The conversion of D-[U-C-13]glucose to proline (Pro), aspartate (Asp), and cysteine (Cys) is limited in premature neonates, implying that these amino acids (AA) are conditionally essential. This study was performed to determine whether these findings resulted from an insufficient precursor dose or intracellular compartmentation of newly synthesized amino acids, rather than inadequate synthesis. In the first phase of this study, seven total parenteral nutrition-fed, premature neonates received IV D-[(UC)-C-13]glucose at 5 mg/kg/min for 4 hr. In the second phase, a separate cohort of eight patients received an identical infusion. Blood was obtained before and at the end of the infusion. Isotopic enrichments of the free plasma AA and glucose were measured using gas chromatography/mass spectrometry in both studies. In phase 2, the isotopic enrichments of the AA bound to the hepatically synthesized proteins, fibrinogen and VLDL-apolipoprotein B-100 (apo B-100), were measured. In phase 1, despite a glucose precursor enrichment greater than 66%, Pro, Asp, and Cys remained the least enriched of all amino acids studied (P < 0.05). Asp, but not Pro, demonstrated very high enrichments in apo B-100 (P < 0.001), reflecting distinct intracellular compartmentation. We conclude that the limited conversion of D-[U-C-13]glucose to Pro, Asp, and Cys did not result from low precursor glucose enrichment and that there is evidence of Asp compartmentation (intracellular) in premature neonates. However, the low Pro enrichment in the free plasma AA pool and the absence of intracellular Pro compartmentation suggest that Pro may be a conditionally essential AA for premature neonates. (C) 1996 Academic Press, Inc.