LAUSR

Significance The human prostate accumulates high luminal citrate levels to serve sperm viability. There is only indirect qualitative evidence about metabolic pathways and carbon sources maintaining these levels. Human citrate-secreting prostate cancer cells were supplied with 13C-labeled substrates, and NMR spectra of extracellular fluid were recorded. We report absolute citrate production rates of prostate cells and direct evidence that glucose is the main carbon source for secreted citrate. Pyruvate carboxylase provides sufficient anaplerotic carbons to support citrate secretion. Glutamine carbons exchange with carbons for secreted citrate but are likely not involved in its net synthesis. Moreover, we developed metabolic models employing the 13C distribution in extracellular citrate as input to assess intracellular pathways followed by carbons toward citrate.