Bilirubin oxidation products (BOXes): synthesis, stability and chemical characteristics

Abstract

SummaryBilirubin oxidation products (BOXes) have been a subject of interest in neurosurgery because they are purported to be involved in subarachnoid hemorrhage induced cerebral vasospasm. There is a growing body of information concerning their putative role in vasospasm; however, there is a dearth of information concerning the chemical and biochemical characteristics of BOXes. A clearer understanding of the synthesis, stability and characteristics of BOXes will be important for a better understanding of the role of BOXes post subarachnoid hemorrhage.We used hydrogen peroxide to oxidize bilirubin and produce BOXes. BOXes were extracted and analyzed using conventional methods such as HPLC and mass spectrometry. Characterization of the stability BOXes demonstrates that light can photodegrade BOXes with a t 1/2 of up to 10 h depending upon conditions. Mixed isomers of BOXes have an apparent extinction coefficient of ε = 6985, and a λ max of 310 nm.BOXes are produced by the oxidation of bilirubin, yielding a mixture of isomers: 4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (BOX A) and 3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (BOX B). The BOXes are photodegraded by ambient light and can be analyzed spectrophotometrically with their extinction coefficient as well as with HPLC or mass spectrometry. Their small molecular weight and photodegradation may have made them difficult to characterize in previous studies. KeywordsBilirubin; reactive oxygen species; HPLC; mass spectrometry; molecular weight; photo-degradation; blood product; photolysis There is extensive study of the oxidations (including peroxidations) of unsaturated lipids and proteins in biological systems, including the resultant compounds produced [1,26,39,41]. There is also evidence that bilirubin is a biologic antioxidant [34,[43][44][45][46]51]. The oxidation of (unconjugated) bilirubin has largely focused on the degradation of bilirubin between the pyrroles [5], with little discussion concerning the putative biological activity of the products of bilirubin oxidation [49]. We recently reported on a new family of bilirubin oxidation products found following subarachnoid hemorrhage [3,22,25,38]. It appears that bilirubin oxidation products (BOXes) can be formed by the direct, nonenzymatic oxidation of bilirubin with hydrogen peroxide [22]. In Fig. 1a Kranc et al. [22]. Cleavage of bilirubin at the pyrrole, rather than between the pyrrole rings produces an apparently reactive monopyrrole amide [22]. However, to date there has been relatively little information concerning the stability or characteristics of BOXes, which is important in understanding the role BOXes may play in subarachnoid haemorrhage induced vasospasm. Here we present our latest findings concerning the chemical characteristics of BOXes produced by the oxidation of bilirubin. The relatively unique characteristics of BOXes and their nonspecific, nonenzymatic production provides important insight into the putative role of unconjugated bilirubin and its oxid...

Impact of Heme and Heme Degradation Products on Vascular Diameter in Mouse Visual Cortex

“…Ultraviolet light–exposed BOX salt solution (254 nm for 5 hours) was not effective in inducing vasoconstriction and converted the response to a nonsignificant increase in vascular diameter (5.8±3.5%; n=5 from 5 animals; P =0.14; 1‐way ANOVA; Figure 4 D), confirming that BOXes lose their biological activity by isomerization or degradation after illumination in the ultraviolet range. 12 , 15 In a next step of compound purification, we determined changes in arteriolar diameter after application of BOX extract (0.03 mg/mL), generated from the dried chloroform extract of the salt mixture. BOX extract induced a substantial decrease in vessel diameter by 31.5 ± 9.4% (n=5 from 4 animals; P <0.001; 1‐way ANOVA; Figure 4 D) within 15 minutes.…”

“…With the exception of the synthetic variant of the Z ‐isomer of BOX A ( Z ‐BOX A syn), all BOX‐containing compounds were prepared by the nonenzymatic oxidative degradation of bilirubin (Figure 3 A), modified after Wurster et al 15 Protected from light, 1 g of bilirubin was dissolved in 500 mL of 5 mol/L sodium hydroxide solution. The mixture was adjusted with 11 mol/L HCl to pH 7.5, and 50% hydrogen peroxide solution was added to a final concentration of 10% by volume.…”

Bilirubin Oxidation Products and Cerebral Vasoconstriction

“…In any case, due to the absence of sufficient purified compound, mole determinations were based upon a collective extinction coefficient (𝜀; 6,985 L/mol-cm -1 ) approximated using the absorption at 310 nm of a saline-reconstituted, chloroform extract of a hydrogen peroxide–bilirubin reaction mixture containing BOX A, BOX B, and MVM ( Wurster et al, 2008 ). Caveats associated with adoption of absorption at 310 nm include that there was some variability in the amounts of these BOXes species formed and, in particular, varied formation of MVM ( Wurster et al, 2008 ). This variability would influence the 𝜀 approximation because the λ max for MVM is 317 or 319 nm, at least as determined in methanol (as noted above; Bonnett and McDonagh, 1969 ; Kurtin, 1978 ).…”

“…A second caveat associated with the approximated 𝜀 ( Wurster et al, 2008 ) is that the determination was based upon an estimated 50% conversion of bilirubin to BOXes by the hydrogen peroxide oxidation of bilirubin, with the remaining 50% unreacted bilirubin and biliverdin as well as “other compounds” that do not absorb at 310 nm ( Kranc et al, 2000 ; Pyne-Geithman et al, 2005 ; Wurster et al, 2008 ).…”

Pyrrolic and Dipyrrolic Chlorophyll Degradation Products in Plants and Herbivores

“…In mammals most of the bilirubin is conjugated and excreted into the bile, but an additional oxidative degradation occurs. This ROS‐mediated breakdown leads to dipyrrolic propentdyopents (PDPs) and monopyrrolic bilirubin oxidation end products (BOXes, Scheme ) . BOX A and B exhibit an intact vinyl pyrrole and differ only in the position of vinyl and methyl group, whereas BOX C derives from the propionic acid pyrrole.…”