the solvent in an evacuated centrifuge, we investigated the stability of the E-fractions, resulting in only 0.6% of Z-BOX B after 10 d at room temperature. We characterized the E-BOXes with HR-MS and NMR and found that chemical shifts are in accordance with the earlier published data of the hitherto proposed E-BOX A structure. (19)
Figure 3
Figure 3. Analysis of BOX isomers using liquid chromatography coupled to mass spectrometry. (A) Preparative HPLC separation of E-BOX A, E-BOX B, Z-BOX A, and Z-BOX B after photoconversion of Z-BOXes; the UV absorption at 280 nm is shown. (B, C) Ultra performance liquid chromatography-tandem mass spectrometer (UHPLC-MS) profiles of E-BOX A and E-BOX B over after preparative HPLC fractionation (t = 0 day) and after 10 days at room temperature in the dark; the ion trace of BOXes, m/z = 179.0815 [M + H]+ ± 5 ppm, is shown.
Structural Evidence for E-BOX A as a Major Photoconversion Product of Z-BOX A
To prove the configuration of the assumed E-BOX A, we crystallized the HPLC-purified photoconversion product of Z-BOX A from methanol as colorless prisms for structural determination. The results from X-ray diffraction confirm that the photoproduct exhibits the molecular backbone of BOX A with E-configurations of the exocyclic double bond (Figure 4; C9H10N2O2, 178.19 g/mol; for crystal data, see Supporting Information). The remaining bond lengths and angles of E-BOX A and Z-BOX A are comparable.
Figure 4
Figure 4. Structure of E-BOX A, the photoconversion product of the naturally occurring Z-BOX A. (A) Ball-and-stick model of E-BOX A, calculated from the structural parameters obtained from X-ray diffraction. (B) Illustration of E-BOX A (left) and Z-BOX A (right) in ball-and-stick models. The carbamide group (blue plane) is rotated by ∼33° toward the main plane of the molecule (red plane) only in E-BOX A. The structure of Z-BOX A was generated from published crystallographic data. (19)
The major impact that results from the isomerization of the exocyclic double bond is the altered spatial orientation of the acetamide side chain with respect to the heterocyclic pyrrole ring (Figure 4B). While the carbamide function of the side chain of Z-BOX A is close enough to form a strong intramolecular hydrogen bond to the pyrrole nitrogen, this interaction and concomitant steric fixation are not possible in E-BOX A. In turn, E-BOX A exhibits more polar molecular moieties outwards and facilitates dipolar intermolecular interactions with other molecules and polar solvents. Thus, E-BOX A forms three-dimensional planes (Supporting Information Figure S3), whereas Z-BOX A may only form a two-dimensional spread (Supporting Information Figure S4). Z-BOX A represents a nearly complete planar structure (with the exception of the vinyl group). In contrast, the carbamide function of E-BOX A is rotated (∼33° torsion angle) toward the plane that contains the pyrrole ring and the exocyclic double bond (Figure 4B).
We recorded the 1H NMR spectrum (Supporting Information Figure S5) and confirmed the identity of the crystallized structure with experimental and computed data on the so far only proposed E-BOX A structure. (19,21) With an elution time of 4.5 min in analytical reversed-phase chromatography, E-BOX A interacts much less with the used octadecylsilyl column material than Z-BOX A, which elutes at 6.5 min. E-BOX A was stable as a solid for more than 4 months when kept protected from light at room temperature, whereas E-BOX B was less stable and slowly reisomerized to the thermodynamically favored Z-isomer.
Preparation and Stability of E-PDP Isomers
In analogy to the photoconversion method to generate E-BOXes, we set up an irradiation procedure for 0.3 mL aliquots of saturated aqueous PDP solutions that were obtained from oxidative bilirubin degradation and subsequent solid-phase extraction with 20% acetonitrile/water elution. After an irradiation time of 30 min, the maximum amount of around 20% of newly formed isomers was reached. UHPLC-MS analysis revealed that six new PDP isomers were formed (Figure 5), suggesting that besides the four corresponding E-PDP isomers, two additional isomeric structures were formed.
Figure 5
Figure 5. Preparative HPLC/UV of the irradiated PDP raw mixture in acidified solvents (A) and stability of E-PDP A2 in acidified solvents directly after separation at room temperature accessed via UHPLC-MS (B). After 30 min, only traces of E-PDP A2 remained in the eluate. The ion trace of PDPs, m/z = 341.11079 [M + Na]+ ± 5 ppm, is shown.
Although the chromatographic separation of the isomers was established, the isolation of these six photoconversion products of Z-PDPs proved to be very difficult as the formed E-PDPs readily reisomerized to the corresponding Z-isomers. We applied different solvent mixtures (neutral acetonitrile/water or buffered with ammonium formate buffer at pH 4, or buffered with ammonium carbonate at pH 8; water/methanol), gradient elution programs, and tested the effects of switching the chromatography column from (lipophilic) reversed-phase to (hydrophilic) normal phase. Attempts to isolate the pure PDP photoproducts failed as the removal of the solvent from chromatographic separation revealed the instability of these structures, which was associated with the reconversion to the initially used Z-PDPs in the range of 2–20% even when the fractions were freeze-dried immediately after elution (see the Supporting Information).
With the knowledge that irradiation leads to equilibria between the Z- and E-isomers, we irradiated the isolated fractions of PDP photoconversion to gain further insight about which photoconversion product would correspond to the individual Z-PDP A1/2 or Z-PDP B1/2 isomers. Thus, we structurally assigned four of the six new compounds to their related Z-PDPs (Supporting Information Figure S6).
To evaluate whether the photoconversion products of PDPs exhibit the E-configuration at the exocyclic double bond, we oxidized these fractions with hydrogen peroxide and assessed the E/Z-configuration of the formed BOXes via UHPLC-MS (Supporting Information Figure S7). The oxidation of two PDP isomers led to E-BOX A, and the oxidation of one further PDP photoisomer led to the formation of E-BOX B. This is a strong indication that these PDP photoisomers exhibit an E-configuration at the exocyclic double bond.
Taken together, E-BOX A was generated via photoconversion and isolated via semipreparative HPLC as a stable compound when protected from light. E-BOX B, E-PDPs, and two additional isomeric PDP structures that formed during irradiation were generated likewise but could not be isolated due to the fast conversion to their Z-configured precursors.
E-BOX A Does Not Induce Morphological Alterations in Cultured Cells but Reisomerizes to Z-BOX A in Cell Culture
To assess whether purified E-BOX A causes morphological alterations of cultured cells as described for Z-BOX A in HepG2 cells, we tested E-BOX A, Z-BOX A (positive control), and 0.25% DMSO in growth medium as vehicle control (negative control) according to a recently published protocol. (14) After an incubation period of 24 h, Z-BOX A induced the formation of spherical cell bodies, whereas its photoconversion product E-BOX A did not cause any morphological alterations in comparison to cells treated with the vehicle control (Figure 6A). When the compound solutions in the cell culture medium were stored for 24 h prior to administration, part of the E-BOX A-treated cells exhibited the spherical morphotype seen in Z-BOX A-treated cells. We assessed the post-experimental E/Z-isomer purity of both treatments using HPLC-MS/MS. While the Z-BOX A solution still contained only the Z-isomer, the E-BOX A solution exhibited only a residual fraction of ∼3% E-BOX A and a high concentration of the Z-isomer. We therefore hypothesized that E-BOX A had reisomerized in the course of the experiment. To test this hypothesis, we assessed the isomeric purities of the individual treatments via the peak area ratios of E-BOX A and Z-BOX A. After 24 h, only 68.9 ± 0.03% and after 48 h 45.3 ± 6.52% residual E-BOX A was detected in the cell culture supernatants (N = 3).
Figure 6
Figure 6. Impact of E-BOX A on cell morphology in vitro and reisomerization in protein-containing media. (A) Morphological effects of E-BOX A and Z-BOX A treatments on HepG2 cells compared to control conditions. Scale bar: 25 μm. (B) Residual fraction of E-BOX A in relation to the initial amount (t = 0 h; 100%) after incubation in different biochemical milieus at 37 °C in the dark after 6, 24, and 48 h. While E-BOX A is stable in water and protein-free, physiologic salt solution for 48 h, the addition of 1% human serum albumin (HSA; a representative for serum proteins present in CSF of SAH patients) causes a time-dependent decrease of the E-BOX A content in favor of Z-BOX A. In CSF of SAH patients and in serum samples of healthy humans, E-BOX A reisomerizes faster to its Z-isomer. Data are presented as mean ± standard deviation (SD).
E-BOX A Reisomerizes to Z-BOX A in Protein-Rich Environments
With the knowledge that E-BOX A may reisomerize to Z-BOX A at 37 °C in the presence of human cells in the cell culture medium and without further light exposure, we asked whether and how long E-BOX A would be stable in (bio-)chemical milieus relevant for the production of BOXes and possible elimination via the systemic circulation. We therefore spiked different media as well as human specimens with E-BOX A and assessed the resulting isomer content of E-BOX A after incubation for up to 48 h at 37 °C.
E-BOX A was fully stable (>99% of initial E-isomer) in water and artificial cerebrospinal fluid (aCSF, physiologic saline solution) over the 48 h incubation time in the dark. Compared with this, the E-BOX A content in 1% human serum albumin (HSA)/aCSF, a matrix resembling even pathologically elevated protein concentrations, was significantly reduced through the formation of 14% Z-BOX A within the first 6 h of incubation and 51% after 48 h. We further investigated the reisomerization in human CSF samples withdrawn from SAH patients via EVD as well as in serum samples of healthy humans. To exclude errors of the measured E-BOX A and Z-BOX A values by potentially pre-existing BOXes in the samples or due to the formation of BOXes via in vitro degradation of heme/hemoproteins, we incubated and analyzed additional aliquots of the samples without E-BOX A spiking. The patient samples did not contain BOXes at the beginning of the experiment and further did not exhibit any production BOXes during incubation. When E-BOX A-spiked human CSF samples and serum samples were incubated at 37 °C, the decrease of the E-BOX A content was even stronger compared to the samples in 1% HSA/aCSF. After an initial reduction to 86 and 92%, respectively, after 6 h post-incubation, the residual relative amount of E-BOX A after 48 h was only ∼14%.
Photoconversion of Z-BOX A to E-BOX A Neutralizes Vasoconstrictive Activity in Mouse In Vitro and In Vivo
Following our hypothesis that the structural differences of E-BOX A compared to Z-BOX A would result in an altered impact on the vascular diameter in cerebral arterioles, we tested the vasoactivity of E-BOX A in comparison to the naturally occurring Z-BOX A in both in vitro and in vivo (Figure 7A,B). For in vitro experiments, coronal brain slices of 350 μm thickness, comprising the occipital and parietal cortex, were cut using a microtome and continuously superfused with aCSF during the optical imaging. Due to the lack of intravascular blood pressure and blood flow in acute brain slices, the physiological resting tone was simulated using the NO synthase inhibitor L-NAME. A previous study demonstrated that the superfusion of mouse brain slices with L-NAME did not change the mean arteriolar diameter (−4.8 ± 5.4%; N = 8) during the application period of 90 min. (15) Application of 5 μmol/L Z-BOX A resulted in the reduction of the mean arteriolar diameter (N = 6; −13.2 ± 2.5%; P < 0.01) compared to the L-NAME control. (15) When the brain slices were superfused with 5 μmol/L E-BOX A (−5.6 ± 5.8%; N = 6), E-BOX A was not vasoactive in the brain slice model and the diameter change differed significantly from the Z-BOX A conditions (P < 0.05). We assessed the ratio of Z- to E-BOX A with UHPLC-MS and found only 0.5 ± 0.2% Z-BOX A at the beginning of the experiment and a minimal increase to 0.8 ± 0.2% after 90 min.
Figure 7
Figure 7. Vasoactive effect of the E-BOX A isomer on intracortical arterioles in vitro and on pial arterioles in vivo. (A) Time course of the normalized diameter of preconstricted, intraparenchymal arterioles after superfusion with Z-BOX A or E-BOX A (5 μmol/L, N = 6) in Slo1-WT mice. (B) Percentage arteriolar diameter changes after 90 min Z-BOX A or E-BOX A superfusion in slices of Slo1-WT mice. In line diagrams, the bold lines represent the mean ± SD (gray and pink regions). Thin lines correspond to the diameter time course of single experiments. Dot plots are shown as mean ± SD. Labels above the plots represent comparisons between the BOX A-induced diameter change and the control group. (C) Time course of the normalized diameter of pial arterioles compared between Z-BOX A and E-BOX A (100 μmol/L, N = 5). Bold lines represent the mean value ± SD (gray and pink regions). (D) Summary of percentage diameter changes after Z-BOX A or E-BOX A application. Dot plots are presented as mean ± SD. Data points correspond to the diameter values of single experiments. Statistical comparison between the E-BOX A time slots (120 min versus 180 min) refers to paired samples. ***P < 0.001; **P < 0.01; and *P < 0.05. ns = nonsignificant.
We further evaluated the in vivo vasoactivity of the photoconversion product E-BOX A in comparison to the naturally occurring and pretested isomer Z-BOX A (Figure 7C,D). Therefore, an acute cranial window of 2.5 mm in diameter over the parietal and occipital cortex was implemented in intact animals. After a 10 min baseline scan, a 100 μmol/L solution of E-BOX A was repeatedly injected with small puffs of around 5 nL volume in 5 min intervals over 180 min. During this time, the selected vessel was scanned every 30 min for 10 min to monitor potential changes in the arteriolar diameter. Joerk and Ritter et al. demonstrated that equally concentrated Z-BOX A (N = 5) significantly narrowed pial arterioles to −17.0 ± 7.2% of the baseline level after an application period of 120 min. (15) The diameter decrease was significantly different in comparison to the aCSF control group (N = 4; P < 0.01). In contrast, the diameter did not change upon application of E-BOX A (N = 5), reached −3.5 ± 4.8%, and were not significantly different from the control group treated with aCSF (P = 0.25; Figure 7C). In contrast, the vasoresponse between the Z-BOX A and E-BOX A treatment group was significantly different (P < 0.01; Figure 7D). Since the mean arteriolar diameter tended to decrease from 90 to 120 min under E-BOX A treatment, we extended the recording time to 180 min. During this additional period, the indicated decrease of the mean diameter was preceded and led to a final diameter change of −7.5 ± 6.0% of the baseline level without a significant difference to the 120 min level. In summary, E-BOX A did not induce arteriolar vasoconstriction in vitro and in vivo in clear contrast to its corresponding Z-isomer.
Discussion
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Z-BOXes and Z-PDPs, the hitherto most widely investigated higher-order heme degradation products, retain the Z-configuration of the exocyclic double bonds from their biological precursors, heme, bilirubin, and biliverdin. Accordingly, in studies investigating the amount of these naturally occurring HDPs in CSF, serum/plasma, and bile, exclusively Z-configured BOXes and PDPs were detected. (12,14,15,21) While recent in vivo and in vitro studies gave evidence that Z-configured HDPs induce a manifest arteriolar vasoconstriction in cerebral arterioles, (15,16) there was so far no data on the biological effects of E-configured HDPs. If an interventional photoconversion of Z-configured heme degradation products should be used for the prevention and therapy of such hemolysate compound-mediated DCI in SAH patients, the following three basic requirements must be fulfilled. The photoconversion products must (1) not be vasoactive or toxic, (2) have physicochemical properties that allow their adequate elimination, and (3) be stable long enough to be excreted.
Conditions of Photoconversion
To examine whether these preconditions are fulfilled, we first investigated the photoconversion process of Z-BOXes and Z-PDPs. While in the course of the discovery, the light sensitivity of BOXes was attributed to photodegradation to methyl vinyl maleimide, (10) we herein confirmed assumptions from HPLC-MS/MS and NMR experiments (19,21) that the prior process at short irradiation times is indeed Z → E photoisomerization. We further demonstrate the same reactivity in favor of Z → E photoisomerization for the PDP A1/2 and PDP B1/2 isomers.
In regard to maximizing the fraction of Z-isomers converted to their corresponding E-isomers via irradiation, the light of the UV-A spectrum is optimal and results in conversions of ∼50% for BOXes and ∼20% for PDPs at 10 or 30 min irradiation periods (Figures 3 and 5). As seen with the biological precursor bilirubin, (22) it is important to include co-occurring photodecomposition into the considerations on the ideal wavelength range for Z → E photoconversion. The energy input of photochemical reactions is inversely proportional to the irradiation wavelength (E = hc/λ, with E = energy, h = Planck constant, c = speed of light). Photoisomerization of the exocyclic double bond of Z-BOX A succeeds already with UV-A light, i.e., the lower-energy wavelengths range (315–380 nm) of UV light (Figure 2), resembling photochemically mild conditions. The light energy of the photons is needed to cross the activation energy barrier of the exocyclic ππ-bond, whereas the energy difference between Z-BOX A and E-BOX A is only about 36 kJ/mol. (19) When Z-BOX A is irradiated with shorter wavelength light, also further bonds, particularly within the conjugated double bond system, will be excited (see the UV absorbance spectrum) (19) and may lead to a variety of further parallel reactions, including the break-up of the molecule (photodegradation). Such often unspecific reactions will consequently decrease the yield of the desired Z–E isomerization, which was shown earlier during an 8 h irradiation of Z-BOX A with sunlight, which also includes UV-B light. (21) Thus, the wavelength ranges in the upper UV-A light should be used to maximize the fraction converted via the desired photoisomerization process even though this may result in slightly longer irradiation periods. In addition, exposure to longer-wavelength light may drastically reduce potential side effects, (23) e.g., tissue damage, which also strongly depends on the way, the envisaged prophylactic or therapeutic irradiation is implemented. To avoid such detrimental side effects, neither irradiation of the whole body via the skin nor of CSF inside the body, e.g., via fiber optics, seem appropriate. We rather propose to photoconvert vasoconstrictive Z-HDPs via UV-A irradiation by means of extracorporeal apheresis and consequent recirculation of the cleared CSF. Photoconversion of Z-HDPs also slowly succeeds with ambient light, which includes near UV-A wavelength. The long-wavelength absorbance shoulder of Z-BOX A reaches even beyond 400 nm (Supporting Information Figure S2A), which makes in particular Z-BOX A photoconvertible with lower-energy light. Since photoconversion of the HDP precursor bilirubin may be markedly more efficient in situ through bypassing thermodynamic barriers via protein binding, (22) further in vitro and in vivo studies are encouraged.
Stability of E-BOXes and E-PDPs
It was not possible to achieve a complete Z → E photoconversion of BOXes and PDPs as the reaction reaches equilibria, where also E-configured compounds are photoconverted back to the precursors with the Z-configuration. Additionally, the purified E-isomers reisomerize thermally to the Z-configuration, which is congruent to density functional theory calculations for BOX A, which showed that the Z-configuration of the exocyclic double bond is thermodynamically favored (>35 kJ/mol) compared to the E-configured isomeric structure. (19) We found that the thermal stability of the individual E-configured HDPs differs substantially. While E-BOX A was stable for several months at room temperature in the dark, E-BOX B started to slowly reisomerize during chromatographic purification and isolation. Due to a faster reisomerization, it was not possible to isolate pure E-PDP isomers, which contained at best ∼2% of the corresponding Z-isomers after chromatography. To monitor the concentrations of E- and Z-configured HDPs in situ, HPLC/MS analytics may be coupled with recently developed techniques of CSF microdialysis. (24)
We demonstrate the labile E-configuration of the dipyrrolic photoconversion products via biomimetic oxidative degradation with hydrogen peroxide to the corresponding E-BOX A and E-BOX B structures, respectively. Using the thermal reisomerization, we further assign four E-PDP isomers to their structural classes PDP A/B, which differ from each other in the position of the methyl and vinyl side chains (Figure 1).
Cell Culture Experiments and Reisomerization of E-BOX A
Z-BOX A alters the spread and brick-shaped morphology of different cell culture models for hepatocytes by inducing a spherical morphotype with condensed cell bodies. (14)E-BOX A does not induce this detrimental condensation of cell bodies. This finding suggests different pharmacodynamic properties of E- and Z-BOX A. The naturally occurring Z-PDP isomer pairs, Z-PDP A1/2 and Z-PDP B1/2, also do not cause this morphological alteration (Supporting Information Figure S8). Based on the observation that cells incubated with E-BOX A slowly began to switch to the spherical morphotype, we revealed that also E-BOX A reisomerizes to its Z-isomer in biological systems. However, this E → Z reconversion is much slower than that of the E-PDP isomers. The thermodynamically driven process is impeded by an activation energy barrier (19,20) and includes presumably unspecific interactions of E-BOX A with proteins, which may interact with E-BOX A, resulting in an accelerated conversion. While E-BOX A is stable for 48 h in water and aCSF in the dark, the presence of serum proteins in CSF and other body fluids enables a slow reisomerization reaction as shown with CSF of SAH patients and serum of healthy humans. The thermodynamic reisomerization has several implications. First, it provides a conclusive explanation that no E-configured HDPs were found in human specimens. Second, in regard to a prophylactic or therapeutic application of photoconversion, it is important that the generated E-isomers can sufficiently be withdrawn from the perivascular space to diminish the elicitation of vasoconstriction after the reisomerization. In the case of E-BOX A, the strongly increased hydrophilicity and stability of >85% for 6 h in CSF of SAH patients leave a time window for redistribution and elimination. The part of E-BOX A reallocated from the site of action to the bloodstream might enter the efficient pathway of active hepatobiliary excretion. (14) Although the elimination pathways for the other HDPs investigated herein still remain unknown, photoconversion may be beneficial to convert the Z-HDPs at the site of action and to achieve at least a reduction of locally high concentrations of the detrimental Z-isomers. Since reisomerization of E-BOX A only takes place when the molecules get in contact with proteins/peptides, we propose a catalytic (but presumably unspecific) mechanism that lowers the energy barrier for the formal flip of the exocyclic double bond.
Structure of E-BOX A
The stability of E-BOX A allowed its crystallization and X-ray diffractometric examination to obtain the precise molecular structure. Due to the changed steric situation of E-BOX A, intramolecular hydrogen bonding, a key feature of Z-BOX A, cannot be sustained in the E-configuration. Consequently, two polar moieties, the acetamide group and the pyrrole N–H functionality, become fully available for intermolecular interactions. Hence, all hydrophilic groups of the molecule are oriented outwardly, which leads to a substantially increased hydrophilicity (confirmed in the markedly earlier elution in reversed-phase chromatography) and makes E-BOX A much less likely to retain in lipophilic compartments such as cell membranes or myelin tissue. Augmented polar intermolecular interactions also become obvious from the crystal structure of E-BOX A, where the individual molecules develop meshed structures (Supporting Information Figure S3), whereas Z-BOX A forms only ribbon-like patterns (Supporting Information Figure S4). Moreover, the photochemical break-up of the intramolecular hydrogen bond of Z-BOX A abrogates the enforced coplanarity of the pyrrole ring and the acetamide group, which exhibit a dihedral angle of about 33° in E-BOX A. As a consequence of the markedly altered steric and distribution of functional groups, E-BOX A may interact distinctly different with the molecular targets of its Z-isomer.
In Vitro and In Vivo Testing of E-BOX A in Mouse Models
In contrast to Z-BOX A, E-BOX A does not cause vasoconstriction in cerebral arterioles in vitro and in vivo. This proves that the structural alterations drastically influence the vasoconstrictive potency and further pinpoints a fine-tuned recognition of these small molecule effectors. Given that the photoconversion neutralizes the detrimental vasoconstrictive properties of BOX A, E-BOX A fulfills the central requirement for the feasibility of a preventive and therapeutic photoconversion of Z-BOX A. Interestingly, we observed a tendency to a slight vascular narrowing in the in vivo experiments after an initial 90 min treatment with E-BOX A. This trend continued until the end of the extended observation time of 3 h without reaching a steady state. In the light of our cell culture experiments and in particular the protein-triggered reisomerization of E-BOX A to Z-BOX A, it is conclusive that fractions of the applied E-BOX A reisomerized upon contact with the mouse CSF and brain tissue. This entails a gentle increase of Z-BOX A concentrations, which subsequently explain the smooth but constant vascular narrowing. By making use of the results of E-BOX A reisomerization in protein-rich biological matrices, we interpolate a conversion of E-BOX A to Z-BOX A of approximately 7% in 3 h. With respect to recently published data on the dose–response characteristics of Z-BOX A, this concentration of about 2 μmol/L would still be sufficient to cause a comparable vasoconstriction in the same assays. (15) Since also Z-configured BOX B and PDP A1/2 and B1/2 contain analogous structural elements, particularly the exocyclic double bond, we anticipate that the photoconversion of these effectors leads to a similar neutralization of vasoconstrictive impact.
Conclusions
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In conclusion, photoconversion of HDPs affects the intramolecular cohesion and alters the positioning of functional groups. At the example of BOX A, we demonstrate that beyond the markedly altered physicochemical properties, photoconversion to the E-isomer particularly changes the pharmacodynamics of the molecule and neutralizes the detrimental vasoconstrictive properties. In light of our findings, an irradiation-based conversion of the naturally occurring Z-HDPs to the transiently stable E-HDPs e.g. via extracorporeal apheresis seems to be a feasible option to prevent and treat HDP-mediated hypoperfusion in SAH patients. Our findings encourage further investigations on such irradiation-based treatment options for DCI.
Experimental Section
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Preparation and Quantification of BOXes and PDPs
Z-BOXes and Z-PDPs were prepared and purified from in vitro degradation of Z,Z-bilirubin with hydrogen peroxide and from chemical synthesis according to the published protocols. (12,16,19,20)
The E-isomers of BOXes and PDPs were prepared via irradiation of the corresponding Z-isomers. Solutions of Z-BOX A, Z-BOX B, Z-PDP A1/2, and Z-PDP B1/2 in acetonitrile/water were exposed for 0.5–60 min to the light of a xenon arc lamp or sunlight at which different K-edge glass filters (SCHOTT, Jena, Germany) were introduced to modulate the irradiation wavelength range (Supporting Information Figure S1). The used filter set is given as an identifier and absorption edge wavelength: WG7 (275 nm), WG3 (335 nm), WG5 (310 nm), WG9 (345 nm), GG13 (360 nm), GG14 (485 nm), OG3 (575 nm), and RG1 (590 nm). After irradiation, the samples were analyzed via HPLC-MS/MS and integrated peak areas were compared.
E-BOX A and B were purified via isocratic high-performance liquid chromatography (HPLC) based on a published protocol (19) but with 17% (v/v) acetonitrile/water and a flow rate of 5 mL/min.
Crystal structure determination and crystal data are reported in the Supporting Information.
The peak areas of Z- and E-BOX A, BOX B, PDP A1/2, and PDP B1/2 were assessed via (ultra) HPLC coupled to (high resolution) mass spectrometry using the described procedures. (15,21)
Reisomerization of E-BOX A in Different Chemical Environments and Human Specimens
Aliquots of HPLC-purified aqueous E-BOX A (5 μL of 100 μmol/L) were added to a final concentration of 5 μmol/L to water, artificial cerebrospinal fluid (aCSF), 1% human serum albumin in aCSF, human CSF, and human serum (N = 3 independent samples each plus three technical replicates of each sample) and incubated under light exclusion at 37 °C. Human CSF and serum samples without E-BOX A addition served as a control for the potential generation of BOXes by the oxidation of heme, biliverdin, or bilirubin. After 6, 24, and 48 h of incubation, one technical replicate of each biological replicate was quenched and extracted (protein precipitation) by 10 min vortex mixing with 200 μL of acetonitrile followed by 10 min centrifugation at 16.100g. The supernatant was transferred to brown glass vials and kept at 4 °C in an autosampler until measurement via HPLC-MS/MS. The samples of the supernatants from cell culture experiments (see below) were taken after 24 h and 48 h of incubation time at 37 °C in the dark and analyzed likewise.
Human Specimens
Human serum and human cerebrospinal fluid (CSF) from EVD were withdrawn with written consent after approval by the ethical committee of the Jena University Hospital (3256-09/11, 3548-08/12). The study was not preregistered.
Animals
All animal procedures were approved by the local regulatory authority (Thüringer Landesamt für Verbraucherschutz; registration numbers 02-040/12, and TWZ-030-2017) and carried out in accordance with the Federation of European Laboratory Animal Science Associations, the German Federal Institute for Risk Assessment and the ARRIVE (25) guidelines. In vitro experiments were performed on acute brain slices from randomly assigned male and female Slo1-WT mice (26) at postnatal day 20–31 (N = 20; P0 is defined as the day of birth). For in vivo studies, adult male and female Slo1-WT at P70-200 (body weight: 20–35 g) were used (N = 14) in experiments during the daytime. Animals were obtained by crossing heterozygous Slo1 mice. All mice had a genetic background of FVB/NJ, (15,26) were imported from the ULAR Diagnostic Services and Rodent QA (Philadelphia), and were genotyped in the second postnatal week. Animals had ad libitum access to food and water. Up to five mice were housed in environmental enrichment cages with a soft filter top.
Brain Slice Preparation, Optical Imaging, and Diameter Calculation In Vitro
Acute mouse brain slice preparation was already reported. (15) The slice samples were illuminated using differential interference contrast video microscopy (Eclipse FN1, Nikon Instruments, Amsterdam, Netherlands) in combination with a Hamamatsu camera, a PCI-RTV24 acquisition board, driven by the software ViewCreatorPro (ADLINK Technology, Taipei, Taiwan). Arterioles located in layer II/III of the parietal cortex and running perpendicular to the cortical surface were selected as structures of interest. The brain slice recording and arteriolar diameter calculation was performed as recently reported. (15)
Cranial Window Implantation and Two-Photon Laser Scanning Microscopy of Cerebral Arterioles
In intact animals, pial arterioles were illuminated by two-photon laser scanning microscopy after acute implantation of a cranial window according to a recently published standard protocol. (15) To minimize animal suffering during the in vivo experiment, animals were pretreated with the analgesic drug meloxicam (1 mg/kg) and anaesthetized with isoflurane. Isoflurane allows a precise control of the respiratory rate and anesthetic state. Before the scalp incision, the skin of the head was infiltrated by the subcutaneous application of 2% lidocaine. To differentiate arterioles from veins, the fluorescent dye Alexa 633 was puff-applied in the subarachnoid space via glass micropipettes (resistance: 1.0–1.5 MΩ, puff length: 100 ms, pressure: 1 bar) penetrating the dura mater. E-BOX A (100 μmol/L) was locally applied by the same procedure as Alexa 633 (<100 μm distance to the arteriolar vessel wall of interest). Fluorescence excitation at 810 nm was provided by a tunable Ti:Sapphire laser (Chameleon Ultra II, Coherent, Santa Clara, California) using a 20× water immersion objective. Changes in the diameter of pial arterioles were monitored at 5 Hz (MPScan). After optical recording, animals were killed by decapitation under deep isoflurane anesthesia. No animals died during the experiments. The study was exploratory and had no exclusion criteria.
Vessel Diameter In Vivo
We selected pial arterioles on the brain surface with an intraluminal diameter ranging between 30 and 80 μm. Owing to intrinsic regulated vasomotions, pial arterioles were monitored over 10 min for each measurement point to subtract out these variations. In detail, the medium arteriolar diameter was calculated as the distance between the opposite labeled vessel walls at the level of the largest diameter and at three defined locations along the vessel course in the field of view. Therefore, we used an orthogonal projection of the current image stack provided by ImageJ (version 1.48, Wayne Rasband, National Institutes of Health).
Cell Culture
To study the impact of E-BOXes and PDP isomers on cell morphology with regard to a cell culture model established for Z-BOXes, (14) HepG2 cells (DSMZ, Braunschweig, Germany; authenticated by short tandem repeats profiling; twelve passages at the maximum) were incubated overnight to attach (30.000 cells/well of a 96-well plate). The cells were treated (N = 3 independent cell culture preparations) either with the freshly prepared individual heme degradation products at 500 μmol/L in Dulbecco’s modified Eagle’s medium/F-12 containing 10% fetal bovine serum for 24 h or with treatment solutions that were kept for 24 h at 37 °C before the 24 h incubation. Morphological changes were monitored via light microscopy.
Statistical Analysis
The animal experiments were single-blinded. The experimenter was masked to the applied BOX A compounds. Within the experimental groups, animals were identified by numbers and a random number generator selected the animals to be treated next. In the case of in vitro experiments, the parameter N represents the number of animals tested. Because only one arteriole per animal was always monitored, N also represents the number of arterioles. For in vivo experiments, N represents the total number of animals. The assessed number of animals used per group was set to N = 7 (in vitro) or N = 5 (in vivo). In dot plots, all marked line values represent means ± standard deviation (SD). Line diagrams depicted the mean ± 95% SD. Group comparisons were performed using Student’s t-test for two independent samples or one-way analysis of variance (ANOVA) for more than two groups with the Bonferroni test applied to correct for multiple comparisons. To check the robustness of the results with regard to parametric test assumptions, the nonparametric equivalent of the parametric test was also applied. As the results did not differ substantially, the description is given as parametric test results. With regard to the animals, no sample calculation was performed. An unadjusted P ≤ 0.05 was considered statistically significant. Data were gathered, statistically analyzed, and displayed using ImageJ (version 1.48, Wayne Rasband, National Institutes of Health), Microsoft Excel (Microsoft Office 2010, Redmond, Washington), SigmaPlot 14 (Systat, Erkrath, Germany), and OriginPro 9.1 (OriginLab, Northampton, Massachusetts).
Supporting Information
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01698.
Transmission spectra of K-edge glass filters employed to adjust the wavelength range of irradiation; UV/Vis absorbance spectra of Z-BOX A and B as well as of Z-PDP A1, Z-PDP B1, Z-PDP A2, and Z-PDP B2 in aqueous solution; E-BOX A forms meshed structures with a three-dimensional spread; Z-BOX A forms ribbon-like structures with a two-dimensional spread; 1H NMR spectrum of E-BOX A in deuterated acetonitrile; irradiation of single PDPs and assessment of newly formed E-isomers by LC-MS; oxidation of isolated E-PDPs to E-BOXes; morphological effects of Z-PDP A1/2 and Z-PDP B1/2 treatments on HepG2 cells compared to control conditions; time-line diagram of the in vivo animal experiments (PDF)
Terms & Conditions
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Author Information
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- Georg Pohnert - Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingstraße 8, 07743 Jena, Germany;
http://orcid.org/0000-0003-2351-6336;
- Raphael A. Seidel - Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingstraße 8, 07743 Jena, Germany; Department of Anesthesiology and Intensive Care Medicine/Center for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, 07747 Jena, Germany; Devie Medical, c/o Jena University Hospital, Bachstraße 18, 07743 Jena, Germany;http://orcid.org/0000-0002-9848-7122
- Matthias Westerhausen - Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Humboldtstraße 8, 07743 Jena, Germany;http://orcid.org/0000-0002-1520-2401
R.A.S. and M.R. contributed equally to this work.
This manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
R.A.S. and M.R. contributed equally to this work.
This manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Acknowledgments
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The authors kindly acknowledge Franziska Röstel, Hannes Richter, and Dr. Marcel Kahnes for technical assistance and financial support by the German Research Foundation within the framework of FOR1738. A.J. and O.W.W. acknowledge funding by the Research Program, “Else Kröner-Forschungskolleg AntiAge”.
| aCSF | artificial cerebrospinal fluid |
| BOX | bilirubin oxidation end product |
| CSF | cerebrospinal fluid |
| DCI | delayed cerebral ischemia |
| EVD | external ventricular drainage |
| HSA | human serum albumin |
| HDP | heme degradation product |
| (U)HPLC | (ultra) high-performance liquid chromatography |
| PDP | propentdyopent |
| MS | mass spectrometry |
| SAH | subarachnoid hemorrhage |
| SD | standard deviation |
References
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This article references 26 other publications.
- 1Steiner, T.; Juvela, S.; Unterberg, A.; Jung, C.; Forsting, M.; Rinkel, G. European Stroke Organization guidelines for the management of intracranial aneurysms and subarachnoid haemorrhage. Cerebrovasc. Dis. 2013, 35, 93– 112, DOI: 10.1159/000346087Google Scholar1European Stroke Organization guidelines for the management of intracranial aneurysms and subarachnoid haemorrhageSteiner Thorsten; Juvela Seppo; Unterberg Andreas; Jung Carla; Forsting Michael; Rinkel GabrielCerebrovascular diseases (Basel, Switzerland) (2013), 35 (2), 93-112 ISSN:.BACKGROUND: Intracranial aneurysm with and without subarachnoid haemorrhage (SAH) is a relevant health problem: The overall incidence is about 9 per 100,000 with a wide range, in some countries up to 20 per 100,000. Mortality rate with conservative treatment within the first months is 50-60%. About one third of patients left with an untreated aneurysm will die from recurrent bleeding within 6 months after recovering from the first bleeding. The prognosis is further influenced by vasospasm, hydrocephalus, delayed ischaemic deficit and other complications. The aim of these guidelines is to provide comprehensive recommendations on the management of SAH with and without aneurysm as well as on unruptured intracranial aneurysm. METHODS: We performed an extensive literature search from 1960 to 2011 using Medline and Embase. Members of the writing group met in person and by teleconferences to discuss recommendations. Search results were graded according to the criteria of the European Federation of Neurological Societies. Members of the Guidelines Committee of the European Stroke Organization reviewed the guidelines. RESULTS: These guidelines provide evidence-based information on epidemiology, risk factors and prognosis of SAH and recommendations on diagnostic and therapeutic methods of both ruptured and unruptured intracranial aneurysms. Several risk factors of aneurysm growth and rupture have been identified. We provide recommendations on diagnostic work up, monitoring and general management (blood pressure, blood glucose, temperature, thromboprophylaxis, antiepileptic treatment, use of steroids). Specific therapeutic interventions consider timing of procedures, clipping and coiling. Complications such as hydrocephalus, vasospasm and delayed ischaemic deficit were covered. We also thought to add recommendations on SAH without aneurysm and on unruptured aneurysms. CONCLUSION: Ruptured intracranial aneurysm with a high rate of subsequent complications is a serious disease needing prompt treatment in centres having high quality of experience of treatment for these patients. These guidelines provide practical, evidence-based advice for the management of patients with intracranial aneurysm with or without rupture. Applying these measures can improve the prognosis of SAH.
- 2Corsten, L.; Raja, A.; Guppy, K.; Roitberg, B.; Misra, M.; Alp, M. S.; Charbel, F.; Debrun, G.; Ausman, J. Contemporary management of subarachnoid hemorrhage and vasospasm: the UIC experience. Surg. Neurol. 2001, 56, 140– 150, DOI: 10.1016/S0090-3019(01)00513-4Google Scholar2Contemporary management of subarachnoid hemorrhage and vasospasm: the UIC experienceCorsten L; Raja A; Guppy K; Roitberg B; Misra M; Alp M S; Charbel F; Debrun G; Ausman JSurgical neurology (2001), 56 (3), 140-8; discussion 148-50 ISSN:0090-3019.BACKGROUND: Cerebral vasospasm is a well-known and serious complication of aneurysmal subarachnoid hemorrhage. The means of monitoring and treatment of vasospasm have been widely studied. Each neurosurgical center develops a protocol based on their experience, availability of equipment and personnel, and cost, so as to keep morbidity and mortality rates as low as possible for their patients with vasospasm. METHODS: At the University of Illinois at Chicago, we have developed algorithms for the diagnosis and management of cerebral vasospasm based on the experience of the senior authors over the past 25 years. This paper describes in detail our approach to diagnosis and treatment of aneurysmal subarachnoid hemorrhage and vasospasm. Our discussion is highlighted with data from a retrospective analysis of 324 aneurysm patients. RESULTS: Over 3 years, 324 aneurysms were treated; 185 (57%) were clipped, 139 (43%) were coiled. The rate of vasospasm for the 324 patients was 27%. The rate of hydrocephalus was 32% for those patients who underwent clipping, and 29% for those coiled. The immediate outcomes for those who underwent clipping was excellent in 35%, good in 38%, poor in 15.5%, vegetative in 3%, and death in 8% of the patients. For those who underwent coiling the immediate outcome was excellent in 64%, good in 14.5%, vegetative in 2.5%, and death in 14.5% of the patients. These statistics include all Hunt and Hess grades. For those patients who underwent clipping, 51% were intact at 6 months follow-up, 15% had a permanent deficit, 10% had a focal cranial nerve deficit, and 2% had died from complications not directly related to the procedure. For those patients who had undergone coiling, 75% were intact at 6 months follow-up, 12.5% had a permanent deficit, and 12.5% had a cranial nerve deficit, with no deaths. CONCLUSIONS: The morbidity and mortality of cerebral vasospasm is significant. A good outcome after aneurysmal subarachnoid hemorrhage is dependent upon careful patient management in the preoperative, perioperative, and postoperative periods. The timely work-up and aggressive treatment of neurological deterioration, whether or not it is because of vasospasm, is paramount.
- 3Uhl, E.; Lehmberg, J.; Steiger, H. J.; Messmer, K. Intraoperative detection of early microvasospasm in patients with subarachnoid hemorrhage by using orthogonal polarization spectral imaging. Neurosurgery 2003, 52, 1307– 1317, DOI: 10.1227/01.NEU.0000065154.04824.9EGoogle Scholar3Intraoperative detection of early microvasospasm in patients with subarachnoid hemorrhage by using orthogonal polarization spectral imagingUhl Eberhard; Lehmberg Jens; Steiger Hans-Jakob; Messmer KonradNeurosurgery (2003), 52 (6), 1307-15; disacussion 1315-7 ISSN:0148-396X.OBJECTIVE: Changes of major cerebral vessels in patients with subarachnoid hemorrhage (SAH) are well known from routine cerebral angiography. Data on changes in the microcirculation do not exist. This study sought to provide a qualitative and quantitative analysis of the cortical microcirculation after SAH. METHODS: By means of orthogonal polarization spectral imaging, a qualitative and quantitative analysis of cortical microcirculation was performed during aneurysm surgery in 3 patients with an incidental intracerebral aneurysm and 10 patients with SAH. Vessel diameters, red blood cell velocity, and functional capillary density were analyzed before and after the aneurysm was clipped. RESULTS: Initial capillary density in patients with an incidental aneurysm was 91.5 +/- 36.5 cm(-1) (mean +/- standard deviation) compared with 30.5 +/- 13.8 in patients with SAH (P < 0.05). In patients with SAH, capillary density increased significantly to 53.9 +/- 29.1 cm(-1) (P < 0.05) during the operation, as did the frequency of venules with a red blood cell velocity greater than 2 mm/s (P < 0.05). No significant change of arteriolar or venular diameters was observed. However, in patients with SAH, mono- and multisegmental microvasospasms in arterioles were observed, with a reduction of vessel diameters up to 75.1%. CONCLUSION: Orthogonal polarization spectral imaging is a suitable method to study cerebral microcirculation during surgery. In patients with SAH, capillary density is significantly decreased and small arteries and arterioles of the cortical surface exhibit vasospasm that cannot be detected by angiography or transcranial Doppler sonography. These changes may contribute to the initial clinical symptoms and may have an influence on the clinical postoperative course.
- 4Giller, C. A.; Giller, A. M.; Landreneau, F. Detection of emboli after surgery for intracerebral aneurysms. Neurosurgery 1998, 42, 490– 494, DOI: 10.1097/00006123-199803000-00010Google Scholar4Detection of emboli after surgery for intracerebral aneurysmsGiller C A; Giller A M; Landreneau FNeurosurgery (1998), 42 (3), 490-3; discussion 493-4 ISSN:0148-396X.BACKGROUND: Neurological change after surgery for cerebral aneurysm caused by embolic events is commonly suspected, but direct detection of emboli has not been possible in the past. Transcranial Doppler ultrasound (TCD) is able to detect emboli, and large numbers of emboli detected in TCD studies have been associated with radiological changes and clinical deterioration. METHODS: During a 2-year period, 11 patients were observed to have emboli during routine TCD studies after aneurysm surgery. The computed tomographic (CT) scans of these patients were reviewed for low-density areas, suggesting ischemia. All patients studied during a 1-year interval (July 1995-July 1996) served as a control group and were reviewed for similar CT findings, and the two groups were compared using Fisher's exact test. RESULTS: Nine of the 11 patients (82%) observed to have emboli developed low-density areas on their CT scans, whereas 30 of the 123 (24%) patients without emboli developed low-density areas on their CT scans. The difference was significant (P < 0.001, Fisher's exact test). Credible sources for emboli were readily identified in each of the 11 patients. CONCLUSION: TCD allows detection of emboli after aneurysm surgery, and this detection is strongly associated with CT evidence of ischemia. Although detection of emboli was relatively rare in this study, rates of emboli occurrence may increase if systematic monitoring is used.
- 5Korbakis, G.; Prabhakaran, S.; John, S.; Garg, R.; Conners, J. J.; Bleck, T. P.; Lee, V. H. MRI Detection of Cerebral Infarction in Subarachnoid Hemorrhage. Neurocrit. Care 2016, 24, 428– 435, DOI: 10.1007/s12028-015-0212-zGoogle Scholar5MRI Detection of Cerebral Infarction in Subarachnoid HemorrhageKorbakis Georgia; Prabhakaran Shyam; John Sayona; Garg Rajeev; Bleck Thomas P; Conners James J; Lee Vivien HNeurocritical care (2016), 24 (3), 428-35 ISSN:.OBJECTIVE: To investigate magnetic resonance imaging (MRI) detection of cerebral infarction (CI) in patients presenting with subarachnoid hemorrhage (SAH). BACKGROUND: CI is a well-known complication of SAH that is typically detected on computed tomography (CT). MRI has improved sensitivity for acute CI over CT, particularly with multiple, small, or asymptomatic lesions. METHODS: With IRB approval, 400 consecutive SAH patients admitted to our institution from August 2006 to March 2011 were retrospectively reviewed. Traumatic SAH and secondary SAH were excluded. Data were collected on demographics, cause of SAH, Hunt Hess and World Federation of Neurosurgical Societies grades, and neuroimaging results. MRIs were categorized by CI pattern as single cortical (SC), single deep (SD), multiple cortical (MC), multiple deep (MD), and multiple cortical and deep (MCD). RESULTS: Among 123 (30.8 %) SAH patients who underwent MRIs during their hospitalization, 64 (52 %) demonstrated acute CI. The mean time from hospital admission to MRI was 5.7 days (range 0-29 days). Among the 64 patients with MRI infarcts, MRI CI pattern was as follows: MC in 20 (31 %), MCD in 18 (28 %), SC in 16 (25 %), SD in 3 (5 %), MD in 2 (3 %), and 5 (8 %) did not have images available for review. Most infarcts detected on MRI (39/64 or 61 %) were not visible on CT. CONCLUSIONS: The use of MRI increases the detection of CI in SAH. Unlike CT studies, MRI-detected CI in SAH tends to involve multiple vascular territories. Studies that rely on CT may underestimate the burden of CI after SAH.
- 6Rodriguez-Régent, C.; Hafsa, M.; Turc, G.; Ben Hassen, W.; Edjlali, M.; Sermet, A.; Laquay, N.; Trystram, D.; Al-Shareef, F.; Meder, J. F. Early quantitative CT perfusion parameters variation for prediction of delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage. Eur. Radiol. 2016, 26, 2956– 2963, DOI: 10.1007/s00330-015-4135-zGoogle Scholar6Early quantitative CT perfusion parameters variation for prediction of delayed cerebral ischemia following aneurysmal subarachnoid hemorrhageRodriguez-Regent Christine; Hafsa Monia; Ben Hassen Wagih; Edjlali Myriam; Trystram Denis; Al-Shareef Fawaz; Meder Jean-Francois; Oppenheim Catherine; Naggara Olivier; Turc Guillaume; Sermet Alain; Laquay Nathalie; Devaux BertrandEuropean radiology (2016), 26 (9), 2956-63 ISSN:.OBJECTIVES: To prospectively evaluate the predictive value of cerebral perfusion-computerized tomography (CTP) parameters variation between day0 and day4 after aneurysmal subarachnoid haemorrhage (aSAH). METHODS: Mean transit time (MTT) and cerebral blood flow (CBF) values were compared between patients with delayed cerebral ischemia (DCI+ group) and patients without DCI (DCI- group) for previously published optimal cutoff values and for variations of MTT (ΔMTT) and of CBF (ΔCBF) values between day0 and day4. DCI+ was defined as a cerebral infarction on 3-months follow-up MRI. RESULTS: Among 47 included patients, 10 suffered DCI+. Published optimal cutoff values did not predict DCI, either at day0 or at day4. Conversely, ΔMTT and ΔCBF significantly differed between the DCI+ and DCI- groups, with optimal ΔMTT and ΔCBF values of 0.91 seconds (83.9 % sensitivity, 79.5 % specificity, AUC 0.84) and -7.6 mL/100 g/min (100 % sensitivity, 71.4 % specificity, AUC 0.86), respectively. In multivariate analysis, ΔCBF (OR = 1.91, IC95% 1.13-3.23 per each 20 % decrease of ΔCBF) and ΔMTT values (OR = 14.70, IC95% 4.85-44.52 per each 20 % increase of ΔMTT) were independent predictors of DCI. CONCLUSIONS: Assessment of MTT and CBF value variations between day0 and day4 may serve as an early imaging surrogate for prediction of DCI in aSAH. KEY POINTS: • CT perfusion values are an imaging surrogate for prediction of DCI. • Early variations (day0-day4) after aneurysmal subarachnoid haemorrhage predicted DCI. • A CBF decrease of 7.6 mL/min/100 g predicted DCI with 100 % sensitivity. • An MTT increase of 0.91 seconds predicted DCI with 83.9 % sensitivity. • DCI risk multiplied by 2 per 20 % ΔCBF decrease and by 15 per 20 % ΔMTT increase.
- 7Balbi, M.; Koide, M.; Wellman, G. C.; Plesnila, N. Inversion of neurovascular coupling after subarachnoid hemorrhage in vivo. J. Cereb. Blood Flow Metab. 2017, 37, 3625– 3634, DOI: 10.1177/0271678X16686595Google Scholar7Inversion of neurovascular coupling after subarachnoid hemorrhage in vivoBalbi Matilde; Plesnila Nikolaus; Balbi Matilde; Plesnila Nikolaus; Koide Masayo; Wellman George C; Plesnila NikolausJournal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism (2017), 37 (11), 3625-3634 ISSN:.Subarachnoid hemorrhage (SAH) induces acute changes in the cerebral microcirculation. Recent findings ex vivo suggest neurovascular coupling (NVC), the process that increases cerebral blood flow upon neuronal activity, is also impaired after SAH. The aim of the current study was to investigate whether this occurs also in vivo. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. Twenty-four hours later NVC was tested by forepaw stimulation and CO2 reactivity by inhalation of 10% CO2. Vessel diameter was assessed in vivo by two-photon microscopy. NVC was also investigated ex vivo using brain slices. Cerebral arterioles of sham-operated mice dilated to 130% of baseline upon CO2 inhalation or forepaw stimulation and cerebral blood flow (CBF) increased. Following SAH, however, CO2 reactivity was completely lost and the majority of cerebral arterioles showed paradoxical constriction in vivo and ex vivo resulting in a reduced CBF response. As previous results showed intact NVC 3 h after SAH, the current findings indicate that impairment of NVC after cerebral hemorrhage occurs secondarily and is progressive. Since neuronal activity-induced vasoconstriction (inverse NVC) is likely to further aggravate SAH-induced cerebral ischemia and subsequent brain damage, inverse NVC may represent a novel therapeutic target after SAH.
- 8Dreier, J. P.; Drenckhahn, C.; Woitzik, J.; Major, S.; Offenhauser, N.; Weber-Carstens, S.; Wolf, S.; Strong, A. J.; Vajkoczy, P.; Hartings, J. A. Spreading ischemia after aneurysmal subarachnoid hemorrhage. Acta Neurochir., Suppl. 2013, 115, 125– 129Google Scholar8Spreading ischemia after aneurysmal subarachnoid hemorrhageDreier Jens P; Drenckhahn Christoph; Woitzik Johannes; Major Sebastian; Offenhauser Nikolas; Weber-Carstens Steffen; Wolf Stefan; Strong Anthony J; Vajkoczy Peter; Hartings Jed AActa neurochirurgica. Supplement (2013), 115 (), 125-9 ISSN:0065-1419.Spreading depolarization (SD) is a wave of mass neuronal and glial depolarization associated with net influx of cations and water. Prolonged SDs facilitate neuronal death. SD induces tone alterations in cerebral resistance arterioles, leading to either transient hyperperfusion (physiological neurovascular coupling) in healthy tissue or hypoperfusion (inverse neurovascular coupling = spreading ischemia) in tissue at risk for progressive damage. Spreading ischemia has been shown experimentally in an animal model replicating the conditions present following aneurysmal subarachnoid hemorrhage (aSAH), in animal models of the ischemic core and penumbra following middle cerebral artery occlusion, and in patients with aSAH. In animals, spreading ischemia produced widespread cortical necrosis. In patients, spreading ischemia occurred in temporal correlation with ischemic lesion development early and late after aSAH. We briefly review important features of SD and spreading ischemia following aSAH.
- 9Vivancos, J.; Gilo, F.; Frutos, R.; Maestre, J.; Garcia-Pastor, A.; Quintana, F.; Roda, J. M.; Ximenez-Carrillo, A. Clinical management guidelines for subarachnoid haemorrhage. Diagnosis and treatment. Neurología 2014, 29, 353– 370, DOI: 10.1016/j.nrl.2012.07.009Google Scholar9Clinical management guidelines for subarachnoid haemorrhage. Diagnosis and treatmentVivancos J; Gilo F; Frutos R; Maestre J; Garcia-Pastor A; Quintana F; Roda J M; Ximenez-Carrillo A; Diez Tejedor E; Fuentes B; Alonso de Lecinana M; Alvarez-Sabin J; Arenillas J; Calleja S; Casado I; Castellanos M; Castillo J; Davalos A; Diaz-Otero F; Egido J A; Fernandez J C; Freijo M; Gallego J; Gil-Nunez A; Irimia P; Lago A; Masjuan J; Marti-Fabregas J; Martinez-Sanchez P; Martinez-Vila E; Molina C; Morales A; Nombela F; Purroy F; Ribo M; Rodriguez-Yanez M; Roquer J; Rubio F; Segura T; Serena J; Simal P; Tejada JNeurologia (Barcelona, Spain) (2014), 29 (6), 353-70 ISSN:.OBJECTIVE: To update the Spanish Society of Neurology's guidelines for subarachnoid haemorrhage diagnosis and treatment. MATERIAL AND METHODS: A review and analysis of the existing literature. Recommendations are given based on the level of evidence for each study reviewed. RESULTS: The most common cause of spontaneous subarachnoid haemorrhage (SAH) is cerebral aneurysm rupture. Its estimated incidence in Spain is 9/100 000 inhabitants/year with a relative frequency of approximately 5% of all strokes. Hypertension and smoking are the main risk factors. Stroke patients require treatment in a specialised centre. Admission to a stroke unit should be considered for SAH patients whose initial clinical condition is good (Grades I or II on the Hunt and Hess scale). We recommend early exclusion of aneurysms from the circulation. The diagnostic study of choice for SAH is brain CT (computed tomography) without contrast. If the test is negative and SAH is still suspected, a lumbar puncture should then be performed. The diagnostic tests recommended in order to determine the source of the haemorrhage are MRI (magnetic resonance imaging) and angiography. Doppler ultrasonography studies are very useful for diagnosing and monitoring vasospasm. Nimodipine is recommended for preventing delayed cerebral ischaemia. Blood pressure treatment and neurovascular intervention may be considered in treating refractory vasospasm. CONCLUSIONS: SAH is a severe and complex disease which must be managed in specialised centres by professionals with ample experience in relevant diagnostic and therapeutic processes.
- 10Kranc, K. R.; Pyne, G. J.; Tao, L.; Claridge, T. D.; Harris, D. A.; Cadoux-Hudson, T. A.; Turnbull, J. J.; Schofield, C. J.; Clark, J. F. Oxidative degradation of bilirubin produces vasoactive compounds. Eur. J. Biochem. 2000, 267, 7094– 7101, DOI: 10.1046/j.1432-1327.2000.01812.xGoogle Scholar10Oxidative degradation of bilirubin produces vasoactive compoundsKranc, Kamil R.; Pyne, Gail J.; Tao, Limei; Claridge, Tim D. W.; Harris, David A.; Cadoux-Hudson, Thomas A. D.; Turnbull, Jonathan J.; Schofield, Christopher J.; Clark, Joseph F.European Journal of Biochemistry (2000), 267 (24), 7094-7101CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)Subarachnoid hemorrhage is often followed by hemolysis and concomitant oxidative stress, and is frequently complicated by pathol. vasoconstriction or cerebral vasospasm. It is known that upregulation of heme oxygenase (HO-1) is induced by oxidative stress and results in release of biliverdin and bilirubin (BR), which are scavengers of reactive oxygen species (ROS). Here the authors report biomimetic studies aimed at modeling pathol. conditions leading to oxidative degrdn. of BR. Oxidative degrdn. products of BR, formed by reaction with hydrogen peroxide (an ROS model system), demonstrated biol. activity by stimulating oxygen consumption and force development in vascular smooth muscle from porcine carotid artery. Analogous biol. activity was obsd. with vasoactive cerebrospinal fluid from subarachnoid hemorrhage patients. Three degrdn. products of BR were isolated: two were assigned as isomeric monopyrrole (C9H11N2O2) derivs., 4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide and 3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide and the third was 4-methyl-3-vinylmaleimide (MVM), a previously isolated photodegrdn. product of biliverdin. Possible mechanisms of oxidative degrdn. of BR are discussed. Tentative assignment of these structures in the cerebrospinal fluid (CSF) of cerebral vasospasm patients has been made. It is proposed that one or more of the degrdn. products of biliverdin or bilirubin are involved in complications such as vasospasm and or pathol. vasoconstriction assocd. with hemorrhage.
- 11Ritter, M.; Neupane, S.; Seidel, R. A.; Steinbeck, C.; Pohnert, G. In vivo and in vitro identification of Z-BOX C—a new bilirubin oxidation end product. Org. Biomol. Chem. 2018, 16, 3553– 3555, DOI: 10.1039/C8OB00164BGoogle Scholar11In vivo and in vitro identification of Z-BOX C - a new bilirubin oxidation end productRitter, Marcel; Neupane, Sandesh; Seidel, Raphael A.; Steinbeck, Christoph; Pohnert, GeorgOrganic & Biomolecular Chemistry (2018), 16 (19), 3553-3555CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)A new bilirubin oxidn. 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 pathol. human bile samples using liq. chromatog./mass spectrometry and has potential relevance for liver dysfunction and cerebral vasospasms.
- 12Ritter, M.; Seidel, R. A.; Bellstedt, P.; Schneider, B.; Bauer, M.; Gorls, H.; Pohnert, G. Isolation and Identification of Intermediates of the Oxidative Bilirubin Degradation. Org. Lett. 2016, 18, 4432– 4435, DOI: 10.1021/acs.orglett.6b02287Google Scholar12Isolation and Identification of Intermediates of the Oxidative Bilirubin DegradationRitter, Marcel; Seidel, Raphael A.; Bellstedt, Peter; Schneider, Bernd; Bauer, Michael; Goerls, Helmar; Pohnert, GeorgOrganic Letters (2016), 18 (17), 4432-4435CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)Four endogenous products of oxidative bilirubin degrdn. were isolated and fully characterized. The constitutional isomers belong to the propentdyopents (PDPs). Their structures and further oxidative transformations to biol. active bilirubin oxidn. end products (Z-BOXes) are reported. Using liq. chromatog.-mass spectrometry protocols, PDPs were detected in human bile and gallstones. Given the recent interest in BOXes as effectors in cerebral vasospasms and liver dysfunction, co-occurring PDPs represent an addnl. potentially active compd. class to be considered.
- 13Clark, J. F.; Sharp, F. R. Bilirubin oxidation products (BOXes) and their role in cerebral vasospasm after subarachnoid hemorrhage. J. Cereb. Blood Flow Metab. 2006, 26, 1223– 1233, DOI: 10.1038/sj.jcbfm.9600280Google Scholar13Bilirubin oxidation products (BOXes) and their role in cerebral vasospasm after subarachnoid hemorrhageClark, Joseph F.; Sharp, Frank R.Journal of Cerebral Blood Flow & Metabolism (2006), 26 (10), 1223-1233CODEN: JCBMDN; ISSN:0271-678X. (Nature Publishing Group)A review. Many factors have been postulated to cause delayed subarachnoid hemorrhage (SAH)-induced vasospasm, including Hb, nitric oxide, endothelin, and free radicals. We propose that free radicals (because of the high levels that are produced in the blood clots surrounding blood vessels after SAH) act on bilirubin, biliverdin, and possibly heme to produce BOXes (Bilirubin OXidized Products). Bilirubin oxidn. products act on vascular smooth muscle cells to produce chronic vasoconstriction and vasospasm combined with a vasculopathy because of smooth muscle cell injury. This review summarizes recent evidence that BOXes play a role in SAH-induced vasospasm. The data supporting a role for BOXes includes (1) identification of mols. in cerebrospinal fluid (CSF) of patients with vasospasm after SAH that have structures consistent with BOXes; (2) BOXes are vasoactive in vitro and mimic the biochem. actions of CSF of patients with vasospasm; (3) BOXes are vasoactive in vivo, constricting rat cerebral vessels; and (4) there is a correlation between clin. occurrence of vasospasm and BOXes concn. in our preliminary study of patients with SAH. Since oxidn. of bilirubin, biliverdin, and perhaps heme is proposed to produce BOXes that contribute to vasospasm, either blocking bilirubin formation, inactivating bilirubin or BOXes, or removing all of the blood clot before vasospasm are potential treatment targets.
- 14Seidel, R. A.; Claudel, T.; Schleser, F. A.; Ojha, N. K.; Westerhausen, M.; Nietzsche, S.; Sponholz, C.; Cuperus, F.; Coldewey, S. M.; Heinemann, S. H. Impact of higher-order heme degradation products on hepatic function and hemodynamics. J. Hepatol. 2017, 67, 272– 281, DOI: 10.1016/j.jhep.2017.03.037Google Scholar14Impact of higher-order heme degradation products on hepatic function and hemodynamicsSeidel Raphael A; Claudel Thierry; Trauner Michael; Schleser Franziska A; Sponholz Christoph; Coldewey Sina M; Ojha Navin K; Heinemann Stefan H; Westerhausen Matthias; Nietzsche Sandor; Cuperus Frans; Pohnert Georg; Bauer MichaelJournal of hepatology (2017), 67 (2), 272-281 ISSN:.BACKGROUND & AIMS: Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions. METHODS: Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia. RESULTS: Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbβ. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton. CONCLUSIONS: Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia. LAY SUMMARY: Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats.
- 15Joerk, A.; Ritter, M.; Langguth, N.; Seidel, R. A.; Freitag, D.; Herrmann, K. H.; Schaefgen, A.; Ritter, M.; Gunther, M.; Sommer, C. Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage. Circ. Res. 2019, 124, e101– e114, DOI: 10.1161/CIRCRESAHA.118.314160Google Scholar15Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid HemorrhageJoerk, Alexander; Ritter, Marcel; Langguth, Niklas; Seidel, Raphael Andreas; Freitag, Diana; Herrmann, Karl-Heinz; Schaefgen, Anna; Ritter, Marvin; Guenther, Milena; Sommer, Charline; Braemer, Dirk; Walter, Jan; Ewald, Christian; Kalff, Rolf; Reichenbach, Juergen Rainer; Westerhausen, Matthias; Pohnert, Georg; Witte, Otto Wilhelm; Holthoff, KnutCirculation Research (2019), 124 (12), e101-e114CODEN: CIRUAL; ISSN:0009-7330. (Lippincott Williams & Wilkins)Delayed ischemic neurol. deficit is the most common cause of neurol. impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidn. end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degrdn., known as PDPs (propentdyopents), have been considered as potential addnl. effectors in the development of arterial vasoconstriction. To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. Via liq. chromatog./mass spectrometry, we measured increased PDP and BOX concns. in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diam. in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concn. range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diam. changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we obsd. a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. For the first time, we have quantified significantly higher concns. of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.
- 16Joerk, A.; Seidel, R. A.; Walter, S. G.; Wiegand, A.; Kahnes, M.; Klopfleisch, M.; Kirmse, K.; Pohnert, G.; Westerhausen, M.; Witte, O. W. Impact of heme and heme degradation products on vascular diameter in mouse visual cortex. J. Am. Heart Assoc. 2014, 3, 1– 13, DOI: 10.1161/JAHA.114.001220Google ScholarThere is no corresponding record for this reference.
- 17Jasprova, J.; Dal Ben, M.; Vianello, E.; Goncharova, I.; Urbanova, M.; Vyroubalova, K.; Gazzin, S.; Tiribelli, C.; Sticha, M.; Cerna, M. The Biological Effects of Bilirubin Photoisomers. PLoS One 2016, 11, e0148126 DOI: 10.1371/journal.pone.0148126Google Scholar17The biological effects of bilirubin photoisomersJasprova, Jana; Dal Ben, Matteo; Vianello, Eleonora; Goncharova, Iryna; Urbanova, Marie; Vyroubalova, Karolina; Gazzin, Silvia; Tiribelli, Claudio; Sticha, Martin; Cerna, Marcela; Vitek, LiborPLoS One (2016), 11 (2), e0148126/1-e0148126/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Although phototherapy was introduced as early as 1950's, the potential biol. effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biol. effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepd. by photo-irradn. of unconjugated bilirubin. The individual photoproducts were chromatog. sepd. (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissocn. of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and CD. The biol. effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding const. to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metab. or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.
- 18Watchko, J. F.; Tiribelli, C. Bilirubin-induced neurologic damage--mechanisms and management approaches. N. Engl. J. Med. 2013, 369, 2021– 2030, DOI: 10.1056/NEJMra1308124Google Scholar18Bilirubin-induced neurologic damage - mechanisms and management approachesWatchko, Jon F.; Tiribelli, ClaudioNew England Journal of Medicine (2013), 369 (21), 2021-2030CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)A review. The complex cascade of mol. and cellular events leading to bilirubin-induced neurotoxicity remains incompletely delineated. This review describes bilirubin-induced brain damage and recent insights into its pathogenesis and prevention.
- 19Klopfleisch, M.; Seidel, R. A.; Gorls, H.; Richter, H.; Beckert, R.; Imhof, W.; Reiher, M.; Pohnert, G.; Westerhausen, M. Total synthesis and detection of the bilirubin oxidation product (Z)-2-(3-ethenyl-4-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide (Z-BOX A). Org. Lett. 2013, 15, 4608– 4611, DOI: 10.1021/ol402221bGoogle Scholar19Total Synthesis and Detection of the Bilirubin Oxidation Product (Z)-2-(3-Ethenyl-4-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide (Z-BOX A)Klopfleisch, Maurice; Seidel, Raphael A.; Gorls, Helmar; Richter, Hannes; Beckert, Rainer; Imhof, Wolfgang; Reiher, Markus; Pohnert, Georg; Westerhausen, MatthiasOrganic Letters (2013), 15 (17), 4608-4611CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)The selective total synthesis of the pure Z-isomer of BOX A I, a product of oxidative heme degrdn. with significant physiol. impact, was achieved in four to six steps starting from 3-bromo-4-methylfuran-2,5-dione. Z-BOX A forms a strong hydrogen bridge framework in the cryst. state. LC-MS techniques allow identification and characterization of isomeric forms of BOX A.
- 20Seidel, R. A.; Schowtka, B.; Klopfleisch, M.; Kuhl, T.; Weiland, A.; Koch, A.; Gorls, H.; Imhof, D.; Pohnert, G.; Westerhausen, M. Total synthesis and characterization of the bilirubin oxidation product (Z)-2-(4-ethenyl-3-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide (Z-BOX B). Tetrahedron Lett. 2014, 55, 6526– 6529, DOI: 10.1016/j.tetlet.2014.09.108Google Scholar20Total synthesis and characterization of the bilirubin oxidation product (Z)-2-(4-ethenyl-3-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide (Z-BOX B)Seidel, Raphael A.; Schowtka, Bjoern; Klopfleisch, Maurice; Kuehl, Toni; Weiland, Andreas; Koch, Alexander; Goerls, Helmar; Imhof, Diana; Pohnert, Georg; Westerhausen, MatthiasTetrahedron Letters (2014), 55 (48), 6526-6529CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)Bilirubin oxidn. end products (BOXes) show significant physiol. effects and are connected to severe diseases such as subarachnoid hemorrhage induced cerebral vasospasm. BOX B [2-(4-ethenyl-3-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide] was prepd. via a five-step synthesis starting from Me [4-bromo-3-methyl-5-oxofuran-2(5H)-ylidene]ethanoate, which was vinylated and then reacted with NH4OAc, LiOH, (COCl)2, and finally with ammonia yielding Z-BOX B. Derivs. of Z-BOX A [(Z)-2-(3-ethenyl-4-methyl-5-oxo-1,5-dihydro-2H-[15N]pyrrol-2-ylidene)ethanamide] and Z-BOX B with [15N]labeled lactam rings were synthesized accordingly.
- 21Seidel, R. A.; Kahnes, M.; Bauer, M.; Pohnert, G. Simultaneous determination of the bilirubin oxidation end products Z-BOX A and Z-BOX B in human serum using liquid chromatography coupled to tandem mass spectrometry. J. Chromatogr. B 2015, 974, 83– 89, DOI: 10.1016/j.jchromb.2014.10.027Google Scholar21Simultaneous determination of the bilirubin oxidation end products Z-BOX A and Z-BOX B in human serum using liquid chromatography coupled to tandem mass spectrometrySeidel, Raphael A.; Kahnes, Marcel; Bauer, Michael; Pohnert, GeorgJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2015), 974 (), 83-89CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)Bilirubin oxidn. end products (BOXes) appear upon endogenous heme degrdn. and can be found in the cerebrospinal fluid after hemorrhagic stroke. BOXes are assumed to contribute to delayed cerebral vasospasm and secondary loss of brain tissue. Here, we present a validated LC-ESI-MS/MS method for the sensitive detn. of the regio-isomers Z-BOX A and Z-BOX B in human serum. We found that Z-BOX A and Z-BOX B appear in serum of healthy volunteers. The sample prepn. includes the addn. of 5-bromonicotinamide as internal std. and protein pptn. with acetonitrile. Baseline-sepn. was achieved on a C-18 column with a binary solvent gradient of formic acid in water/acetonitrile at 1 mL/min within a total anal. time of 17 min. Using single reaction monitoring in the pos. ion mode, the linear working ranges were 2.74-163 pg/μL (Z-BOX A) and 2.12-162.4 pg/μL (Z-BOX B) with R2 > 0.995. Intra- and inter-day precisions were <10%. The inherent analyte concns. of Z-BOX A (14.4 ± 5.1 nM) and Z-BOX B (10.9 ± 3.1 nM) in pooled human serum were detd. by std. addn. The photolability of both analytes was demonstrated. This method enables to monitor Z-BOX A and Z-BOX B as a prerequisite to systematically study the biol. significance of higher order metabolites of heme degrdn.
- 22Goncharova, I.; Jasprova, J.; Vitek, L.; Urbanova, M. Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding. Anal. Biochem. 2015, 490, 34– 45, DOI: 10.1016/j.ab.2015.08.001Google Scholar22Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin bindingGoncharova, Iryna; Jasprova, Jana; Vitek, Libor; Urbanova, MarieAnalytical Biochemistry (2015), 490 (), 34-45CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)Bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidn. to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. Here, the authors present a novel anal. concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, CD spectroscopy, and mol. modeling methods was used for recognition of the binding site for BR, its derivs. (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidn. (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of Trp-HSA were used to calc. the binding consts. The results of the CD displacement expts. performed with hemin were interpreted together with the findings of mol. docking performed on the pigment-HSA complexes. The authors estd. that Z,Z-BR and its metabolic products bound to 2 independent binding sites. The findings supported the existence of a reversible antioxidant redox cycle for BR and explained an addnl. pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR could be converted and also bound to HSA).
- 23Kielbassa, C.; Roza, L.; Epe, B. Wavelength dependence of oxidative DNA damage induced by UV and visible light. Carcinogenesis 1997, 18, 811– 816, DOI: 10.1093/carcin/18.4.811Google Scholar23Wavelength dependence of oxidative DNA damage induced by UV and visible lightKielbassa, Christopher; Roza, Len; Epe, BerndCarcinogenesis (1997), 18 (4), 811-816CODEN: CRNGDP; ISSN:0143-3334. (Oxford University Press)DNA damage induced by UV radiation and visible light (290-500 nm) in AS52 Chinese hamster cells was analyzed by an alk. elution assay with specific repair endonucleases. Cells were exposed to extensively filtered monochrome or broad-band radiation. Between 290 and 315 nm, the ratio of base modifications sensitive to Fpg protein (i.e., 8-hydroxyguanine and formamidopyrimidines) and T4 endonuclease V (i.e., cyclobutane pyrimidine dimers) was const. (∼1:200), indicating that the direct excitation of DNA is responsible for both types of damage in this range of the spectrum. While the yield of pyrimidine dimers per unit dose continued to decrease exponentially beyond 315 nm, the yield of Fpg-sensitive modifications increased to a second max. between 400 and 450 nm. The damage spectrum in this wavelength range consisted of only a few other modifications (strand breaks, abasic sites and pyrimidine modifications sensitive to endonuclease III) and is attributed to endogenous photosensitizers that give rise to oxidative DNA damage via singlet oxygen and/or type I reactions. The generation of Fpg-sensitive modifications by visible light was not linear with dose but followed a satn. curve. It is calcd. that the exposure of the cells to low doses of solar radiation results in the formation of cyclobutane pyrimidine dimers and Fpg-sensitive modifications in a ratio of 10:1.
- 24Thelin, E. P.; Nelson, D. W.; Ghatan, P. H.; Bellander, B. M. Microdialysis Monitoring of CSF Parameters in Severe Traumatic Brain Injury Patients: A Novel Approach. Front. Neurol. 2014, 5, 159, DOI: 10.3389/fneur.2014.00159Google Scholar24Microdialysis Monitoring of CSF Parameters in Severe Traumatic Brain Injury Patients: A Novel ApproachThelin Eric P; Bellander Bo-Michael; Nelson David W; Ghatan Per HamidFrontiers in neurology (2014), 5 (), 159 ISSN:1664-2295.BACKGROUND: Neuro-intensive care following traumatic brain injury (TBI) is focused on preventing secondary insults that may lead to irreversible brain damage. Microdialysis (MD) is used to detect deranged cerebral metabolism. The clinical usefulness of the MD is dependent on the regional localization of the MD catheter. The aim of this study was to analyze a new method of continuous cerebrospinal fluid (CSF) monitoring using the MD technique. The method was validated using conventional laboratory analysis of CSF samples. MD-CSF and regional MD-Brain samples were correlated to patient outcome. MATERIALS AND METHODS: A total of 14 patients suffering from severe TBI were analyzed. They were monitored using (1) a MD catheter (CMA64-iView, n = 7448 MD samples) located in a CSF-pump connected to the ventricular drain and (2) an intraparenchymal MD catheter (CMA70, n = 8358 MD samples). CSF-lactate and CSF-glucose levels were monitored and were compared to MD-CSF samples. MD-CSF and MD-Brain parameters were correlated to favorable (Glasgow Outcome Score extended, GOSe 6-8) and unfavorable (GOSe 1-5) outcome. RESULTS: Levels of glucose and lactate acquired with the CSF-MD technique could be correlated to conventional levels. The median MD recovery using the CMA64 catheter in CSF was 0.98 and 0.97 for glucose and lactate, respectively. Median MD-CSF (CMA 64) lactate (p = 0.0057) and pyruvate (p = 0.0011) levels were significantly lower in the favorable outcome group compared to the unfavorable group. No significant difference in outcome was found using the lactate:pyruvate ratio (LPR), or any of the regional MD-Brain monitoring in our analyzed cohort. CONCLUSION: This new technique of global MD-CSF monitoring correlates with conventional CSF levels of glucose and lactate, and the MD recovery is higher than previously described. Increase in lactate and pyruvate, without any effect on the LPR, correlates to unfavorable outcome, perhaps related to the presence of erythrocytes in the CSF.
- 25Kilkenny, C.; Browne, W. J.; Cuthill, I. C.; Emerson, M.; Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010, 8, e1000412 DOI: 10.1371/journal.pbio.1000412Google ScholarThere is no corresponding record for this reference.
- 26Meredith, A. L.; Thorneloe, K. S.; Werner, M. E.; Nelson, M. T.; Aldrich, R. W. Overactive bladder and incontinence in the absence of the BK large conductance Ca2+-activated K+ channel. J. Biol. Chem. 2004, 279, 36746– 36752, DOI: 10.1074/jbc.M405621200Google Scholar26Overactive Bladder and Incontinence in the Absence of the BK Large Conductance Ca2+-activated K+ ChannelMeredith, Andrea L.; Thorneloe, Kevin S.; Werner, Matthias E.; Nelson, Mark T.; Aldrich, Richard W.Journal of Biological Chemistry (2004), 279 (35), 36746-36752CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)BK large conductance voltage- and calcium-activated potassium channels respond to elevations in intracellular calcium and membrane potential depolarization, braking excitability of smooth muscle. BK channels are thought to have a particularly prominent role in urinary bladder smooth muscle function and therefore are candidate targets for overactive bladder therapy. To address the role of the BK channel in urinary bladder function, the gene mSlo1 for the pore-forming subunit of the BK channel was deleted. Slo-/- mice were viable but exhibited moderate ataxia. Urinary bladder smooth muscle cells of Slo-/- mice lacked calcium- and voltage-activated BK currents, whereas local calcium transients ("calcium sparks") and voltage-dependent potassium currents were unaffected. In the absence of BK channels, urinary bladder spontaneous and nerve-evoked contractions were greatly enhanced. Consistent with increased urinary bladder contractility caused by the absence of BK currents, Slo-/- mice demonstrate a marked elevation in urination frequency. These results reveal a central role for BK channels in urinary bladder function and indicate that BK channel dysfunction leads to overactive bladder and urinary incontinence.
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Abstract
Figure 1
Figure 1. Heme degradation products (HDPs), obtained upon nonenzymatic degradation of heme, biliverdin, or bilirubin, may occur in the Z- and E-configurations. Naturally occurring HDPs with Z-configuration (white background) cause vasoconstriction of cerebral arteries, whereas the effect of their isomers with E-configuration (red background) is to be investigated. BOX: bilirubin oxidation end product and PDP: propentdyopent.
Figure 2
Figure 2. Relative peak areas of the Z- and E-isomers of BOXes (A) and PDPs (B) in relation to the transmission cut-off wavelengths after irradiation of Z-isomers.
Figure 3
Figure 3. Analysis of BOX isomers using liquid chromatography coupled to mass spectrometry. (A) Preparative HPLC separation of E-BOX A, E-BOX B, Z-BOX A, and Z-BOX B after photoconversion of Z-BOXes; the UV absorption at 280 nm is shown. (B, C) Ultra performance liquid chromatography-tandem mass spectrometer (UHPLC-MS) profiles of E-BOX A and E-BOX B over after preparative HPLC fractionation (t = 0 day) and after 10 days at room temperature in the dark; the ion trace of BOXes, m/z = 179.0815 [M + H]+ ± 5 ppm, is shown.
Figure 4
Figure 4. Structure of E-BOX A, the photoconversion product of the naturally occurring Z-BOX A. (A) Ball-and-stick model of E-BOX A, calculated from the structural parameters obtained from X-ray diffraction. (B) Illustration of E-BOX A (left) and Z-BOX A (right) in ball-and-stick models. The carbamide group (blue plane) is rotated by ∼33° toward the main plane of the molecule (red plane) only in E-BOX A. The structure of Z-BOX A was generated from published crystallographic data. (19)
Figure 5
Figure 5. Preparative HPLC/UV of the irradiated PDP raw mixture in acidified solvents (A) and stability of E-PDP A2 in acidified solvents directly after separation at room temperature accessed via UHPLC-MS (B). After 30 min, only traces of E-PDP A2 remained in the eluate. The ion trace of PDPs, m/z = 341.11079 [M + Na]+ ± 5 ppm, is shown.
Figure 6
Figure 6. Impact of E-BOX A on cell morphology in vitro and reisomerization in protein-containing media. (A) Morphological effects of E-BOX A and Z-BOX A treatments on HepG2 cells compared to control conditions. Scale bar: 25 μm. (B) Residual fraction of E-BOX A in relation to the initial amount (t = 0 h; 100%) after incubation in different biochemical milieus at 37 °C in the dark after 6, 24, and 48 h. While E-BOX A is stable in water and protein-free, physiologic salt solution for 48 h, the addition of 1% human serum albumin (HSA; a representative for serum proteins present in CSF of SAH patients) causes a time-dependent decrease of the E-BOX A content in favor of Z-BOX A. In CSF of SAH patients and in serum samples of healthy humans, E-BOX A reisomerizes faster to its Z-isomer. Data are presented as mean ± standard deviation (SD).
Figure 7
Figure 7. Vasoactive effect of the E-BOX A isomer on intracortical arterioles in vitro and on pial arterioles in vivo. (A) Time course of the normalized diameter of preconstricted, intraparenchymal arterioles after superfusion with Z-BOX A or E-BOX A (5 μmol/L, N = 6) in Slo1-WT mice. (B) Percentage arteriolar diameter changes after 90 min Z-BOX A or E-BOX A superfusion in slices of Slo1-WT mice. In line diagrams, the bold lines represent the mean ± SD (gray and pink regions). Thin lines correspond to the diameter time course of single experiments. Dot plots are shown as mean ± SD. Labels above the plots represent comparisons between the BOX A-induced diameter change and the control group. (C) Time course of the normalized diameter of pial arterioles compared between Z-BOX A and E-BOX A (100 μmol/L, N = 5). Bold lines represent the mean value ± SD (gray and pink regions). (D) Summary of percentage diameter changes after Z-BOX A or E-BOX A application. Dot plots are presented as mean ± SD. Data points correspond to the diameter values of single experiments. Statistical comparison between the E-BOX A time slots (120 min versus 180 min) refers to paired samples. ***P < 0.001; **P < 0.01; and *P < 0.05. ns = nonsignificant.
References
This article references 26 other publications.
- 1Steiner, T.; Juvela, S.; Unterberg, A.; Jung, C.; Forsting, M.; Rinkel, G. European Stroke Organization guidelines for the management of intracranial aneurysms and subarachnoid haemorrhage. Cerebrovasc. Dis. 2013, 35, 93– 112, DOI: 10.1159/0003460871European Stroke Organization guidelines for the management of intracranial aneurysms and subarachnoid haemorrhageSteiner Thorsten; Juvela Seppo; Unterberg Andreas; Jung Carla; Forsting Michael; Rinkel GabrielCerebrovascular diseases (Basel, Switzerland) (2013), 35 (2), 93-112 ISSN:.BACKGROUND: Intracranial aneurysm with and without subarachnoid haemorrhage (SAH) is a relevant health problem: The overall incidence is about 9 per 100,000 with a wide range, in some countries up to 20 per 100,000. Mortality rate with conservative treatment within the first months is 50-60%. About one third of patients left with an untreated aneurysm will die from recurrent bleeding within 6 months after recovering from the first bleeding. The prognosis is further influenced by vasospasm, hydrocephalus, delayed ischaemic deficit and other complications. The aim of these guidelines is to provide comprehensive recommendations on the management of SAH with and without aneurysm as well as on unruptured intracranial aneurysm. METHODS: We performed an extensive literature search from 1960 to 2011 using Medline and Embase. Members of the writing group met in person and by teleconferences to discuss recommendations. Search results were graded according to the criteria of the European Federation of Neurological Societies. Members of the Guidelines Committee of the European Stroke Organization reviewed the guidelines. RESULTS: These guidelines provide evidence-based information on epidemiology, risk factors and prognosis of SAH and recommendations on diagnostic and therapeutic methods of both ruptured and unruptured intracranial aneurysms. Several risk factors of aneurysm growth and rupture have been identified. We provide recommendations on diagnostic work up, monitoring and general management (blood pressure, blood glucose, temperature, thromboprophylaxis, antiepileptic treatment, use of steroids). Specific therapeutic interventions consider timing of procedures, clipping and coiling. Complications such as hydrocephalus, vasospasm and delayed ischaemic deficit were covered. We also thought to add recommendations on SAH without aneurysm and on unruptured aneurysms. CONCLUSION: Ruptured intracranial aneurysm with a high rate of subsequent complications is a serious disease needing prompt treatment in centres having high quality of experience of treatment for these patients. These guidelines provide practical, evidence-based advice for the management of patients with intracranial aneurysm with or without rupture. Applying these measures can improve the prognosis of SAH.
- 2Corsten, L.; Raja, A.; Guppy, K.; Roitberg, B.; Misra, M.; Alp, M. S.; Charbel, F.; Debrun, G.; Ausman, J. Contemporary management of subarachnoid hemorrhage and vasospasm: the UIC experience. Surg. Neurol. 2001, 56, 140– 150, DOI: 10.1016/S0090-3019(01)00513-42Contemporary management of subarachnoid hemorrhage and vasospasm: the UIC experienceCorsten L; Raja A; Guppy K; Roitberg B; Misra M; Alp M S; Charbel F; Debrun G; Ausman JSurgical neurology (2001), 56 (3), 140-8; discussion 148-50 ISSN:0090-3019.BACKGROUND: Cerebral vasospasm is a well-known and serious complication of aneurysmal subarachnoid hemorrhage. The means of monitoring and treatment of vasospasm have been widely studied. Each neurosurgical center develops a protocol based on their experience, availability of equipment and personnel, and cost, so as to keep morbidity and mortality rates as low as possible for their patients with vasospasm. METHODS: At the University of Illinois at Chicago, we have developed algorithms for the diagnosis and management of cerebral vasospasm based on the experience of the senior authors over the past 25 years. This paper describes in detail our approach to diagnosis and treatment of aneurysmal subarachnoid hemorrhage and vasospasm. Our discussion is highlighted with data from a retrospective analysis of 324 aneurysm patients. RESULTS: Over 3 years, 324 aneurysms were treated; 185 (57%) were clipped, 139 (43%) were coiled. The rate of vasospasm for the 324 patients was 27%. The rate of hydrocephalus was 32% for those patients who underwent clipping, and 29% for those coiled. The immediate outcomes for those who underwent clipping was excellent in 35%, good in 38%, poor in 15.5%, vegetative in 3%, and death in 8% of the patients. For those who underwent coiling the immediate outcome was excellent in 64%, good in 14.5%, vegetative in 2.5%, and death in 14.5% of the patients. These statistics include all Hunt and Hess grades. For those patients who underwent clipping, 51% were intact at 6 months follow-up, 15% had a permanent deficit, 10% had a focal cranial nerve deficit, and 2% had died from complications not directly related to the procedure. For those patients who had undergone coiling, 75% were intact at 6 months follow-up, 12.5% had a permanent deficit, and 12.5% had a cranial nerve deficit, with no deaths. CONCLUSIONS: The morbidity and mortality of cerebral vasospasm is significant. A good outcome after aneurysmal subarachnoid hemorrhage is dependent upon careful patient management in the preoperative, perioperative, and postoperative periods. The timely work-up and aggressive treatment of neurological deterioration, whether or not it is because of vasospasm, is paramount.
- 3Uhl, E.; Lehmberg, J.; Steiger, H. J.; Messmer, K. Intraoperative detection of early microvasospasm in patients with subarachnoid hemorrhage by using orthogonal polarization spectral imaging. Neurosurgery 2003, 52, 1307– 1317, DOI: 10.1227/01.NEU.0000065154.04824.9E3Intraoperative detection of early microvasospasm in patients with subarachnoid hemorrhage by using orthogonal polarization spectral imagingUhl Eberhard; Lehmberg Jens; Steiger Hans-Jakob; Messmer KonradNeurosurgery (2003), 52 (6), 1307-15; disacussion 1315-7 ISSN:0148-396X.OBJECTIVE: Changes of major cerebral vessels in patients with subarachnoid hemorrhage (SAH) are well known from routine cerebral angiography. Data on changes in the microcirculation do not exist. This study sought to provide a qualitative and quantitative analysis of the cortical microcirculation after SAH. METHODS: By means of orthogonal polarization spectral imaging, a qualitative and quantitative analysis of cortical microcirculation was performed during aneurysm surgery in 3 patients with an incidental intracerebral aneurysm and 10 patients with SAH. Vessel diameters, red blood cell velocity, and functional capillary density were analyzed before and after the aneurysm was clipped. RESULTS: Initial capillary density in patients with an incidental aneurysm was 91.5 +/- 36.5 cm(-1) (mean +/- standard deviation) compared with 30.5 +/- 13.8 in patients with SAH (P < 0.05). In patients with SAH, capillary density increased significantly to 53.9 +/- 29.1 cm(-1) (P < 0.05) during the operation, as did the frequency of venules with a red blood cell velocity greater than 2 mm/s (P < 0.05). No significant change of arteriolar or venular diameters was observed. However, in patients with SAH, mono- and multisegmental microvasospasms in arterioles were observed, with a reduction of vessel diameters up to 75.1%. CONCLUSION: Orthogonal polarization spectral imaging is a suitable method to study cerebral microcirculation during surgery. In patients with SAH, capillary density is significantly decreased and small arteries and arterioles of the cortical surface exhibit vasospasm that cannot be detected by angiography or transcranial Doppler sonography. These changes may contribute to the initial clinical symptoms and may have an influence on the clinical postoperative course.
- 4Giller, C. A.; Giller, A. M.; Landreneau, F. Detection of emboli after surgery for intracerebral aneurysms. Neurosurgery 1998, 42, 490– 494, DOI: 10.1097/00006123-199803000-000104Detection of emboli after surgery for intracerebral aneurysmsGiller C A; Giller A M; Landreneau FNeurosurgery (1998), 42 (3), 490-3; discussion 493-4 ISSN:0148-396X.BACKGROUND: Neurological change after surgery for cerebral aneurysm caused by embolic events is commonly suspected, but direct detection of emboli has not been possible in the past. Transcranial Doppler ultrasound (TCD) is able to detect emboli, and large numbers of emboli detected in TCD studies have been associated with radiological changes and clinical deterioration. METHODS: During a 2-year period, 11 patients were observed to have emboli during routine TCD studies after aneurysm surgery. The computed tomographic (CT) scans of these patients were reviewed for low-density areas, suggesting ischemia. All patients studied during a 1-year interval (July 1995-July 1996) served as a control group and were reviewed for similar CT findings, and the two groups were compared using Fisher's exact test. RESULTS: Nine of the 11 patients (82%) observed to have emboli developed low-density areas on their CT scans, whereas 30 of the 123 (24%) patients without emboli developed low-density areas on their CT scans. The difference was significant (P < 0.001, Fisher's exact test). Credible sources for emboli were readily identified in each of the 11 patients. CONCLUSION: TCD allows detection of emboli after aneurysm surgery, and this detection is strongly associated with CT evidence of ischemia. Although detection of emboli was relatively rare in this study, rates of emboli occurrence may increase if systematic monitoring is used.
- 5Korbakis, G.; Prabhakaran, S.; John, S.; Garg, R.; Conners, J. J.; Bleck, T. P.; Lee, V. H. MRI Detection of Cerebral Infarction in Subarachnoid Hemorrhage. Neurocrit. Care 2016, 24, 428– 435, DOI: 10.1007/s12028-015-0212-z5MRI Detection of Cerebral Infarction in Subarachnoid HemorrhageKorbakis Georgia; Prabhakaran Shyam; John Sayona; Garg Rajeev; Bleck Thomas P; Conners James J; Lee Vivien HNeurocritical care (2016), 24 (3), 428-35 ISSN:.OBJECTIVE: To investigate magnetic resonance imaging (MRI) detection of cerebral infarction (CI) in patients presenting with subarachnoid hemorrhage (SAH). BACKGROUND: CI is a well-known complication of SAH that is typically detected on computed tomography (CT). MRI has improved sensitivity for acute CI over CT, particularly with multiple, small, or asymptomatic lesions. METHODS: With IRB approval, 400 consecutive SAH patients admitted to our institution from August 2006 to March 2011 were retrospectively reviewed. Traumatic SAH and secondary SAH were excluded. Data were collected on demographics, cause of SAH, Hunt Hess and World Federation of Neurosurgical Societies grades, and neuroimaging results. MRIs were categorized by CI pattern as single cortical (SC), single deep (SD), multiple cortical (MC), multiple deep (MD), and multiple cortical and deep (MCD). RESULTS: Among 123 (30.8 %) SAH patients who underwent MRIs during their hospitalization, 64 (52 %) demonstrated acute CI. The mean time from hospital admission to MRI was 5.7 days (range 0-29 days). Among the 64 patients with MRI infarcts, MRI CI pattern was as follows: MC in 20 (31 %), MCD in 18 (28 %), SC in 16 (25 %), SD in 3 (5 %), MD in 2 (3 %), and 5 (8 %) did not have images available for review. Most infarcts detected on MRI (39/64 or 61 %) were not visible on CT. CONCLUSIONS: The use of MRI increases the detection of CI in SAH. Unlike CT studies, MRI-detected CI in SAH tends to involve multiple vascular territories. Studies that rely on CT may underestimate the burden of CI after SAH.
- 6Rodriguez-Régent, C.; Hafsa, M.; Turc, G.; Ben Hassen, W.; Edjlali, M.; Sermet, A.; Laquay, N.; Trystram, D.; Al-Shareef, F.; Meder, J. F. Early quantitative CT perfusion parameters variation for prediction of delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage. Eur. Radiol. 2016, 26, 2956– 2963, DOI: 10.1007/s00330-015-4135-z6Early quantitative CT perfusion parameters variation for prediction of delayed cerebral ischemia following aneurysmal subarachnoid hemorrhageRodriguez-Regent Christine; Hafsa Monia; Ben Hassen Wagih; Edjlali Myriam; Trystram Denis; Al-Shareef Fawaz; Meder Jean-Francois; Oppenheim Catherine; Naggara Olivier; Turc Guillaume; Sermet Alain; Laquay Nathalie; Devaux BertrandEuropean radiology (2016), 26 (9), 2956-63 ISSN:.OBJECTIVES: To prospectively evaluate the predictive value of cerebral perfusion-computerized tomography (CTP) parameters variation between day0 and day4 after aneurysmal subarachnoid haemorrhage (aSAH). METHODS: Mean transit time (MTT) and cerebral blood flow (CBF) values were compared between patients with delayed cerebral ischemia (DCI+ group) and patients without DCI (DCI- group) for previously published optimal cutoff values and for variations of MTT (ΔMTT) and of CBF (ΔCBF) values between day0 and day4. DCI+ was defined as a cerebral infarction on 3-months follow-up MRI. RESULTS: Among 47 included patients, 10 suffered DCI+. Published optimal cutoff values did not predict DCI, either at day0 or at day4. Conversely, ΔMTT and ΔCBF significantly differed between the DCI+ and DCI- groups, with optimal ΔMTT and ΔCBF values of 0.91 seconds (83.9 % sensitivity, 79.5 % specificity, AUC 0.84) and -7.6 mL/100 g/min (100 % sensitivity, 71.4 % specificity, AUC 0.86), respectively. In multivariate analysis, ΔCBF (OR = 1.91, IC95% 1.13-3.23 per each 20 % decrease of ΔCBF) and ΔMTT values (OR = 14.70, IC95% 4.85-44.52 per each 20 % increase of ΔMTT) were independent predictors of DCI. CONCLUSIONS: Assessment of MTT and CBF value variations between day0 and day4 may serve as an early imaging surrogate for prediction of DCI in aSAH. KEY POINTS: • CT perfusion values are an imaging surrogate for prediction of DCI. • Early variations (day0-day4) after aneurysmal subarachnoid haemorrhage predicted DCI. • A CBF decrease of 7.6 mL/min/100 g predicted DCI with 100 % sensitivity. • An MTT increase of 0.91 seconds predicted DCI with 83.9 % sensitivity. • DCI risk multiplied by 2 per 20 % ΔCBF decrease and by 15 per 20 % ΔMTT increase.
- 7Balbi, M.; Koide, M.; Wellman, G. C.; Plesnila, N. Inversion of neurovascular coupling after subarachnoid hemorrhage in vivo. J. Cereb. Blood Flow Metab. 2017, 37, 3625– 3634, DOI: 10.1177/0271678X166865957Inversion of neurovascular coupling after subarachnoid hemorrhage in vivoBalbi Matilde; Plesnila Nikolaus; Balbi Matilde; Plesnila Nikolaus; Koide Masayo; Wellman George C; Plesnila NikolausJournal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism (2017), 37 (11), 3625-3634 ISSN:.Subarachnoid hemorrhage (SAH) induces acute changes in the cerebral microcirculation. Recent findings ex vivo suggest neurovascular coupling (NVC), the process that increases cerebral blood flow upon neuronal activity, is also impaired after SAH. The aim of the current study was to investigate whether this occurs also in vivo. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. Twenty-four hours later NVC was tested by forepaw stimulation and CO2 reactivity by inhalation of 10% CO2. Vessel diameter was assessed in vivo by two-photon microscopy. NVC was also investigated ex vivo using brain slices. Cerebral arterioles of sham-operated mice dilated to 130% of baseline upon CO2 inhalation or forepaw stimulation and cerebral blood flow (CBF) increased. Following SAH, however, CO2 reactivity was completely lost and the majority of cerebral arterioles showed paradoxical constriction in vivo and ex vivo resulting in a reduced CBF response. As previous results showed intact NVC 3 h after SAH, the current findings indicate that impairment of NVC after cerebral hemorrhage occurs secondarily and is progressive. Since neuronal activity-induced vasoconstriction (inverse NVC) is likely to further aggravate SAH-induced cerebral ischemia and subsequent brain damage, inverse NVC may represent a novel therapeutic target after SAH.
- 8Dreier, J. P.; Drenckhahn, C.; Woitzik, J.; Major, S.; Offenhauser, N.; Weber-Carstens, S.; Wolf, S.; Strong, A. J.; Vajkoczy, P.; Hartings, J. A. Spreading ischemia after aneurysmal subarachnoid hemorrhage. Acta Neurochir., Suppl. 2013, 115, 125– 1298Spreading ischemia after aneurysmal subarachnoid hemorrhageDreier Jens P; Drenckhahn Christoph; Woitzik Johannes; Major Sebastian; Offenhauser Nikolas; Weber-Carstens Steffen; Wolf Stefan; Strong Anthony J; Vajkoczy Peter; Hartings Jed AActa neurochirurgica. Supplement (2013), 115 (), 125-9 ISSN:0065-1419.Spreading depolarization (SD) is a wave of mass neuronal and glial depolarization associated with net influx of cations and water. Prolonged SDs facilitate neuronal death. SD induces tone alterations in cerebral resistance arterioles, leading to either transient hyperperfusion (physiological neurovascular coupling) in healthy tissue or hypoperfusion (inverse neurovascular coupling = spreading ischemia) in tissue at risk for progressive damage. Spreading ischemia has been shown experimentally in an animal model replicating the conditions present following aneurysmal subarachnoid hemorrhage (aSAH), in animal models of the ischemic core and penumbra following middle cerebral artery occlusion, and in patients with aSAH. In animals, spreading ischemia produced widespread cortical necrosis. In patients, spreading ischemia occurred in temporal correlation with ischemic lesion development early and late after aSAH. We briefly review important features of SD and spreading ischemia following aSAH.
- 9Vivancos, J.; Gilo, F.; Frutos, R.; Maestre, J.; Garcia-Pastor, A.; Quintana, F.; Roda, J. M.; Ximenez-Carrillo, A. Clinical management guidelines for subarachnoid haemorrhage. Diagnosis and treatment. Neurología 2014, 29, 353– 370, DOI: 10.1016/j.nrl.2012.07.0099Clinical management guidelines for subarachnoid haemorrhage. Diagnosis and treatmentVivancos J; Gilo F; Frutos R; Maestre J; Garcia-Pastor A; Quintana F; Roda J M; Ximenez-Carrillo A; Diez Tejedor E; Fuentes B; Alonso de Lecinana M; Alvarez-Sabin J; Arenillas J; Calleja S; Casado I; Castellanos M; Castillo J; Davalos A; Diaz-Otero F; Egido J A; Fernandez J C; Freijo M; Gallego J; Gil-Nunez A; Irimia P; Lago A; Masjuan J; Marti-Fabregas J; Martinez-Sanchez P; Martinez-Vila E; Molina C; Morales A; Nombela F; Purroy F; Ribo M; Rodriguez-Yanez M; Roquer J; Rubio F; Segura T; Serena J; Simal P; Tejada JNeurologia (Barcelona, Spain) (2014), 29 (6), 353-70 ISSN:.OBJECTIVE: To update the Spanish Society of Neurology's guidelines for subarachnoid haemorrhage diagnosis and treatment. MATERIAL AND METHODS: A review and analysis of the existing literature. Recommendations are given based on the level of evidence for each study reviewed. RESULTS: The most common cause of spontaneous subarachnoid haemorrhage (SAH) is cerebral aneurysm rupture. Its estimated incidence in Spain is 9/100 000 inhabitants/year with a relative frequency of approximately 5% of all strokes. Hypertension and smoking are the main risk factors. Stroke patients require treatment in a specialised centre. Admission to a stroke unit should be considered for SAH patients whose initial clinical condition is good (Grades I or II on the Hunt and Hess scale). We recommend early exclusion of aneurysms from the circulation. The diagnostic study of choice for SAH is brain CT (computed tomography) without contrast. If the test is negative and SAH is still suspected, a lumbar puncture should then be performed. The diagnostic tests recommended in order to determine the source of the haemorrhage are MRI (magnetic resonance imaging) and angiography. Doppler ultrasonography studies are very useful for diagnosing and monitoring vasospasm. Nimodipine is recommended for preventing delayed cerebral ischaemia. Blood pressure treatment and neurovascular intervention may be considered in treating refractory vasospasm. CONCLUSIONS: SAH is a severe and complex disease which must be managed in specialised centres by professionals with ample experience in relevant diagnostic and therapeutic processes.
- 10Kranc, K. R.; Pyne, G. J.; Tao, L.; Claridge, T. D.; Harris, D. A.; Cadoux-Hudson, T. A.; Turnbull, J. J.; Schofield, C. J.; Clark, J. F. Oxidative degradation of bilirubin produces vasoactive compounds. Eur. J. Biochem. 2000, 267, 7094– 7101, DOI: 10.1046/j.1432-1327.2000.01812.x10Oxidative degradation of bilirubin produces vasoactive compoundsKranc, Kamil R.; Pyne, Gail J.; Tao, Limei; Claridge, Tim D. W.; Harris, David A.; Cadoux-Hudson, Thomas A. D.; Turnbull, Jonathan J.; Schofield, Christopher J.; Clark, Joseph F.European Journal of Biochemistry (2000), 267 (24), 7094-7101CODEN: EJBCAI; ISSN:0014-2956. (Blackwell Science Ltd.)Subarachnoid hemorrhage is often followed by hemolysis and concomitant oxidative stress, and is frequently complicated by pathol. vasoconstriction or cerebral vasospasm. It is known that upregulation of heme oxygenase (HO-1) is induced by oxidative stress and results in release of biliverdin and bilirubin (BR), which are scavengers of reactive oxygen species (ROS). Here the authors report biomimetic studies aimed at modeling pathol. conditions leading to oxidative degrdn. of BR. Oxidative degrdn. products of BR, formed by reaction with hydrogen peroxide (an ROS model system), demonstrated biol. activity by stimulating oxygen consumption and force development in vascular smooth muscle from porcine carotid artery. Analogous biol. activity was obsd. with vasoactive cerebrospinal fluid from subarachnoid hemorrhage patients. Three degrdn. products of BR were isolated: two were assigned as isomeric monopyrrole (C9H11N2O2) derivs., 4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide and 3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide and the third was 4-methyl-3-vinylmaleimide (MVM), a previously isolated photodegrdn. product of biliverdin. Possible mechanisms of oxidative degrdn. of BR are discussed. Tentative assignment of these structures in the cerebrospinal fluid (CSF) of cerebral vasospasm patients has been made. It is proposed that one or more of the degrdn. products of biliverdin or bilirubin are involved in complications such as vasospasm and or pathol. vasoconstriction assocd. with hemorrhage.
- 11Ritter, M.; Neupane, S.; Seidel, R. A.; Steinbeck, C.; Pohnert, G. In vivo and in vitro identification of Z-BOX C—a new bilirubin oxidation end product. Org. Biomol. Chem. 2018, 16, 3553– 3555, DOI: 10.1039/C8OB00164B11In vivo and in vitro identification of Z-BOX C - a new bilirubin oxidation end productRitter, Marcel; Neupane, Sandesh; Seidel, Raphael A.; Steinbeck, Christoph; Pohnert, GeorgOrganic & Biomolecular Chemistry (2018), 16 (19), 3553-3555CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)A new bilirubin oxidn. 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 pathol. human bile samples using liq. chromatog./mass spectrometry and has potential relevance for liver dysfunction and cerebral vasospasms.
- 12Ritter, M.; Seidel, R. A.; Bellstedt, P.; Schneider, B.; Bauer, M.; Gorls, H.; Pohnert, G. Isolation and Identification of Intermediates of the Oxidative Bilirubin Degradation. Org. Lett. 2016, 18, 4432– 4435, DOI: 10.1021/acs.orglett.6b0228712Isolation and Identification of Intermediates of the Oxidative Bilirubin DegradationRitter, Marcel; Seidel, Raphael A.; Bellstedt, Peter; Schneider, Bernd; Bauer, Michael; Goerls, Helmar; Pohnert, GeorgOrganic Letters (2016), 18 (17), 4432-4435CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)Four endogenous products of oxidative bilirubin degrdn. were isolated and fully characterized. The constitutional isomers belong to the propentdyopents (PDPs). Their structures and further oxidative transformations to biol. active bilirubin oxidn. end products (Z-BOXes) are reported. Using liq. chromatog.-mass spectrometry protocols, PDPs were detected in human bile and gallstones. Given the recent interest in BOXes as effectors in cerebral vasospasms and liver dysfunction, co-occurring PDPs represent an addnl. potentially active compd. class to be considered.
- 13Clark, J. F.; Sharp, F. R. Bilirubin oxidation products (BOXes) and their role in cerebral vasospasm after subarachnoid hemorrhage. J. Cereb. Blood Flow Metab. 2006, 26, 1223– 1233, DOI: 10.1038/sj.jcbfm.960028013Bilirubin oxidation products (BOXes) and their role in cerebral vasospasm after subarachnoid hemorrhageClark, Joseph F.; Sharp, Frank R.Journal of Cerebral Blood Flow & Metabolism (2006), 26 (10), 1223-1233CODEN: JCBMDN; ISSN:0271-678X. (Nature Publishing Group)A review. Many factors have been postulated to cause delayed subarachnoid hemorrhage (SAH)-induced vasospasm, including Hb, nitric oxide, endothelin, and free radicals. We propose that free radicals (because of the high levels that are produced in the blood clots surrounding blood vessels after SAH) act on bilirubin, biliverdin, and possibly heme to produce BOXes (Bilirubin OXidized Products). Bilirubin oxidn. products act on vascular smooth muscle cells to produce chronic vasoconstriction and vasospasm combined with a vasculopathy because of smooth muscle cell injury. This review summarizes recent evidence that BOXes play a role in SAH-induced vasospasm. The data supporting a role for BOXes includes (1) identification of mols. in cerebrospinal fluid (CSF) of patients with vasospasm after SAH that have structures consistent with BOXes; (2) BOXes are vasoactive in vitro and mimic the biochem. actions of CSF of patients with vasospasm; (3) BOXes are vasoactive in vivo, constricting rat cerebral vessels; and (4) there is a correlation between clin. occurrence of vasospasm and BOXes concn. in our preliminary study of patients with SAH. Since oxidn. of bilirubin, biliverdin, and perhaps heme is proposed to produce BOXes that contribute to vasospasm, either blocking bilirubin formation, inactivating bilirubin or BOXes, or removing all of the blood clot before vasospasm are potential treatment targets.
- 14Seidel, R. A.; Claudel, T.; Schleser, F. A.; Ojha, N. K.; Westerhausen, M.; Nietzsche, S.; Sponholz, C.; Cuperus, F.; Coldewey, S. M.; Heinemann, S. H. Impact of higher-order heme degradation products on hepatic function and hemodynamics. J. Hepatol. 2017, 67, 272– 281, DOI: 10.1016/j.jhep.2017.03.03714Impact of higher-order heme degradation products on hepatic function and hemodynamicsSeidel Raphael A; Claudel Thierry; Trauner Michael; Schleser Franziska A; Sponholz Christoph; Coldewey Sina M; Ojha Navin K; Heinemann Stefan H; Westerhausen Matthias; Nietzsche Sandor; Cuperus Frans; Pohnert Georg; Bauer MichaelJournal of hepatology (2017), 67 (2), 272-281 ISSN:.BACKGROUND & AIMS: Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions. METHODS: Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia. RESULTS: Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbβ. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton. CONCLUSIONS: Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia. LAY SUMMARY: Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats.
- 15Joerk, A.; Ritter, M.; Langguth, N.; Seidel, R. A.; Freitag, D.; Herrmann, K. H.; Schaefgen, A.; Ritter, M.; Gunther, M.; Sommer, C. Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage. Circ. Res. 2019, 124, e101– e114, DOI: 10.1161/CIRCRESAHA.118.31416015Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid HemorrhageJoerk, Alexander; Ritter, Marcel; Langguth, Niklas; Seidel, Raphael Andreas; Freitag, Diana; Herrmann, Karl-Heinz; Schaefgen, Anna; Ritter, Marvin; Guenther, Milena; Sommer, Charline; Braemer, Dirk; Walter, Jan; Ewald, Christian; Kalff, Rolf; Reichenbach, Juergen Rainer; Westerhausen, Matthias; Pohnert, Georg; Witte, Otto Wilhelm; Holthoff, KnutCirculation Research (2019), 124 (12), e101-e114CODEN: CIRUAL; ISSN:0009-7330. (Lippincott Williams & Wilkins)Delayed ischemic neurol. deficit is the most common cause of neurol. impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidn. end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degrdn., known as PDPs (propentdyopents), have been considered as potential addnl. effectors in the development of arterial vasoconstriction. To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. Via liq. chromatog./mass spectrometry, we measured increased PDP and BOX concns. in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diam. in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concn. range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diam. changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we obsd. a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. For the first time, we have quantified significantly higher concns. of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.
- 16Joerk, A.; Seidel, R. A.; Walter, S. G.; Wiegand, A.; Kahnes, M.; Klopfleisch, M.; Kirmse, K.; Pohnert, G.; Westerhausen, M.; Witte, O. W. Impact of heme and heme degradation products on vascular diameter in mouse visual cortex. J. Am. Heart Assoc. 2014, 3, 1– 13, DOI: 10.1161/JAHA.114.001220There is no corresponding record for this reference.
- 17Jasprova, J.; Dal Ben, M.; Vianello, E.; Goncharova, I.; Urbanova, M.; Vyroubalova, K.; Gazzin, S.; Tiribelli, C.; Sticha, M.; Cerna, M. The Biological Effects of Bilirubin Photoisomers. PLoS One 2016, 11, e0148126 DOI: 10.1371/journal.pone.014812617The biological effects of bilirubin photoisomersJasprova, Jana; Dal Ben, Matteo; Vianello, Eleonora; Goncharova, Iryna; Urbanova, Marie; Vyroubalova, Karolina; Gazzin, Silvia; Tiribelli, Claudio; Sticha, Martin; Cerna, Marcela; Vitek, LiborPLoS One (2016), 11 (2), e0148126/1-e0148126/16CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Although phototherapy was introduced as early as 1950's, the potential biol. effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biol. effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepd. by photo-irradn. of unconjugated bilirubin. The individual photoproducts were chromatog. sepd. (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissocn. of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and CD. The biol. effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding const. to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metab. or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.
- 18Watchko, J. F.; Tiribelli, C. Bilirubin-induced neurologic damage--mechanisms and management approaches. N. Engl. J. Med. 2013, 369, 2021– 2030, DOI: 10.1056/NEJMra130812418Bilirubin-induced neurologic damage - mechanisms and management approachesWatchko, Jon F.; Tiribelli, ClaudioNew England Journal of Medicine (2013), 369 (21), 2021-2030CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)A review. The complex cascade of mol. and cellular events leading to bilirubin-induced neurotoxicity remains incompletely delineated. This review describes bilirubin-induced brain damage and recent insights into its pathogenesis and prevention.
- 19Klopfleisch, M.; Seidel, R. A.; Gorls, H.; Richter, H.; Beckert, R.; Imhof, W.; Reiher, M.; Pohnert, G.; Westerhausen, M. Total synthesis and detection of the bilirubin oxidation product (Z)-2-(3-ethenyl-4-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide (Z-BOX A). Org. Lett. 2013, 15, 4608– 4611, DOI: 10.1021/ol402221b19Total Synthesis and Detection of the Bilirubin Oxidation Product (Z)-2-(3-Ethenyl-4-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide (Z-BOX A)Klopfleisch, Maurice; Seidel, Raphael A.; Gorls, Helmar; Richter, Hannes; Beckert, Rainer; Imhof, Wolfgang; Reiher, Markus; Pohnert, Georg; Westerhausen, MatthiasOrganic Letters (2013), 15 (17), 4608-4611CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)The selective total synthesis of the pure Z-isomer of BOX A I, a product of oxidative heme degrdn. with significant physiol. impact, was achieved in four to six steps starting from 3-bromo-4-methylfuran-2,5-dione. Z-BOX A forms a strong hydrogen bridge framework in the cryst. state. LC-MS techniques allow identification and characterization of isomeric forms of BOX A.
- 20Seidel, R. A.; Schowtka, B.; Klopfleisch, M.; Kuhl, T.; Weiland, A.; Koch, A.; Gorls, H.; Imhof, D.; Pohnert, G.; Westerhausen, M. Total synthesis and characterization of the bilirubin oxidation product (Z)-2-(4-ethenyl-3-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide (Z-BOX B). Tetrahedron Lett. 2014, 55, 6526– 6529, DOI: 10.1016/j.tetlet.2014.09.10820Total synthesis and characterization of the bilirubin oxidation product (Z)-2-(4-ethenyl-3-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide (Z-BOX B)Seidel, Raphael A.; Schowtka, Bjoern; Klopfleisch, Maurice; Kuehl, Toni; Weiland, Andreas; Koch, Alexander; Goerls, Helmar; Imhof, Diana; Pohnert, Georg; Westerhausen, MatthiasTetrahedron Letters (2014), 55 (48), 6526-6529CODEN: TELEAY; ISSN:0040-4039. (Elsevier Ltd.)Bilirubin oxidn. end products (BOXes) show significant physiol. effects and are connected to severe diseases such as subarachnoid hemorrhage induced cerebral vasospasm. BOX B [2-(4-ethenyl-3-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)ethanamide] was prepd. via a five-step synthesis starting from Me [4-bromo-3-methyl-5-oxofuran-2(5H)-ylidene]ethanoate, which was vinylated and then reacted with NH4OAc, LiOH, (COCl)2, and finally with ammonia yielding Z-BOX B. Derivs. of Z-BOX A [(Z)-2-(3-ethenyl-4-methyl-5-oxo-1,5-dihydro-2H-[15N]pyrrol-2-ylidene)ethanamide] and Z-BOX B with [15N]labeled lactam rings were synthesized accordingly.
- 21Seidel, R. A.; Kahnes, M.; Bauer, M.; Pohnert, G. Simultaneous determination of the bilirubin oxidation end products Z-BOX A and Z-BOX B in human serum using liquid chromatography coupled to tandem mass spectrometry. J. Chromatogr. B 2015, 974, 83– 89, DOI: 10.1016/j.jchromb.2014.10.02721Simultaneous determination of the bilirubin oxidation end products Z-BOX A and Z-BOX B in human serum using liquid chromatography coupled to tandem mass spectrometrySeidel, Raphael A.; Kahnes, Marcel; Bauer, Michael; Pohnert, GeorgJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (2015), 974 (), 83-89CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)Bilirubin oxidn. end products (BOXes) appear upon endogenous heme degrdn. and can be found in the cerebrospinal fluid after hemorrhagic stroke. BOXes are assumed to contribute to delayed cerebral vasospasm and secondary loss of brain tissue. Here, we present a validated LC-ESI-MS/MS method for the sensitive detn. of the regio-isomers Z-BOX A and Z-BOX B in human serum. We found that Z-BOX A and Z-BOX B appear in serum of healthy volunteers. The sample prepn. includes the addn. of 5-bromonicotinamide as internal std. and protein pptn. with acetonitrile. Baseline-sepn. was achieved on a C-18 column with a binary solvent gradient of formic acid in water/acetonitrile at 1 mL/min within a total anal. time of 17 min. Using single reaction monitoring in the pos. ion mode, the linear working ranges were 2.74-163 pg/μL (Z-BOX A) and 2.12-162.4 pg/μL (Z-BOX B) with R2 > 0.995. Intra- and inter-day precisions were <10%. The inherent analyte concns. of Z-BOX A (14.4 ± 5.1 nM) and Z-BOX B (10.9 ± 3.1 nM) in pooled human serum were detd. by std. addn. The photolability of both analytes was demonstrated. This method enables to monitor Z-BOX A and Z-BOX B as a prerequisite to systematically study the biol. significance of higher order metabolites of heme degrdn.
- 22Goncharova, I.; Jasprova, J.; Vitek, L.; Urbanova, M. Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding. Anal. Biochem. 2015, 490, 34– 45, DOI: 10.1016/j.ab.2015.08.00122Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin bindingGoncharova, Iryna; Jasprova, Jana; Vitek, Libor; Urbanova, MarieAnalytical Biochemistry (2015), 490 (), 34-45CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)Bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidn. to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. Here, the authors present a novel anal. concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, CD spectroscopy, and mol. modeling methods was used for recognition of the binding site for BR, its derivs. (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidn. (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of Trp-HSA were used to calc. the binding consts. The results of the CD displacement expts. performed with hemin were interpreted together with the findings of mol. docking performed on the pigment-HSA complexes. The authors estd. that Z,Z-BR and its metabolic products bound to 2 independent binding sites. The findings supported the existence of a reversible antioxidant redox cycle for BR and explained an addnl. pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR could be converted and also bound to HSA).
- 23Kielbassa, C.; Roza, L.; Epe, B. Wavelength dependence of oxidative DNA damage induced by UV and visible light. Carcinogenesis 1997, 18, 811– 816, DOI: 10.1093/carcin/18.4.81123Wavelength dependence of oxidative DNA damage induced by UV and visible lightKielbassa, Christopher; Roza, Len; Epe, BerndCarcinogenesis (1997), 18 (4), 811-816CODEN: CRNGDP; ISSN:0143-3334. (Oxford University Press)DNA damage induced by UV radiation and visible light (290-500 nm) in AS52 Chinese hamster cells was analyzed by an alk. elution assay with specific repair endonucleases. Cells were exposed to extensively filtered monochrome or broad-band radiation. Between 290 and 315 nm, the ratio of base modifications sensitive to Fpg protein (i.e., 8-hydroxyguanine and formamidopyrimidines) and T4 endonuclease V (i.e., cyclobutane pyrimidine dimers) was const. (∼1:200), indicating that the direct excitation of DNA is responsible for both types of damage in this range of the spectrum. While the yield of pyrimidine dimers per unit dose continued to decrease exponentially beyond 315 nm, the yield of Fpg-sensitive modifications increased to a second max. between 400 and 450 nm. The damage spectrum in this wavelength range consisted of only a few other modifications (strand breaks, abasic sites and pyrimidine modifications sensitive to endonuclease III) and is attributed to endogenous photosensitizers that give rise to oxidative DNA damage via singlet oxygen and/or type I reactions. The generation of Fpg-sensitive modifications by visible light was not linear with dose but followed a satn. curve. It is calcd. that the exposure of the cells to low doses of solar radiation results in the formation of cyclobutane pyrimidine dimers and Fpg-sensitive modifications in a ratio of 10:1.
- 24Thelin, E. P.; Nelson, D. W.; Ghatan, P. H.; Bellander, B. M. Microdialysis Monitoring of CSF Parameters in Severe Traumatic Brain Injury Patients: A Novel Approach. Front. Neurol. 2014, 5, 159, DOI: 10.3389/fneur.2014.0015924Microdialysis Monitoring of CSF Parameters in Severe Traumatic Brain Injury Patients: A Novel ApproachThelin Eric P; Bellander Bo-Michael; Nelson David W; Ghatan Per HamidFrontiers in neurology (2014), 5 (), 159 ISSN:1664-2295.BACKGROUND: Neuro-intensive care following traumatic brain injury (TBI) is focused on preventing secondary insults that may lead to irreversible brain damage. Microdialysis (MD) is used to detect deranged cerebral metabolism. The clinical usefulness of the MD is dependent on the regional localization of the MD catheter. The aim of this study was to analyze a new method of continuous cerebrospinal fluid (CSF) monitoring using the MD technique. The method was validated using conventional laboratory analysis of CSF samples. MD-CSF and regional MD-Brain samples were correlated to patient outcome. MATERIALS AND METHODS: A total of 14 patients suffering from severe TBI were analyzed. They were monitored using (1) a MD catheter (CMA64-iView, n = 7448 MD samples) located in a CSF-pump connected to the ventricular drain and (2) an intraparenchymal MD catheter (CMA70, n = 8358 MD samples). CSF-lactate and CSF-glucose levels were monitored and were compared to MD-CSF samples. MD-CSF and MD-Brain parameters were correlated to favorable (Glasgow Outcome Score extended, GOSe 6-8) and unfavorable (GOSe 1-5) outcome. RESULTS: Levels of glucose and lactate acquired with the CSF-MD technique could be correlated to conventional levels. The median MD recovery using the CMA64 catheter in CSF was 0.98 and 0.97 for glucose and lactate, respectively. Median MD-CSF (CMA 64) lactate (p = 0.0057) and pyruvate (p = 0.0011) levels were significantly lower in the favorable outcome group compared to the unfavorable group. No significant difference in outcome was found using the lactate:pyruvate ratio (LPR), or any of the regional MD-Brain monitoring in our analyzed cohort. CONCLUSION: This new technique of global MD-CSF monitoring correlates with conventional CSF levels of glucose and lactate, and the MD recovery is higher than previously described. Increase in lactate and pyruvate, without any effect on the LPR, correlates to unfavorable outcome, perhaps related to the presence of erythrocytes in the CSF.
- 25Kilkenny, C.; Browne, W. J.; Cuthill, I. C.; Emerson, M.; Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010, 8, e1000412 DOI: 10.1371/journal.pbio.1000412There is no corresponding record for this reference.
- 26Meredith, A. L.; Thorneloe, K. S.; Werner, M. E.; Nelson, M. T.; Aldrich, R. W. Overactive bladder and incontinence in the absence of the BK large conductance Ca2+-activated K+ channel. J. Biol. Chem. 2004, 279, 36746– 36752, DOI: 10.1074/jbc.M40562120026Overactive Bladder and Incontinence in the Absence of the BK Large Conductance Ca2+-activated K+ ChannelMeredith, Andrea L.; Thorneloe, Kevin S.; Werner, Matthias E.; Nelson, Mark T.; Aldrich, Richard W.Journal of Biological Chemistry (2004), 279 (35), 36746-36752CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)BK large conductance voltage- and calcium-activated potassium channels respond to elevations in intracellular calcium and membrane potential depolarization, braking excitability of smooth muscle. BK channels are thought to have a particularly prominent role in urinary bladder smooth muscle function and therefore are candidate targets for overactive bladder therapy. To address the role of the BK channel in urinary bladder function, the gene mSlo1 for the pore-forming subunit of the BK channel was deleted. Slo-/- mice were viable but exhibited moderate ataxia. Urinary bladder smooth muscle cells of Slo-/- mice lacked calcium- and voltage-activated BK currents, whereas local calcium transients ("calcium sparks") and voltage-dependent potassium currents were unaffected. In the absence of BK channels, urinary bladder spontaneous and nerve-evoked contractions were greatly enhanced. Consistent with increased urinary bladder contractility caused by the absence of BK currents, Slo-/- mice demonstrate a marked elevation in urination frequency. These results reveal a central role for BK channels in urinary bladder function and indicate that BK channel dysfunction leads to overactive bladder and urinary incontinence.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c01698.
Transmission spectra of K-edge glass filters employed to adjust the wavelength range of irradiation; UV/Vis absorbance spectra of Z-BOX A and B as well as of Z-PDP A1, Z-PDP B1, Z-PDP A2, and Z-PDP B2 in aqueous solution; E-BOX A forms meshed structures with a three-dimensional spread; Z-BOX A forms ribbon-like structures with a two-dimensional spread; 1H NMR spectrum of E-BOX A in deuterated acetonitrile; irradiation of single PDPs and assessment of newly formed E-isomers by LC-MS; oxidation of isolated E-PDPs to E-BOXes; morphological effects of Z-PDP A1/2 and Z-PDP B1/2 treatments on HepG2 cells compared to control conditions; time-line diagram of the in vivo animal experiments (PDF)
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