Quantitative Analysis of Folate and NADPH Metabolism

Abstract

Folic acid and nicotinamide adenine dinucleotide phosphate (NADPH) play important roles in supporting cell growth. Folic acid, known as vitamin B9, is critical for nucleotide and amino acid synthesis by mediating the transfer and transformation of a one-carbon unit. NADPH, derived from niacin or vitamin B3, is the high-energy electron carrier that is used for antioxidant defense, as well as the biosynthesis of deoxyribonucleotides, fatty acid and sterols. It is thought that synergy of the two pathways for cell growth may include direct metabolic interactions, and cancer cells may hijack these pathways for uncontrolled proliferation. However, a comprehensive understanding of folate and NADPH metabolism are hindered by the instability and low abundance of related species.

Here, on the folate side, we developed an analytical method to measure intracellular folate species from mammalian cells, using an isotope-labeling strategy combined with liquid chromatography-mass spectrometry (LC-MS). This new method has enabled a CRISPR knockout folate metabolism study that showed a reversal of the cytosolic folate pathway is capable of compensating mitochondrial folate deficiency. On the NADPH side, we quantitatively demonstrated that the oxidative pentose phosphate pathway (oxPPP) is the main source of NADPH’s high energy electrons across cancer cell lines, through a deuterium (2H) isotope tracing analysis and a CRISPR knockout study. In the 2H tracing analysis, we found the redox-active hydrogen on NADPH can exchange with ambient water through a Flavin-enzyme catalyzed mechanism. Analysis of major NADPH production pathway knockout cells also suggested any of the oxPPP, malic enzyme 1 (ME1) or isocitrate dehydrogenase 1 (IDH1) is enough to support cell growth, but oxPPP is uniquely required to maintain a normal NADPH/NADP ratio in cancer cell lines. Previous literature showed that the folate pathway could be an important NADPH production pathway, especially in combating oxidative stress for human melanoma cells during metastasis. We discovered a second NADPH-folate connection that high NADP inhibits dihydrofolate reductase (DHFR), resulting in impaired folate-mediated biosynthesis across different cancer cell lines. To explore the terrain of metabolism, we developed a computational network analysis approach for new metabolites discovery by connecting mass from untargeted metabolomics data and known metabolite database. The concept has been validated by analyzing an annotated yeast metabolomics dataset, and preliminary results identified a cluster of potential metabolites specifically accumulated in kidney that may relate to thiamine (vitamin B1) metabolism.

In summary, this study provides novel methods and insights to study folate and NADPH metabolism, shedding light on the intricacy of metabolism for cell growth and potential cancer therapeutics.