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

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 alpha-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 dogs 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 less than or equal to 0.05. Ad hoc comparisons were performed, using the KW procedure for individual comparisons, with alpha 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.