Large enrichments in fatty acid ² H/ ¹ H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae

Shifts in the hydrogen stable isotopic composition ( 2 H/ 1 H ratio) of lipids relative to water (lipid/water 2 H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2 H-fractionation ( 2 ε fattyacid/water ) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2 ε fattyacid/water . In chemostats, fatty acids from glycerol-respiring cells were >550‰ 2 H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2 H/ 1 H ratios, particularly in glycerol-respiring cells by 200‰. Variations in the activities and kinetic isotope effects among NADP + -reducing enzymes indicate cytosolic NADPH supply as the primary control on 2 ε fattyacid/water . Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2 H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2 ε fattyacid/water variations (<30‰) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2 H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2 H/ 1 H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.