r,-Unsaturated Aldehydes Accelerate Oxymyoglobin Oxidation C. Faustman,* ,† D. C. Liebler, ‡ T. D. McClure, ‡ and Q. Sun † Department of Animal Science, Box U-40, 3636 Horsebarn Road Ext., University of Connecticut, Storrs, Connecticut 06269-4040, and Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721 This study investigates the potential basis for enhancement of oxymyoglobin (OxyMb) oxidation by lipid oxidation products. Aldehydes known to be formed as secondary lipid oxidation products were combined with OxyMb in aqueous solution at 37 °C and pH 7.4. Metmyoglobin (MetMb) formation was greater in the presence of R,-unsaturated aldehydes than their saturated counterparts of equivalent carbon chain length. Additionally, increasing chain length from hexenal through nonenal resulted in increased MetMb formation (P < 0.05). Electrospray ionization mass spectrometry (ESI- MS) revealed that OxyMb incubated with 4-hydroxynonenal (HNE) at pH 7.4 at 37 °C yielded myoglobin molecules adducted with one to three molecules of HNE from 0.5 to 2 h of incubation, respectively. A prooxidant effect of HNE was noted at pH 7.4 but was not apparent at pH 5.6 when compared to the control (P < 0.05). This appeared to be due to rapid OxyMb autoxidation at this pH compared to pH 7.4. ESI-MS demonstrated that adduction of HNE to OxyMb occurred at pH 5.6. This research demonstrates that R,-unsaturated aldehydes accelerate OxyMb oxidation and appear to do so via covalent attachment. Keywords: Oxymyoglobin; metmyoglobin; aldehydes; hydroxynonenal INTRODUCTION The red to brown color change that occurs in fresh beef during retail display results from oxidation of ferrous oxymyoglobin (OxyMb) to ferric metmyoglobin (MetMb). The process of lipid oxidation is one of a variety of factors that influence the rate at which this occurs (Faustman and Cassens, 1990). Heme proteins are capable of binding to membranes (Szebeni et al., 1988) and fatty acids (Gotz et al., 1994), and this proximity would enhance the likelihood of interaction with products released as a consequence of lipid oxida- tion. Haurowitz et al. (1941) were among the first investigators to demonstrate that lipid oxidation prod- ucts could be deleterious toward heme proteins. OxyMb oxidation appears to be linked with that of lipids in meat (Greene, 1969; Greene et al., 1971); as concentrations of the lipid soluble antioxidant R-tocopherol are in- creased in beef, the rates of lipid oxidation and OxyMb oxidation are decreased (Faustman et al., 1989). Ad- ditionally, OxyMb stability in liposome and microsome models is improved with elevated concentrations of R-tocopherol (Yin and Faustman, 1993; Yin et al., 1994). Chan et al. (1997) attempted to better understand the potential basis for this oxidative interaction. Incubation of OxyMb in dialysis sacs (MW cutoff 500 Da) placed in solutions of fresh and oxidized liposomes revealed that thiobarbituric acid reactive substances (TBARS) were measurable within the dialysis sacs after 5 h at 30 °C. The concentration of TBARS increased with the extent of oxidation of the lipid solution in which the sacs were incubated. Historically, investigators of meat lipid oxidation have focused on the production of malonal- dehyde. However, several aldehyde products can be formed from membrane lipids (Esterbauer et al., 1982). Chan et al. (1997) utilized several aldehydes, known to be secondary lipid oxidation products of meat lipid oxidation, to demonstrate prooxidative activity toward oxymyoglobin. Of the aldehydes tested, the R,-unsatur- ated aldehydes nonenal and heptenal were found to be especially prooxidative. It has been reported that R,-unsaturated aldehydes are very reactive toward protein (Witz, 1989). Several of these are produced from fatty acids typically found in beef products (Esterbauer, 1982). Among these, 4-hydroxynonenal (HNE) has received the most atten- tion (Esterbauer et al., 1991). HNE has been reported to inactivate protein sulfhydryls (Esterbauer et al., 1991), insulin (Uchida and Stadtman, 1992), and glucose- 6-phosphate dehydrogenase (Szweda et al., 1993). It appears to increase in concentration during perfusion of cardiac tissue and increases dramatically during postischemic reperfusion injury (Blasig et al., 1995). The presence of HNE has been documented in beef and pork at concentrations of 14-150 and 1-152 nmol/g, respec- tively (Sakai et al., 1995). In our attempts to better understand the oxidative interaction between lipids and OxyMb, our working hypothesis has been that secondary lipid oxidation products are more polar than their parent compounds and could diffuse from membranes into the surrounding sarcoplasm. Many of these compounds have reactive groups that would allow them to interact with sarco- plasmic constituents including myoglobin. The objective of this study was to determine the relative reactivities of different aldehyde oxidation products and assess the ability of HNE to react with myoglobin. * Author to whom correspondence should be addressed [telephone (860) 486-2542; fax (860) 486-4375; e-mail cfaustma@ ansc1.cag.uconn.edu]. † University of Connecticut. ‡ University of Arizona. 3140 J. Agric. Food Chem. 1999, 47, 3140-3144 10.1021/jf990016c CCC: $18.00 © 1999 American Chemical Society Published on Web 07/28/1999