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

Bilirubin oxidation end products (BOXes) appear upon endogenous heme degradation 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 determination 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 preparation includes the addition of 5-bromonicotinamide as internal standard and protein precipitation with acetonitrile. Baseline-separation 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 analysis time of 17 min. Using single reaction monitoring in the positive ion mode, the linear working ranges were 2.74-163 pg/mu L (Z-BOX A) and 2.12-162.4 pg/mu L (Z-BOX B) with R-2 > 0.995. Intra- and inter-day precisions were <10%. The inherent analyte concentrations of Z-BOX A (14.4 +/- 5.1 nM) and Z-BOX B (10.9 +/- 3.1 nM) in pooled human serum were determined by standard addition. 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 biological significance of higher order metabolites of heme degradation. (C) 2014 Elsevier B.V. All rights reserved

Impact of heme and heme degradation products on vascular diameter in mouse visual cortex

Background Delayed cerebral vasospasm is the most common cause of mortality and severe neurological impairment in patients who survive subarachnoid hemorrhage. Despite improvements in the field of diagnostic imaging, options for prevention and medical treatment—primarily with the calcium channel antagonist nimodipine or hemodynamic manipulations—are insufficient. Previous studies have suggested that heme and bilirubin oxidation end products, originating from degraded hemoglobin around ruptured blood vessels, are involved in the development of vasospasm by inhibiting large conductance BKCa potassium channels in vascular smooth muscle cells. In this study, we identify individual heme degradation products regulating arteriolar diameter in dependence of BKCa channel activity. Methods and Results Using differential interference contrast video microscopy in acute brain slices, we determined diameter changes of intracerebral arterioles in mouse visual cortex. In preconstricted vessels, the specific BKCa channel blockers paxilline and iberiotoxin as well as iron‐containing hemin caused vasoconstriction. In addition, the bilirubin oxidation end product Z‐BOX A showed a stronger vasoconstrictive potency than its regio‐isomer Z‐BOX B. Importantly, Z‐BOX A had the same vasoconstrictive effect, independent of its origin from oxidative degradation or chemical synthesis. Finally, in slices of Slo1‐deficient knockout mice, paxilline and Z‐BOX A remained ineffective in changing arteriole diameter. Conclusions We identified individual components of the oxidative bilirubin degradation that led to vasoconstriction of cerebral arterioles. The vasoconstrictive effect of Z‐BOX A and Z‐BOX B was mediated by BKCa channel activity that might represent a signaling pathway in the occurrence of delayed cerebral vasospasm in subarachnoid hemorrhage patients

Mixed Alkyl Hydrido Complexes of Zinc : Synthesis, Structure, and Reactivity

International audienceThe (NNNN)-type macrocycle 1,4,7-trimethyl-1,4,7,10-tetraazacyclododecane (Me3TACD, 1,4,7-Me3[12]aneN4) reacted with 1 equiv of ZnEt2 under ethane elimination to give the mononuclear ethyl complex [(Me3TACD)ZnEt] (1). Upon treatment of (Me3TACD)H with 2 equiv of ZnEt2, the dinuclear complex [(Me3TACD)(ZnEt)(ZnEt2)] (2) was formed, which was converted with an additional 1 equiv of (Me3TACD)H to 1. Reaction of 1 with PhSiH3 led to the formation of a tetranuclear ethyl hydrido complex [{(Me3TACD)ZnEt}2(ZnEtH)2] (3). Single-crystal X-ray diffraction study revealed 3 to be a centrosymmetric dimer featuring two [(Me3TACD)ZnEt] units coordinated to a [Zn(μ-H)2Zn] core via amido nitrogen atoms of the Me3TACD ligands. Substitution of the two [(Me3TACD)ZnEt] units in 3 by N-heterocyclic carbene IMes [1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene] gave [(IMes)ZnEtH]2 (4b). The mixed alkyl hydrido complexes [(IMes)ZnRH]2 (R = Me, 4a; Et, 4b) were alternatively synthesized in quantitative yield by reacting [(IMes)ZnR2] (R = Me, Et) with [(IMes)ZnH2]2 in 2:1 ratio. Methyl complex 4a reacted with CO2 (p(CO2) = 0.5 bar) under facile insertion of CO2 into Zn–H bonds to give dinuclear formate complex [(IMes)ZnMe(O2CH)]2 (5a). Treatment of 4b with CO2 (p(CO2) = 0.5 bar) afforded a mixture of di- and trinuclear formate complexes [(IMes)ZnEt(O2CH)]2 (5b) and [(IMes)2Zn3Et3(O2CH)3] (6) under elimination of one IMes as CO2 adduct IMes·CO2