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

Abstract

Purpose: To investigate flux through the polyol pathway in the dog lens by 19F-nuclear magnetic resonance (19F-NMR) spectroscopy, using 3-fluoro-3-deoxy-D-glucose (3-FG) as a substrate.

Methods: 3-FG metabolism was monitored by 19F-NMR analysis. Dog lenses were incubated in Dulbecco's modified Eagle's medium containing 10 mM 3-FG. Enzymatic reductase and dehydrogenase activities were spectrophotometrically determined, whereas the analyses of 3-FG metabolites were conducted by 19F-NMR analysis. Aldose reductase (AR) was immunohistochemically localized in dog lens with antibodies raised against dog kidney AR.

Results: 19F-NMR spectra indicate that incubation of purified dog lenses AR 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 results 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 affinity (Km) of AR for 3-FG is approximately 20-fold better than that for D-glucose, whereas the Km 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 formed. 3-FS formation was reduced by the AR inhibitor AL 1576, and 3-FF formation was eliminated by the SDH inhibitor CP-166,572. In dog lens epithelial cells cultured with 3-FG, only 3-FS is formed. Similarly, only 3-FS is formed when lens capsule containing primarily epithelial lens contaminated with superficial epithelial cells was incubated in 3-FG. Similar incubation of the remaining cortex resulted primarily in the formation of 3-FS and 3-FGA. This enzymatic distribution was confirmed by spectrophotometric activity analysis and the immunohistochemical localization of AR.

Conclusions: The data confirm that flux through the polyol pathway primarily results in sorbitol accumulation. The absence of fructose and gluconic acid from cultured lens epithelium suggests that the epithelial cells primarily contain AR, whereas differentiated fiber cells also contain SDH and glucose dehydrogenase.