Lizak M J, Secchi E F, Lee J W, Sato S, Kubo E, Akagi Y, Kador P F
Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-1850, USA.
Invest Ophthalmol Vis Sci. 1998 Dec;39(13):2688-95.
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.
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.
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.
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.
以3-氟-3-脱氧-D-葡萄糖（3-FG）为底物，通过19F-核磁共振（19F-NMR）光谱研究犬晶状体中多元醇途径的通量。
通过19F-NMR分析监测3-FG的代谢。将犬晶状体在含有10 mM 3-FG的杜尔贝科改良伊格尔培养基中孵育。用分光光度法测定酶促还原酶和脱氢酶活性，而通过19F-NMR分析对3-FG代谢物进行分析。用针对犬肾醛糖还原酶（AR）产生的抗体，通过免疫组织化学方法在犬晶状体中定位AR。
19F-NMR光谱表明，纯化的犬晶状体AR与3-FG孵育会导致3-氟-3-脱氧-D-山梨醇（3-FS）的形成，而犬肝山梨醇脱氢酶（SDH）与3-FS孵育会导致3-氟-3-脱氧-D-果糖（3-FF）的形成。这证实了3-FG通过多元醇途径酶代谢为3-FF。AR对3-FG的亲和力（Km）比对D-葡萄糖的亲和力高约20倍，而SDH对3-FS的Km比对D-山梨醇的Km小四倍。培养的犬晶状体中的3-FG主要代谢为3-FS；然而，也会形成少量的3-FF和3-氟-3-脱氧-D-葡萄糖酸（3-FGA）。AR抑制剂AL 1576减少了3-FS的形成，SDH抑制剂CP-166,572消除了3-FF的形成。在用3-FG培养的犬晶状体上皮细胞中，仅形成3-FS。同样，当将主要含有被浅表上皮细胞污染的晶状体上皮的晶状体囊在3-FG中孵育时，仅形成3-FS。其余皮质的类似孵育主要导致3-FS和3-FGA的形成。通过分光光度活性分析和AR的免疫组织化学定位证实了这种酶分布。
数据证实通过多元醇途径的通量主要导致山梨醇积累。培养的晶状体上皮中不存在果糖和葡萄糖酸，这表明上皮细胞主要含有AR，而分化的纤维细胞也含有SDH和葡萄糖脱氢酶。
