**6. Conclusions**

*Autism Spectrum Disorders - Advances at the End of the Second Decade of the 21st Century*

nine pairs of mice [43].

is further converted to stercobilinogen.

stercobilinogen levels of TS2-NEO mice (*p* = 0.07). A larger sample set will be necessary to determine the significance of depletion in stercobilinogen. Furthermore, the *p*-values determined were improved upon since our last report with a study of

The depletion of stercobilin in the ASD model of mice relative to controls at a greater than 99.9% confidence level suggests that stercobilin depletion in fecal material may have potential value as a biomarker for ASD in humans. Although less statistically significant, stercobilinogen, the metabolic precursor to stercobilin, is also depleted in fecal samples. The observation of these depletions suggests that there may be interference in the metabolic pathway that allows for the differences. As shown in **Figure 6**, stercobilin and stercobilinogen are products of heme catabolism. As bilirubin glucuronides enter the intestines, the action of enzyme systems by anaerobic bacterial flora converts the glucuronides to mesobilirubinogen, which

Our results are also intriguing in the context of a discovery decades ago by Gustafsson and Lanke in which they observed no bilins present in the feces or urine of germfree rats [52]. Once the germfree animals were exposed to fecal matter from control animals, they too began to produce bilins to the same extent as the controls (when both groups were given identical diets). Moreover, they observed that the negative urobilin test (note: urobilin is a metabolic product derived from urobilinogen and is primarily excreted through urine, as shown in **Figure 6**) turned positive in germfree animals infected with a single *Clostridium*-like microorganism that had been isolated from the intestinal contents of rats that showed the presence of bilins in fecal matter. The bilin output increased in these animals after infection

*A depiction of the catabolism of heme into stercobilin. The enterohepatic cycle in which stercobilin can be recirculated back and excreted instead through the urine is also shown. The line shows the point in which* 

*bacterial interaction takes over in the metabolic pathway to create stercobilin.*

**58**

**Figure 6.**

The discovery that stercobilin, and to a lesser extent stercobilinogen, are depleted in the fecal matter from a murine ASD model gives promise of the potential of these substances to serve as clinical biomarkers for ASD. Work to understand the relationship between the depletion of these bilins and the identity of the microbiota responsible is intriguing, as is the possibility that microbiota may play a role in the etiology of ASD; if this is true, it means that fecal transplants may have impact in the treatment of ASD, as recent clinical evidence suggests [57].

### **Acknowledgements**

The authors would like to thank all of their coworkers who contributed to the results described in this chapter, including Kevin Quinn, Charmion Cruickshank-Quinn, Jordan Coffey, Anthony Vadas, Thomas Puleo, Katelyn Lewis, Gregory Pirrone, Eric Helms, Alessandra Dettori, Emanuela Azara, Mauro Marchetti, Paolo Tomasi, Giuseppe Delitala, Emma Fenude, Maria Piera Demontis, Elisabetta Alberico, Vittorio Anania, Silvia Gianorso, Will Friesen, Nhu Nguyen-Dudziak, Stephen Carro, and Michael Wach. Financial support for this research has come from the United Kingdom Legal Services Commission, the Fondazione Banco di Sardegna, the California Scottish Rite, the Mark Diamond Research Fund, and the National Institutes of Health through the Center for Research Resources (Grant #S10-RR029517).

*Autism Spectrum Disorders - Advances at the End of the Second Decade of the 21st Century*
