3-Fluoro-3-deoxy-D-galactose: a new probe for studies on sugar cataract.
OBJECTIVE
Aldose reductase (AR) activity and flux through the polyol pathway can conveniently be monitored in dog lenses by measuring the metabolism of 3-fluoro-3-deoxy-D-glucose by 19F nuclear magnetic resonance (NMR) spectroscopy. Since AR has broad substrate specificity and preferentially utilizes galactose over glucose as substrate, the ability of AR to utilize 3-fluoro-3-deoxy-D-galactose (3-FDGal) as substrate as well as the metabolism of 3-FDGal in intact dog lens and cultured lens epithelial cells has been investigated.


METHODS
The suitableness of 3FDGal as a substrate was examined by incubating 3FDGal with purified dog lens aldose reductase in the presence of an NADPH generating system or with galactitol dehydrogenase in the presence of NAD+. Dog lenses and dog lens epithelial cells were cultured in 3-FDGal medium with and without the AR inhibitor AL 1576. Metabolism was studied using 19F NMR.


RESULTS
AR activity with 3-FDGal as substrate is higher than that with D-galactose and its Km of 4.2 mM is ca 10-fold higher than that of D-galactose. Purified dog lens AR incubated with 3-FDGal resulted in the formation of 3-fluoro-3-deoxy-D-galactitol. Galactitol formation was prevented by the addition of AL 1576. Incubation of 3-FDGal with galactitol dehydrogenase resulted in the formation of 3-fluoro-3-deoxy-D-galactonic acid. Dog lenses cultured in 3-FDGal medium formed NMR peaks corresponding to 3-fluoro-3-deoxy-D-galactitol and 3-fluoro-3-deoxy-D-galactonic acid. The presence of AL 1576 inhibited the formation of galactitol but not galactonic acid. Lens epithelial cells cultured in 3-FDGal medium formed only 3-fluoro-3-deoxy-D-galactitol. These cells developed multiple cytoplasmic vacuoles which was prevented by the aldose reductase inhibitor AL 1576.


CONCLUSIONS
The high affinity of this fluorinated sugar for aldose reductase makes this an excellent probe for investigating aldose reductase activity in dog lens tissues.
Secchi
E F
EF
Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-1850, USA.
Lizak
M J
MJ
Sato
S
S
Kador
P F
PF
D000449:Aldehyde Reductase / Q000378:metabolism; D000818:Animals; D002386:Cataract / Q000139:chemically induced*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000648:ultrastructure; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005643:Fucose / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism; D014617:Vacuoles / Q000648:ultrastructure
C120253:3-fluoro-3-deoxygalactose; D005643:Fucose; D013401:Sugar Alcohol Dehydrogenases; C095946:galactitol 2-dehydrogenase; D000449:Aldehyde Reductase