Stercobilin's depletion in autistic' urines: A study of bile metabolites tetrapyrrole compounds using ESI-MS/MS
Nguyen, Nhu Quynh T.
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Autism spectrum disorders (ASD) are characterized by deficits in social interactions and languages skills. Currently, 1 in 110 American children are considered to have ASD, according the latest report published by the Centers for Disease Control and Prevention. The disorder has become very common in the United States and is a serious public medical concern. The cure for ASD is unknown, but it has been strongly shown that early intervention, especially before age 3 can reduce the severity of the symptoms. Since the diagnosis of autism relies heavily on behavioral observation, a biological approach, or biomarker(s), is/are needed for early diagnosis. LC-ESI-MS study on urine samples indicates that there is lack of peak 595 m/z in autistic urine samples compared to a control group. The peak at 595 m/z has been identified by mass spectrometry as stercobilin, a tetrapyrrole compound produced by the oxidation of urobilinogen, a bilirubin metabolite, by gut flora. Two hypotheses were considered for this phenomenon: mercury-stercobilin complexation and oxidative stress. Mercury, a controversial agent contributing to ASD, was shown to make stoichiometric 1:1 ratio complexes with stercobilin in both aqueous 60%MeOH solution and artificial urine solution. The oxidative stress theory was simulated by oxidizing stercobilin in 30% H2O2 solution, which leads to production of species with differences of one oxygen atom. Experiments for these two hypotheses produced unique MS patterns that can be used for detection of mercury and/or oxidative stress from screening urine samples. Also, to positively confirm the presence of stercobilin and its metabolite precursors in urine samples, stercobilin and urobilin standard were fragmented using increasing energy from 0V to 100V. A very similar fragmentation pathway was shown with the successive removal of outer pyrrole rings, then the two identical inner pyrrole ring cores of both structures broken into various pieces at 70V to 100V energy. These studies of stercobilin and its precursor ion ultimately offer molecular information for the detection of biomarker(s) that can help reduce the ever increasing rate of ASD in the future.