Treatment with p-hydroxyphenylpyruvate in the absence of resuscitation extends survival in rats subjected to lethal blood loss

Introduction

We tested the hypothesis that treatment with an enone-containing compound, p-hydroxyphenylpyruvate (pHPP), the first intermediate in the tyrosine catabolism pathway, could ameliorate the survival time in rats subjected to profound hemorrhagic shock (HS) in the absence of resuscitation. We based this hypothesis on the knowledge that ethyl pyruvate (EP) and related enone-containing compounds are protective in numerous in vitro and in vivo models of oxidant-mediated and cytokine-mediated cellular and organ injury.

Methods

We compared the anti-inflammatory effects of EP and pHPP using U373 cells exposed to IL-1β and RAW 264.7 cells exposed to lipopolysaccharide. Volume-controlled HS was initiated by withdrawing 21.7 ml blood per kg body weight over 20 minutes, followed by an additional 14 ml/kg over 40 minutes. A continuous infusion was delivered during the final 20 minutes of hemorrhage. Groups of six rats randomly received: (1) 2.8 ml saline per kg or 4% of total estimated blood volume (EBV); (2) 2.8 ml/kg of 0.25 nM pHPP; (3) 1.4 ml/kg of 0.50 nM pHPP; (4) 1.4 ml/kg (2% EBV) of 0.25 nM homogentisic acid (HGA); (5) 1 g dichloroacetic acid (DCA) in 2.8 ml/kg; or (6) DCA plus 0.25 nM pHPP in 2.8 ml/kg.

Results

Cell culture studies revealed that pHPP blocked expression of CD54 in U373 cells and blocked release of NO and decreased induction of inducible nitric oxide synthase mRNA in RAW cells at lower concentrations compared with EP. We therefore tested its efficacy in a profound hemorrhagic shock model. Rats in both groups receiving pHPP survived significantly longer (P < 0.001) than rats receiving vehicle alone. All rats in the vehicle group were dead by 140 minutes. Five out of 12 animals treated with pHPP survived longer than 4 hours and one rat survived for 455 minutes. Prompted by the recognition that pHPP is an endogenous metabolite converted to HGA in the tyrosine catabolism pathway, we sought to determine whether treatment with HGA could extend survival in the same HS model. The percentage of treated rats that survived until 5 hours in groups 2 or 4 was 33.3%. No statistical significance was observed among rats in groups 2, 3, 4. In order to obtain additional information regarding the mechanism responsible for the protective effects, we administered an inhibitor of the tyrosine catabolism, dichloroacetic acid alone or in addition to pHPP. Accordingly, the data showed that all rats in groups 5 or 6 died within 4 hours.

Conclusion

These findings show that administration of a nanomolar dose of pHPP markedly improves survival following lethal HS. This improvement is very probably related to a direct pharmacologic effect of pHPP on inflammation that is shared with EP and together with pHPP acting as an anaerobic carbon source. Infusion of drug in only 2% of the EBV strongly suggests that these effects are not related to resuscitation.