Evaluation of the presence of reduced nicotinamide adenine dinucleotide phosphate in bacterial metabolites used as immunostimulators and its role in nitric oxide induction

Abstract

Bacterial metabolites that act as immunostimulators have aroused interest because of their therapeutic potential in several immune disorders. These metabolites are complex, heterogeneous, and comprise numerous immune-boosting biomolecules. To better understand their immune stimulatory properties, characterization of their components is essential. An ether extract of metabolites from nine bacterial species was analyzed for the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) or other fluorophores. This metabolite in combination with bile lipids is a licensed immune stimulatory drug. Excitation of the extract at 340nm resulted in fluorescence with an emission maximum of around 410nm, which is fairly specific for NADH and NADPH. Reverse-phase-HPLC and electro-spray ionization-mass analysis confirmed the presence of NADPH in the bacterial metabolites. Quantification by glutathione reductase assay indicated 11.90±0.01μM of NADPH in the metabolites. Further characterization of the individual bacterial extracts of the metabolite confirmed the presence of NADPH. Subsequently, studies were performed to evaluate the role/s of NADPH in immune-stimulatory drugs. NADPH is known to be involved in production of nitric oxide (NO), which has versatile roles in the immune system. The biological function of NADPH in NO induction by RAW 264.7 (mouse macrophage) cells was evaluated and it was found that bacterial NADPH has a significant role in inducing NO and that NADPH from individual bacterial extracts is capable of inducing NO. Investigation on the stability and biological potency of NADPH in bacterial metabolites is important because of NADPH's wide therapeutic applications, most of which are associated with its role in NO induction.