Bilirubin (BR) is a tetrapyrrolic compound stemming from heme catabolism with diverse physiological functions. It can be oxidized by H₂O₂ to form several degradation products, some of which have been detected in vivo and may contribute to the pathogenesis of certain diseases. However, the oxidative degradation of BR is complex and the conditions that BR degradation occurs pathophysiologically remain obscure. Neutrophils are known to generate large amounts of reactive oxygen species, including H₂O₂, upon activation and they are mobilized to inflammatory sites; therefore, we hypothesized that activated neutrophils could cause BR degradation, which could occur at inflammatory sites. In the present study, we investigated BR degradation by H₂O₂ and identified hematinic acid (BHP1) and a new product BHP2, whose structure was characterized as 2,5-diformyl-4-methyl-1H-pyrrole-3-propanoic acid. An LC-MS/MS method for the quantitation of the two compounds was then established. Using the LC-MS/MS method, we observed the concentration-dependent formation of BHP1 and BHP2 in mouse neutrophils incubated with 10 and 30 μM of BR with the yields being 16 ± 3.2 and 31 ± 5.9 pmol/10⁶ cells for BHP1, and 25 ± 4.4 and 71 ± 26 pmol/10⁶ cells for BHP2, respectively. After adding phorbol 12-myristate 13-acetate, a neutrophil agonist, to 30 μM of BR-treated cells, the BHP1 yield increased to 43 ± 6.6 pmol/10⁶ cells, whereas the BHP2 one decreased to 47 ± 9.2 pmol/10⁶ cells. The two products were also detected in hemorrhagic skins of mice with dermal inflammation and hemorrhage at levels of 4.5 ± 1.9 and 0.18 ± 0.10 nmol/g tissue, respectively, which were significantly higher than those in the non-hemorrhagic skins. BHP2 was neurotoxic starting at 0.10 μM but BHP1 was not, as assessed using Caenorhabditis elegans as the animal model. Neutrophil-mediated BR degradation may be a universally pathophysiological process in inflammation and can be particularly important under pathological conditions concerning hemorrhage.

Levoglucosan is a thermal decomposition product of cellulose in particulate matter. δ¹³C value of levoglucosan could be used in studying the combustion mechanisms and chemical pathways. In order to introduce a minimum number of carbon atoms, based on the stereostructure of levoglucosan, a two-step derivatization method with methylboronic acid and MSTFA was developed and carefully optimized. The recommended reaction temperature is 70°C; the reaction time is 60 min for MBA and 120 min for MSTFA derivatization; and the molar ratio of levoglucosan : MBA : MSTFA is 1 : 1: 100 and 1 : 1: 120 and the reagent volume ratio of MSTFA : pyridine is between 1 : 3 and 1 : 4. The developed method achieved excellent reproducibility and high accuracy. The differences in the carbon isotopic compositions of the target boronate trimethysilylated derivative between the measured and calculated ranged from 0.09 to 0.36‰. The standard deviation of measured δ¹³C value of levoglucosan was between 0.22 and 0.48‰. The method was applied to particle samples collected from the combustion of cellulose at four different temperatures. δ¹³C values of levoglucosan in particle samples generated from a self-made combustion setup suggesting that combustion temperature play a little role on isotope fractionation of levoglucosan, although ¹³C enriched in levoglucosan during the combustion process.