The redox stability of myoglobin (Mb) is compromised by many factors, including lipid oxidation and its products. 4-Hydroxy-2-nonenal (HNE) is an alpha,beta-unsaturated aldehyde derived from the oxidation of omega-6 polyunsaturated fatty acids and is highly reactive and cytotoxic. Our objective was to study potential binding of HNE to Mb and determine how it affects redox stability. OxyMb (0.15 mM) was incubated with HNE (1 mM) at 4, 25, and 37 degrees C at pH 7.4 or 5.6. Samples were analyzed for MetMb formation and by Western blot analyses, LC-MS, LC-MS-MS, circular dichroism (CD), and differential scanning calorimetry (DSC). MetMb formation increased with increasing temperature and was greater at pH 5.6 than at pH 7.4 (P < 0.05). At 37 degrees C, HNE accelerated oxidation at pH 7.4 but not at pH 5.6 (P < 0.05). At both 25 and 4 degrees C, HNE accelerated oxidation at pH 7.4 and 5.6 (P < 0.05). LC-MS revealed the covalent binding of HNE to Mb at both pH values via Michael addition, while Western blot analysis indicated that HNE was bound to histidine (HIS) residues. LC-MS-MS identified six histidine residues of Mb that were readily adducted by HNE, including the proximal (HIS 93) and distal (HIS 64) histidine associated with the heme group. Secondary structure differences between control Mb and Mb incubated with HNE were not detected by CD. However, DSC revealed a decreased T(m) for Mb reacted with HNE at pH 7.4, indicating Mb tertiary structure was altered in a manner consistent with destabilization. These results suggest that HNE accelerates bovine skeletal muscle OxyMb oxidation in vitro by covalent modification at histidine residues.

The purpose of this study was to evaluate the rationale for using the velocity associated with VO2max (vVO2max) and the time that vVO2max can be sustained (tMAX) in exercise prescription. Six male runners performed a 0%-slope incremental treadmill test to determine their VO2max (61.6 +/- 9.1 ml x kg(-1) x min(-1)) and the velocity at which it was elicited (vVO2max; 271 +/- 18 m x min(-1)). Then, they performed exhaustive tests at vVO2max and at 92% of vO2max. VO2max in these tests (4.56 +/- 0.55 and 4.57 +/- 0.50 l x min(-1)) were not different from the incremental value (4.31 +/- 0.83 l x min(-1)). At vVO2max, VO2max was attained after 299 +/- 74 s and sustained for 32 +/- 41 s. At 92% of vVO2max, it took longer to attain VO2max (491 +/- 156 s), but it was sustained longer (130 +/- 66 s). Since no athlete reached VO2max in the first 60% of the runs, the results did not provide a rationale to support the recommendation that athletes train at vVO2max for a fixed percentage of tMAX when the goal in the training session is to maintain VO2max.

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