F-19 AND C-13 NMR-STUDIES OF POLYOL METABOLISM IN FREEZE-TOLERANT PUPAE OF HYALOPHORA-CECROPIA

Sorbitol biosynthesis and regulation in freeze tolerant pupae of Hyalo phora cecropia have been investigated as a function of temperature by F-19 and C-13 nuclear magnetic resonance (NMR) spectroscopy using seve ral C-13-labeled and/or fluorine-substituted carbohydrates. 3-Deoxy-3- fluoro-D-glucose (3DFG) was metabolized to 3-deoxy-3-fluoro-D-sorbitol (3DFS), 3-deoxy-3-fluoro-D-fructose (3DFF), and 3-deoxy-3-fluoro-D-gl uconic acid (3DFGA), indicating that the enzymes required for sorbitol biosynthesis and metabolism are active in H. cecropia at warm (22-deg rees-C) and cold (4 and -10-degrees-C) temperatures. Two additional me tabolites were produced when pupae were injected with either 3DFG, 3DF S, 3DFF, or 3-deoxy-3-fluoro-D-mannose (3DFM). One of these was identi fied as 3-deoxy-3-fluoro-D-mannitol (3DFML) by C-13 NMR using [1-C-13] 3DFM and [1-C-13]3DFG as metabolic probes. H. cecropia pupae injected with D-glucose labeled with C-13 at C-1, C-2, or C-3 and subsequently analyzed by C-13 NMR clearly demonstrated the ability to generate sorb itol and fructose. In contrast, gas chromatography/mass spectrometric analysis of hemolymph failed to detect sorbitol in pupae reared under natural conditions (i.e. in the absence of injected enriched sugars). Thus, although H. cecropia pupae have the enzymic machinery to biosynt hesize sorbitol, they do not appear to accumulate high steady-state co ncentrations of this polyol over the temperature range studied. The sp ecificity of the enzymes involved in alditol biosynthesis in H. cecrop ia was examined by C-13 NMR with a wide range of aldoses enriched with C-13 at C-1. Pupae were capable of converting these sugars to their c orresponding [1-C-13]alditols, indicating that nonspecific dehydrogena se(s), in addition to aldose reductase, is(are) involved in polyol bio synthesis in H. cecropia pupae.