3-FG as substrate for investigating flux through the polyol pathway in doglens by F-19-NMR spectroscopy

PURPOSE. TO investigate nux through the polyol pathway in the dog lens by F -19-nuclear magnetic resonance (F-19-NMR) spectroscopy, using 3-fluoro-3-de oxyl-D-glucose (3-FG) as a substrate. METHODs. 3-FG metabolism was monitored by F-19-NMR analysis. Dog lenses wer e incubated in Dulbecco's modified Eagle's medium containing 10 mM 3-FG. En zymatic reductase and dehydrogenase activities were spectrophotometrically determined, whereas the analyses of 3-FG metabolites were conducted by F-19 -NMR analysis. Aldose reductase (AR) was immunohistochemically localized in dog lens with antibodies raised against dog kidney AR. RESULTS. F-19-NMR spectra indicate that incubation of purified dog lenses A R with 3-FG results in the formation of 3-fluoro-3-deoxy-D-sorbitol (3-FS) and that incubation of dog liver sorbitol dehydrogenase (SDH) with 3-FS res ults in the formation of 3-fluoro-3-deoxy-D-fructose (3-FF). This confirms that 3-FG is metabolized to 3-FF by the polyol pathway enzymes. The affinit y (K-m) of AR for 3-FG is similar to 20-fold better than that for D-glucose , whereas the K-m of SDH for 3-FS was fourfold less than for D-sorbitol. 3- FG in cultured dog lenses is metabolized primarily to 3-FS; however, small amounts of 3-FF and 3-fluoro-3-deoxy-D-gluconic acid (3-FGA) are also forme d. 3-FS formation was reduced by the AR inhibitor AL 1576, and 3-FF formati on was eliminated by the SDH inhibitor CP-166,572. In dog lens epithelial c ells cultured with 3-FG, only 3-FS is formed. Similarly, only 3-FS is forme d when lens capsule containing primarily epithelial lens contaminated with superficial epithelial cells was incubated in 3-FG. Similar incubation of t he remaining cortex resulted primarily in the formation of 3-FS and 3-FGA. This enzymatic distribution was confirmed by spectrophotometric activity an alysis and the immunohistochemical localization of AR. CONCLUSIONS. The data confirm that flux through the polyol pathway primaril y results in sorbitol accumulation. The absence of fructose and gluconic ac id from cultured lens epithelium suggests that the epithelial cells primari ly contain AR, whereas differentiated fiber cells also contain SDH and gluc ose dehydrogenase.