Conclusion

From the experimental evidence, we can conclude that NADPH coupled assay can be used for studying the FMO’s kinetics, but these always give some measurement errors. The amount of NADPH consumed in the reaction is not directly proportional to the amount of substrate consumed, possibly because NADPH is unstable and a common cofactor in biological systems. We set out to find a specific method for our enzyme. Despite numerous available methods for monitoring TMAO, most are unreliable for enzymatic assay without using any sophisticated instruments. The technique reported here can be widely applied to measure the amount of TMAO independent of trimethylamine interference, which has not been the case previously except by (Hefni et al. , 2021).
This method can also be applied for testing urine samples for TMAO concentrations in a healthy individual within a normal range of urea and uric acid concentrations. Our current reported method, which explicitly monitors TMAO using UV spectrophotometry, is thus versatile and gives reproducible results. TMAO formed in the human body is excreted via urine. In a healthy individual, the TMAO concentration is within 10 µM (Hefni et al. , 2021), and hence detection is possible using our method, and the interference is within ± 5% in the presence of urea and uric acid. This study is awaiting ethical clearance.