Lack of Inhibition of Intestinal Heme Oxygenase by Antibiotics and Tin-Protoporphyrin

Abstract

A B S T R A a . W e assessed the in vivo and in vitro effects of antibiotics and tin-protoporphyrin (TP) on intestinal heme oxygenase (HO) activity using a gas chromatographic assay. This method measures the carbon monoxide produced from heme in the presence of NADPH. After in vivo administration of kanamycin (10 mg/kg body weight), ampicillin (200 mg/kg body weight) or neomycin (60 mg/ kg body weight) with or without T P (65 pmol/kg body weight) to suckling rats, no significant difference in HO activity along the small intestine was observed. I n vitro exposure of adult rat intestinal preparations to the antibiotics showed no significant decrease in H O activity between control and experimental tissue preparations. A concentration-dependent stimulatory effect of neomycin was observed. Subcutaneous administration of T P (25 pmol/kg body weight) to adult male Wistar rats revealed no significant inhibition of the intestine. However, in vitro addition of T P (12.5 pM) to the control tissue preparations of adult Wistar rats revealed highly significant inhibition in liver and spleen when compared to the unexposed control tissues. In contrast, when T P was added to control intestinal preparations no inhibition was observed. These findings suggest that suckling rat intestinal heme oxygenase is not inhibited by in vivo treatment with high concentrations of kanamycin, ampicillin, or neomycin. Furthermore, these antibiotics are not in vitro inhibitors of adult rat intestinal HO. Finally, adult rat intestinal H O is not inhibited either in vivo or in vitro by a concentration of TP that significantly inhibits liver and spleen activity. (Pediatr Res 23: 50-53, 1988) Abbreviations CO, carbon monoxide TP, tin-protoporphyrin HO, heme oxygenase VeCO, excretion rate of C O TP, a synthetic heme analog, has been reported to be a competitive inhibitor of HO (1-3). This rate-limiting enzyme in the heme catabolic pathway (4) catalyzes the equimolar formation of bilirubin and CO. In vivo studies with TP have demonstrated the drug's effectiveness in lowering serum bilirubin levels in rats ( 3 , in mice with severe hemolytic anemia (6) in rhesus monkeys (7), and in man (8). Preliminary studies in our laboratory suggested that a single dose of TP has no suppressive effect on the excretion rate of CO (VeCO) in suckling rats (9, 10) unless

In Vitro Inhibition of Adult Rat Intestinal Heme Oxygenase by Metalloporphyrins

“…This phenomenon is less prominent with ZnPP, which accounts most likely for ability of ZnPP to inhibit intestinal HO at light fluxes capable of overcoming inhibition by SnPP. It is not possible to estimate, in retrospect, the light quality and intensity encountered during the previous studies (18,20,21), but our results indicate that the previously observed lack of inhibition of intestinal HO with SnPP could be explained by the fact that HO activity determinations were performed under ambient light (a mixture of indirect summer daylight and cool white fluorescent light ranging in radiant flux from 0.5 to approximately 5 pW/cm2/nm) in clear glass vials. Current activity measurements are performed in amber vials in the dark.…”

“…In fact, a higher dose of ZnPP (100 pM) appeared to inhibit intestinal HO (21). Considering our results with tin protoporphyrin (18,20,21), we questioned this finding and further queried whether conditions peculiar to intestinal tissue itself or to our experimental procedure could have influenced the results. Our findings suggest that both possibilities may have played a role and show that all four metalloporphyrins tested can significantly inhibit intestinal HO activity (as measured by CO generation) in vitro, at concentrations as low as 1 pM, under strictly light-limited conditions.…”

“…Inhibition of intestinal HO may be of clinical importance, because heme reaching the intestine can potentially be degraded by intestinal tissue HO (1 5-18) to CO and bilirubin, where the latter may enter the body via the enterohepatic circulation in the neonate (1 9). Previously we did not observe significant inhibition of intestinal HO in vivo or in vitro at SnPP concentrations (40 pM) which inhibited hepatic and splenic HO (18,(20)(21). We could not explain this lack of inhibition, but considered the existence of an isoform of HO (22), specific to the intestine, which is relatively less inhibitable by SnPP or some mechanism (natural or experimental) by which access of SnPP to HO is limited or by which extraneous CO is generated.…”

The in vitro and in vivo Inhibition of Intestinal Heme Oxygenase by Tin-Protoporphyrin

“…These results contrast with those in the recent report of Hintz et al [ 18] in which no inhibition of intestinal heme oxygenase ac tivity, as determined by carbon monoxide (CO) production in the 13,000-# superna tant, was identified either in vitro or in vivo after SnPP administration at the doses used in this study [ 18]. The disparity between the findings in the previous report [18] and our present results may be due, among other considerations, to the fact that we used mi crosomes in our studies together with exper imental conditions as described by Stohs et al [14], rather than a 13,000-# supernatant, thereby protecting the integrity of the subcel lular fractions prepared from this tissue.…”

“…This dose was selected to match that pre viously used by other investigators [18], Fol lowing oral treatment, heme oxygenase ac tivity was rapidly (within 3 h) inhibited (50% of controls), but the levels returned to normal within 16-24 h after treatment. Fol lowing parenteral treatment, enzyme activity was inhibited to an even greater extent (65% inhibition), and these markedly decreased levels of enzyme activity persisted for at least 48 h after a single dose of the metalloporphyrin.…”