Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme required for diverse biological processes, rendering it indispensable for cellular function. NAD+ can be synthesized through the _de novo_ pathway from tryptophan (TRP) and the salvage pathway from the three forms of vitamin B3, namely nicotinamide (NAM), nicotinic acid (NA) and nicotinamide riboside (NR). Accurately assessing the contributions of these pathways is critical for elucidating molecular mechanisms that govern NAD⁺\-dependent processes, including aging and organismal health.
We employ the tiny nematode _Caenorhabditis elegans_ to elucidate how the interplay between genes and nutrients affects health and aging both acutely and over the lifespan trajectories. It has been well acknowledged that various studies in _C. elegans_ are significantly hindered by the metabolic activity of its food source, _E. coli_ bacteria, despite a large body of research conducted under these conditions. To circumvent these interruptions from bacterial metabolism, we developed and utilized an axenic culture system to dissect the gene-nutrient interplays and their role in health and aging.
Using the axenic culture system, we have developed various nutrient-deficient models. Among these, we observed a distinct phenotype: despite sufficient amounts of _de novo_ synthesis precursor TRP in the medium, vitamin B3 deprivation still led to severe NAD+ deficiency, resulting in developmental and reproductive defects in _C. elegans_. This phenomenon significantly differs from that in mammals, where NAD+ demand can be effectively met through either pathway. This suggested that the _de novo_ NAD+ synthesis pathway in _C. elegans_ might be inefficient or ineffective.
To explore this question further, we examined the effects of other intermediates of the _de novo_ NAD+ synthesis pathway (kynurenine pathway). Our results demonstrated that only the final product of kynurenine pathway, quinolinic acid (QA), rescued the NAD+ deficiency. Initially, we hypothesized that other intermediates in the kynurenine pathway could not effectively convert to QA.
_C. elegans_ lacks a homolog of the enzyme quinolinate phosphoribosyltransferase (QPRT), which converts QA to nicotinic acid mononucleotide (NAMN). However, in 2017, McReynolds _et al._ reported that _C. elegans_ uridine monophosphate synthetase-1 (UMPS-1) can function as QPRT, converting QA to NAMN and subsequently synthesizing NAD+. Based on this mechanism, a _umps-1_ mutant would obstruct the conversion of QA to NAD+.
Further experiments demonstrated that _umps-1_ mutant exhibits severe developmental arrest in axenic culture. However, uridine, but not vitamin B3, effectively rescued the developmental arrest, indicating that _umps-1_ primarily functions in pyrimidine synthesis rather than _de novo_ NAD+ synthesis.
Surprisingly, in the presence of uridine to ensure normal development, QA rescued NAD+ deficiency irrespective of the _umps-1_ defect. This led us to hypothesize the existence of an unknown, potentially novel _de novo_ NAD+ synthesis pathway. However, our subsequent efforts to identify this pathway were unsuccessful over an extended period.
During multiple repetitions of experiments, we noticed an overlooked detail that the concentration of QA required to rescue NAD+ deficiency was much higher than that of vitamin B3, exceeding 1000 times. Given the chemical similarity between QA and NA, we considered whether contamination during synthesis could occur. Could the so-called 99% pure QA contain sufficient NA to rescue NAD+ deficiency?
We measured the NA levels in QA solutions and were surprised to find that NA was widely present in commercial QA reagents. This impurity was likely responsible for QA's ability to rescue NAD+ deficiency. Purified QA significantly reduced the NA content. As expected, the purified QA no longer rescued NAD+ deficiency, confirming the absence of an effective _de novo_ NAD+ synthesis mechanism in _C. elegans_.
We emphasize that _de novo_ NAD+ synthesis is ineffective in _C. elegans_, a critical consideration for future related studies. Additionally, researchers should be mindful of the potential impact of impurities in reagents on experimental outcomes.