Uridine diphospho sugars and related hexose phosphates in the liver of hexosamine-treated rats: identification using phosphorus-31-{proton} two-dimensional NMR with HOHAHA relay

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Abstract
The effects of administration of galactosamine (GalN) and glucosamine (GlcN) on the levels of UDP-sugars and hexose monophosphates in rat livers were studied by a variety of 31P NMR methods. The flux of metabolites in the liver was monitored by in vivo NMR and showed elevated levels of UDP-sugars, and even greater increase in resonances at 4.6 ppm for GlcN treatment and at 2.0 ppm for GalN treatment. The individual compounds corresponding to these changes were identified in PCA liver extracts by 31P-{1H} two-dimensional relay spectroscopy with a HOHAHA-type 1H spin-lock. This method of transferring proton magnetization allows for nearly all of the proton chemical shifts to be observed for the hexose moiety of a UDP-sugar present in a complex mixture. The UDP-sugars in the extracts from treated rats were predominantly UDP-hexosamines. Relay spectra were also used to determine that GalN-1-P was the major component (16.0 .mu.mol/g of liver) of the GalN-treated liver, while both .alpha. and .beta. anomers of GlcNAc-6-P were readily identified as the major hexose monophosphates in the GlcN experiment. Spectra from the 1H dimension of relay experiments conducted on extracts were nearly superimposable on relay spectra obtained under the same conditions for mixtures of standard compounds of known structure. UDP-GlcN and UDP-GalN were not commercially available, but their presence was established in the extracts after GalN treatment by obtaining relay spectra for a mixture of the compounds produced in situ enzymatically, without purification. These compounds, as well as GalN-1-P, were found to be partially converted to the corresponding carbamic acids by the K2CO3 used for PCA neutralization. 31P-{1H} relay spectroscopy should thus be of general utility for the characterization of organophosphate sin tissue extracts.