Great strides have been made over the past decade toward comprehensive study of metabolism. Mass spectrometry (MS) has played a central role by enabling measurement of many metabolites simultaneously. Tracking metabolite labeling from stable isotope tracers can in addition reveal pathway activities. Here, we describe the basics of metabolite measurement by MS, including sample preparation, metabolomic analysis, and data interpretation. In addition, drawing on examples of successful experiments, we highlight the ways in which metabolomics and isotope tracing can illuminate biology.

SummaryOne-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. CRISPR-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-THF, and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis with the mitochondrial pathway required in nutrient poor conditions. eTOC blurbUsing genetic and metabolomic approaches, Ducker et al. dissect the roles of cytosolic and mitochondrial folate metabolism in cell proliferation, revealing that most cells default to mitochondria for making 1C units, simultaneously generating glycine, NADH and NADPH. Upon loss of the mitochondrial pathway, however, cytosolic metabolism supports tumor growth.

Extraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

Much understanding of metabolism is based on monitoring chemical reactions in cells with isotope tracers. For this purpose, 13C is well suited due to its stable incorporation into bio-molecules and minimal kinetic isotope effect. For redox reactions, however, deuterium tracing can provide valuable additional information. To date, studies examining NADPH production with deuterated carbon sources have failed to account for roughly half of NADPH’s redox active hydrogen. Here we show that the missing hydrogen is the result of enzyme-catalyzed H-D exchange between water and NADPH. While isolated NADPH does not undergo H-D exchange with water, such exchange is catalyzed by Flavin enzymes and occurs rapidly in cells. Correction for H-D exchange is required for accurate assessment of the biological sources of NADPH’s high energy electrons. Deuterated water (D2O) is frequently used to monitor fat synthesis in vivo, but the chemical pathway of the deuteron into fat remains unclear. We show that D2O labels fatty acids primarily via NADPH. Knowledge of this labeling route enables calculation, without any fitting parameters, of the mass isotope distributions of fatty acids from cells grown in D2O. Thus, knowledge of enzyme-catalyzed H-D exchange between water and NADPH enables chemically accurate interpretation of deuterium tracing studies of redox cofactor and fatty acid metabolism.

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