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.