In vitro detection of endothelial cell damage using 2-deoxy-D-3H-glucose: comparison with chromium 51, 3H-leucine, 3H-adenine, and lactate dehydrogenase.
To develop a sensitive in vitro assay for detecting endothelial cell damage, we radiolabeled endothelial cell monolayers with tracer amounts of 2-deoxy-D-[1-3H]-glucose (3HDOG). We damaged identical cohorts of endothelial cells radiolabeled with 3HDOG or chromium 51 by exposing monolayers to toxic oxygen radicals generated by xanthine-xanthine oxidase or phorbol myristate acetate (PMA)-activated polymorphonuclear leukocytes (PMNs), a surface active agent (Triton X-100), and anti-HLA antibodies and complement. With each mechanism of injury, the 3HDOG assay detected significant (P less than 0.01) endothelial cell damage at lower concentrations of the injurious agent than the 51Cr assay. When endothelial monolayers were damaged by xanthine-xanthine oxidase or PMA-activated PMNs, efflux of 3HDOG was reduced (range 71% to 94% reduction) by superoxide dismutase and catalase, indicating that efflux of 3HDOG was mediated by toxic oxygen radicals. When monolayers were damaged with xanthine oxidase in the absence of glucose, a much lower concentration of xanthine oxidase was required to initiate efflux of 3HDOG as compared with xanthine oxidase injury in the presence of glucose. Additional studies compared the 3HDOG assay with 3H-adenine, 3H-leucine, and lactate dehydrogenase (LDH) release when endothelial cells were exposed to toxic oxygen radicals generated by PMA-activated PMNs and xanthine-xanthine oxidase. Again, the 3HDOG assay was more sensitive in detecting in vitro endothelial cell damage. We conclude that the 3HDOG assay is more sensitive than the 51Cr, 3H-adenine, 3H-leucine, or LDH release assays in detecting endothelial cell damage in vitro.
D000225:Adenine / Q000378:metabolism*; D002470:Cell Survival; D002854:Chromatography, Paper; D002857:Chromium / Q000378:metabolism*; D003167:Complement Activation; D003837:Deoxy Sugars / Q000378:metabolism*; D003847:Deoxyglucose / Q000378:metabolism*; D004727:Endothelium / Q000166:cytology*; D005260:Female; D005609:Free Radicals; D006801:Humans; D007527:Isoenzymes; D007553:Isotope Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism*; D008722:Methods; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D017830:Octoxynol; D010695:Phlorhizin / Q000494:pharmacology; D011092:Polyethylene Glycols; D011247:Pregnancy; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019820:Xanthine; D014969:Xanthine Oxidase / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D003837:Deoxy Sugars; D005609:Free Radicals; D007527:Isoenzymes; D014970:Xanthines; D002857:Chromium; D019820:Xanthine; D011092:Polyethylene Glycols; D017830:Octoxynol; D003847:Deoxyglucose; D010695:Phlorhizin; D007770:L-Lactate Dehydrogenase; D014969:Xanthine Oxidase; D007930:Leucine; D000225:Adenine; D013755:Tetradecanoylphorbol Acetate