Chemical Basis for Deuterium Labeling of Fat and NADPH

Abstract

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.

A small molecule G6PD inhibitor reveals immune dependence on pentose phosphate pathway

“…We next aimed to directly monitor G6PD mediated hydride transfer to NADPH. Specifically, we traced the transfer of deuterium from [1- 2 H]glucose, via glucose-6-phosphate, to the NADPH’s active hydride ( Figure 1d ) 24 . Consistent with this labeling arising primarily from the G6PD reaction, G6PD knockout cells demonstrated a nearly complete loss of active hydride labeling ( Figure 1f ).…”

Serine Catabolism Feeds NADH when Respiration Is Impaired

“…In normal physiological conditions, 4% of NADH in pancreas is labeled by 2 H-serine infusion. The real contribution of serine catabolism is, however, larger, as labeling is limited by the deuterium kinetic isotope effect (slower reactivity of deuterated, compared to unlabeled, methylene-THF) and by 1 H-2 H exchange between NADH and water (Ducker et al, 2016;Schmidt et al, 2000;Wiberg, 1955;Zhang et al, 2017). The magnitude of both of these effects is on the order of 2-fold (perhaps substantially larger for 1 H-2 H exchange), suggesting that ~20% or more of pancreas NADH is made by serine catabolism, with serine's contribution less in other tissues.…”

“…1A). While the raw extent of malate labeling was small, due to tracer loss via H-D exchange, even modest 2 H labeling is often biologically meaningful (Zhang et al, 2017).…”

2 H/ 1 H variation in microbial lipids is controlled by NADPH metabolism

“…The redox-active H on NADPH is moderately acidic and can potentially exchange with water. Indeed, labeling experiments with 2 H2O in different transformed human cell lines revealed that ∼40% to ∼70% of the redox active hydrogen of NADPH comes from water (48). It was shown that NADPH-dependent flavin enzymes such as glutathione reductase that hold the hydride in a N-H bond instead of a C-H bond catalyze this exchange.…”