Donald P. Hollis

Detection of regional ischemia in perfused beating hearts by phosphorus nuclear magnetic resonance

Abstract Phosphorus-31 NMR spectra of a perfused, beating rabbit heart regionally ischemic following ligation of the left anterior descending coronary artery revealed two peaks arising from inorganic phosphate. During the pre-ligation period there was only a single orthophosphate signal at a position corresponding to one of the two peaks noted during regional ischemia. Since the resonance frequency of the orthophosphate peak is determined by environmental pH and since ischemia results in intracellular acidosis, the two signals in the regionally ischemic heart result from tissue orthophosphate residing at different intracellular pH values in the normal (pH 7.4) and ischemic zones (pH 6.6). To document that one signal was derived from normal tissue and the other from the ischemic area, the pH dependent shift in the inorganic phosphate resonance was followed during global ischemia following the period of regional ischemia. After 20 minutes of total ischemia, only a single peak was observed at the more acidic region, pH 6.4. We conclude that it is possible to identify regional myocardial ischemia by this entirely non-invasive and non-destructive technique. In the future it is likely that rapid and repeated ischemic zone sizing and localization will be possible using existing NMR imaging methods.

Phosphorus nuclear magnetic resonance studies of heart physiology

Abstract 31 P NMR at 72.9 MHz using a 25-mm-diameter phosphorus probe has been used to study correlations among cardiac metabolism, tissue pH, and contractile performance. In all studies, isovolumic left ventricular pressure (LVP) was measured in the paced hearts. Under these conditions, excellent spectra were collected in 5 min, on 6-g rabbit hearts. Good spectra were obtained in 30-sec intervals. Rapid sequential spectra illustrate the metabolic and pH events associated with the onset and recovery of total, global ischemia. Tissue pH was stable for the first minute, but then fell from 7.4 to 6.9 by 6 min, and progressed to a value of 6.4 after 40 min. During the first minute, LVP fell 80%. Metabolites and tissue pH recovered within 6 min of reperfusion, a time when ventricular pressure remained depressed by 50%. These results suggest that both the early fall and initial postischernic recovery of ventricular pressure may not be exclusively regulated by tissue pH, as estimated by NMR. We also used NMR to investigate the metabolic changes associated with regional ischemia. The 31 P NMR spectrum of a regionally ischemic, perfused rabbit heart showed two inorganic phosphate (P i ) peaks. Before ligation there was only a single P i signal at a position corresponding to one of the peaks noted during regional ischemia. Since the resonance frequency of the P i peak is determined by pH and since ischemia causes acidosis the two signals in the regionally ischemic heart result from P i at different intracellular pH values in the normal (pH 7.4) and ischemic zones (pH 6.4). And finally, we compared the status of KCl-arrested, ischemic rabbit hearts and non-KCl-treated hearts and correlated the 31 P NMR spectra with the ability of the heart to return to normal function following a period of ischemia. A rabbit heart was arrested by perfusing it with 30 m M KCl and was then made globally ischemic; a second heart was made ischemic without KCl arrest. After 40 min of global ischemia the KCl-arrested heart showed a near-normal level of ATP, low P i and a pH of 7.0. The nonarrested heart, on the other hand, showed low ATP, high P i , and a pH of 6.4. On reperfusion, the KCl-arrested heart recovered 100% of control function within 5 min but the control heart recovered only 70% of control function after 30 min. The 31 P NMR confirms that KCl arrest preserves ischemic myocardial metabolites and suggests that it can be used to test currently untried treatments for functional protection.

A nuclear magnetic resonance study of cobalt II alcohol dehydrogenase: Substrate analog-metal interactions☆

Abstract Two models for the active site of liver alcohol dehydrogenase (EC 1.1.1.1) have been proposed. Results of X-ray diffraction studies ( B.V. Plapp, H. Eklund, and C.-I. Branden, 1978 , J. Mol. Biol.122, 23–32) on the native enzyme indicate that substrates are directly coordinated to the active site zinc ion, while NMR studies (D. L. Sloan, J. M. Young, and A. S. Mildvan, Biochemistry14, 1998–2008) on the Co II enzyme indicate that substrates are not bound directly to the metal. It was unclear whether the basis for this difference was structural or technical. Therefore, this NMR study has been done with wellcharacterized zinc and cobalt enzymes, and to facilitate comparison with X-ray diffraction data, the substrate analogs chosen were dimethyl sulfoxide and trifluoroethanol. Binding of either analog to the zinc enzyme in the presence of the appropriate cofactor produced unique changes in the T1 and T2 relaxation rates of the 1 H and 19 F nuclei. Similar results were obtained when cobalt enzyme was used for T1 measurements, but relaxation was more rapid due to the presence of the paramagnetic ion. From these data, the distances between the analog nuclei and the catalytic site cobalt ion were calculated to be 8.9 ± 0.9 and 10.5 ± 1.2 A for the cobalt enzyme-NADH-dimethyl sulfoxide and the cobalt enzyme-NAD+-F3CCH2OH complexes, respectively. The distances are comparable and the magnitudes indicate that the functional groups are not directly coordinated to the active site cobalt ion. These values are in good agreement with those previously reported by Sloan et al. (1975) for the cobalt enzyme-NADH-isobutyramide complex, and are consistent with their model in which a metal water ligand forms a bridge between the substrate and the metal. Therefore, there must be a structural basis for the differences observed in magnetic resonance versus X-ray diffraction studies.

Binding of metal ions to apoalkaline phosphatase from E. Coli: Effect of ionic radius☆

Abstract Binding of metal ions to E. Coli apoalkaline phosphatase causes 1) chromophoric changes in tyrosine absorption, 2) changes in enzymatic activity and 3) the release of protons from the enzyme. Investigation of these effects for a selection of metal ions from the Group 11A, Group 11B and transition series revealed that only those ions having crystal ionic radii in the range of 0.72 – 0.99A are able to produce changes in these three properties. The hydrated ionic radii, which are in the range of 4.0 – 4.5A for the ions examined do not correlate well with the ability of the ion to affect the three properties studied here. The size requirement therefore would seem not to apply to the initial binding step which undoubtedly involves the hydrated ion. Rather, the size requirement reflects the size range of a binding site generated by the folding of the protein around the metal ion with concomitant displacement of water molecules from the coordination sphere of the metal.