Effect of intravenous administration of hydroxyethyl-starch-deferoxamine on oxygen-derived free radical generation in cancellous bone specimens obtained from dogs

Daniel D. Lewis From the Department of Veterinary Clinical Sciences, School of Veterinary Medicine (Lewis, Hosgood), and the Department of Chemistry (Church), Louisiana State University, Baton Rouge, LA 70803.

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Daniel F. Church From the Department of Veterinary Clinical Sciences, School of Veterinary Medicine (Lewis, Hosgood), and the Department of Chemistry (Church), Louisiana State University, Baton Rouge, LA 70803.

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Giselle Hosgood From the Department of Veterinary Clinical Sciences, School of Veterinary Medicine (Lewis, Hosgood), and the Department of Chemistry (Church), Louisiana State University, Baton Rouge, LA 70803.

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 BVSc, MS

Summary

The ability of iv administered hydroxyethyl-starch-deferoxamine to attenuate radical production in freshly procured cancellous bone specimens was investigated, using spin-trapping and electron spin resonance (esr) techniques. A core cancellous bone specimen 10 mm long and 5.6 mm in diameter was obtained, using aseptic technique, from the proximal portion of the humerus of 30 adult mixed-breed dogs. After procurement of the initial bone specimen, 10 dogs received a 10 % solution of hydroxyethyl-starch-deferoxamine in 0.9% NaCl (50 mg/kg of body weight, iv), 10 dogs received an equivalent volume (5 ml/kg, iv) of a 10% solution of hydroxyethyl-starch in 0.9 % NaCl, and 10 dogs received 0.9 % saline solution (5 ml/kg, iv). A second core cancellous bone specimen was obtained from the contralateral humerus of each dog 45 minutes after treatment. All specimens were individually incubated in the spin trap α-phenyl-N-tert-butylnitrone in Eagle's minimum essential medium, at 26 C for 45 minutes, then were frozen at -20 C until they were prepared for analysis by esr spectroscopy. Each specimen was thawed, homogenized, and extracted in a low-dielectric organic solvent prior to obtaining an esr spectrum, which was analyzed for hyperfine splitting constants for radical identification. Each first-derivative spectrum was digitally double-integrated to obtain an area; these areas were used to compare intensities of the spin adducts. Difference in the area obtained before and after treatment for each dog was expressed as a ratio of that dog's pretreatment area ([pretreatment-posttreatment ]/pretreatment). The calculated ratios for saline-, hydroxyethyl-starch-, and hydroxyethyl-starch-deferoxamine-treated dogs were compared, using a Kruskal-Wallis (KW) nonparametric test for multiple comparisons of ranked data. Significance was determined at P ≤ 0.05. Ad hoc comparisons were performed, using the KW procedure for individual comparisons, with a set at 0.05.

The mean ± sd and median ratio for each of the treatment groups were: saline-treated dogs, 0.005 ± 0.40 and 0.045; hydroxyethyl-starch-treated dogs, -0.063 0.27 and -0.025; hydroxyethyl-starch-deferoxamine-treated dogs, 0.261 ± 0.278 and 0.335, respectively. There was a significant (P < 0.01, KW) difference in the ratios between treatment groups. Ratios for hydroxyethyl-starch-deferoxamine-treated dogs were significantly (P < 0.05, KW) higher than that for hydroxyethyl-starch-treated dogs but not for saline-treated dogs. The ratios for saline- and hydroxyethyl-starch-treated dogs were not significantly different. We could not associate significant attenuation of radical generation in freshly harvested core cancellous bone specimens with iv administration of hydroxyethyl-starch-deferoxamine. The potential for unconjugated hydroxyethyl-starch to function as an oxidant must considered.

Summary

The ability of iv administered hydroxyethyl-starch-deferoxamine to attenuate radical production in freshly procured cancellous bone specimens was investigated, using spin-trapping and electron spin resonance (esr) techniques. A core cancellous bone specimen 10 mm long and 5.6 mm in diameter was obtained, using aseptic technique, from the proximal portion of the humerus of 30 adult mixed-breed dogs. After procurement of the initial bone specimen, 10 dogs received a 10 % solution of hydroxyethyl-starch-deferoxamine in 0.9% NaCl (50 mg/kg of body weight, iv), 10 dogs received an equivalent volume (5 ml/kg, iv) of a 10% solution of hydroxyethyl-starch in 0.9 % NaCl, and 10 dogs received 0.9 % saline solution (5 ml/kg, iv). A second core cancellous bone specimen was obtained from the contralateral humerus of each dog 45 minutes after treatment. All specimens were individually incubated in the spin trap α-phenyl-N-tert-butylnitrone in Eagle's minimum essential medium, at 26 C for 45 minutes, then were frozen at -20 C until they were prepared for analysis by esr spectroscopy. Each specimen was thawed, homogenized, and extracted in a low-dielectric organic solvent prior to obtaining an esr spectrum, which was analyzed for hyperfine splitting constants for radical identification. Each first-derivative spectrum was digitally double-integrated to obtain an area; these areas were used to compare intensities of the spin adducts. Difference in the area obtained before and after treatment for each dog was expressed as a ratio of that dog's pretreatment area ([pretreatment-posttreatment ]/pretreatment). The calculated ratios for saline-, hydroxyethyl-starch-, and hydroxyethyl-starch-deferoxamine-treated dogs were compared, using a Kruskal-Wallis (KW) nonparametric test for multiple comparisons of ranked data. Significance was determined at P ≤ 0.05. Ad hoc comparisons were performed, using the KW procedure for individual comparisons, with a set at 0.05.

The mean ± sd and median ratio for each of the treatment groups were: saline-treated dogs, 0.005 ± 0.40 and 0.045; hydroxyethyl-starch-treated dogs, -0.063 0.27 and -0.025; hydroxyethyl-starch-deferoxamine-treated dogs, 0.261 ± 0.278 and 0.335, respectively. There was a significant (P < 0.01, KW) difference in the ratios between treatment groups. Ratios for hydroxyethyl-starch-deferoxamine-treated dogs were significantly (P < 0.05, KW) higher than that for hydroxyethyl-starch-treated dogs but not for saline-treated dogs. The ratios for saline- and hydroxyethyl-starch-treated dogs were not significantly different. We could not associate significant attenuation of radical generation in freshly harvested core cancellous bone specimens with iv administration of hydroxyethyl-starch-deferoxamine. The potential for unconjugated hydroxyethyl-starch to function as an oxidant must considered.

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