Stephen J. Kopp

P‐31 Nuclear Magnetic Resonance Analysis of Brain: The Perchloric Acid Extract Spectrum

Abstract: Perchloric acid (PCA) extracts were prepared from liquid‐N2‐frozen guinea pig brains and their organophosphate profiles examined by P‐31 nuclear magnetic resonance (NMR) spectroscopy. Thirty‐two phosphorus‐containing brain metabolites were characterized and quantitated. A distinctive feature of brain tissue metabolism relative to that of other tissues probed by P‐31 NMR is its pronounced ribose 5‐phosphate content. Comparison of brain metabolite levels following control or sublethal cyanide treatment (4 mg/kg) revealed specific cyanide‐induced changes in brain metabolism. Brains from cyanidetreated animals were characterized by a reduced phosphocreatine content and elevated α‐glycerolphosphate and inorganic orthophosphate contents relative to control. P‐31 NMR spectra of brain PCA extracts at pH 7.2 were also obtained under conditions that approximate those used for in vivo and intact tissue in vitro P‐31 spectroscopic analyses. The spectra reveal nine separate resonance bands corresponding to: sugar phosphates, principally ribose 5‐phosphate (3.7δ); inorganic orthophosphate (2.2δ); glycerol 3‐phosphorylethanolamine (0.3δ); glycerol 3‐phosphorylcholine (−0.1δ); phosphocreatine (−3.2δ); adenosine tri‐(β‐ATP) and di‐(β‐ADP) phosphate ionized end‐groups (−6.2δ); α‐ATP, α‐ADP, and nicotinamide adenine dinucleotides esterified end‐groups (−11.1δ); uridine diphosphohexose, hexose esterified end‐groups (−13.0δ); and β‐ATP ionized middle group (−21.6δ). Knowledge of the phosphatic molecules that contribute resonances to the brain P‐31 NMR spectrum as well as understanding their magnetic resonance properties is essential for the interpretation of in vivo brain spectroscopic data as well as brain extract data, since these same compounds contribute to the intact brain P‐31 spectrum.

Chemical characterization of a prominent phosphomonoester resonance from mammalian brain. 31P and 1H NMR analysis at 4.7 and 14.1 tesla

A prominent 31P NMR resonance at 3.84 ppm in mammalian brain has been identified as ethanolamine phosphate. The identification was based on 1H and 31P NMR findings (including pH titrations) at 4.7 and 14.1 T, as well as thin-layer chromatography studies. We previously incorrectly assigned the 3.84 ppm resonance to ribose-5-phosphate. The incorrect assignment occurred because the two compounds have very similar 31P chemical shifts, and because we did not carefully consider the effects of counter ions and ionic strengths when interpreting the 31P chemical shifts. In separate preliminary studies we have demonstrated ethanolamine phosphate to be high in immature developing brain and in the degenerating brain of Alzheimers and Huntingtons disease patients. Ethanolamine phosphate may therefore serve as a sensitive marker of membrane phospholipid turnover for both in vitro and in vivo 31P NMR studies.

P-31 nuclear magnetic resonance analysis of brain. II: Effects of oxygen deprivation on isolated perfused and nonperfused rat brain

Phosphatic metabolite (perchloric acid extractable) concentrations of cerebral tissues were analyzed by phosphorus‐31 nuclear magnetic resonance (P‐31 NMR) spectfoscopy following external perfusion of the isolated rat brain (30 min or 60 min) under the following conditions: (a) constant perfusion pressure with either fluorocarbon‐or erythrocyte‐based medium, and (b) constant perfusate flow rate (3 ml/min) with the erythrocyte‐based medium. Metabolite concentrations of control perfused brains were compared with those in nonperfused controls to provide a basis for detecting any qualitative or quantitative changes in cerebral metabolite composition. Metabolic responses of perfused brains to ischemia (incomplete ischemia, 83% reduction in flow for 10 min; transient complete ischemia for 1.5 or 2 min) were evaluated immediately after the ischemic episode and at selected time points during reperfusion (3 and 15 min). Alterations in cerebral metabolite levels induced by hypoxia were analyzed using a nonperfused rat brain model. Irrespective of the perfusion method employed, the phosphatic metabolites of control perfused rat brains were identical quantitatively to those of the nonperfused controls. Cerebral ischemia resulted in significantly increased levels of ADP, AMP + IMP, Pi, fructose 1,6‐diphosphate, and glycerol 3‐phosphate (global ischemia only), whereas ATP and phosphocreatine (PCr) levels declined significantly. The magnitude of these changes varied with the severity of the ischemia; however, following 15 min of control reperfusion metabolite levels had reverted to preischemic values. Significant perturbations in tissue phosphoethanolamine (3.848 resonance) content were evident at various time points during ischemia and postischemic recovery, which varied according to the perfusion conditions. In contrast to the changes observed in response to ischemia, hypoxia affected only cerebral high‐energy phosphate levels. ATP and PCr levels were reduced, while a concomitant, essentially equimolar, increase in Pi and ADP was observed. The present studies indicate that in terms of phosphatic metabolites, the control equilibrated isolated perfused rat brain is quantitatively and qualitatively indistinguishable from the non‐perfused rat brain in vivo regardless of the perfusion conditions (constant flow versus constant pressure). The metabolic responses to ischemia and hypoxia, as measured by P‐31 NMR, were consistent with the pattern of changes reported elsewhere. Overall, P‐31 NMR spectroscopic evaluation of the intact rat brain provides a potential experimental context for dynamic measures of cerebral metabolism under exogenously controlled conditions. The results reported herein from brain PCA extracts contribute a spectroscopic reference for understanding and interpreting the metabolic information contained in the P‐31 profiles of the intact brain.

P-31 nuclear magnetic resonance analysis of brain: Normoxic and anoxic brain slices

Perchloric acid extracts were prepared from liquid-N2-frozen gerbil and guinea pig brain slices studied under one of three conditions: O2-incubated, N2-incubated or O2-incubated recovery following N2 incubation. Mole percentages of the various phosphatic components contained in the extracts were determined by phosphorus-31 nuclear magnetic resonance spectroscopy. The brain slice extract spectrum revealed a previously unreported group of brain phosphodiesters at −0.73 δ relative to 85% orthophosphoric acid Although the phosphatic profiles from O2-incubated slices fromgerbils and guinea pigs revealed only minor species variations, which differed quantitatively rather than qualitatively, species-specific differences were made readily apparent and amplified by incubating brain slices under oxygen-deficient conditions. Despite these differences which were most prevalent during the recovery phase, the overall metabolic changes described herein in response to N2-incubation were in accord with the results obtained by other analytical techniques. Inorganic orthophosphate (2.63 δ) was increased, while nucleoside (principally, adenosine) triphosphate (α-, −10.92 δ, β-, −21.45 δ, and γ-, −5.80 δ) and phosphocreatine (−3.12 δ) levels were decreased in response to N2 incubation. In addition, inosine monophosphate (3.78 δ) was increased and the levels of a partially characterized acid-labile phosphate (0.85 δ, guinea pig) were decreased upon N2 incubation. Phosphoglyceride metabolism also appeared to be altered by oxygen deprivation (gerbil). These latter findings provide additional information concerning the metabolic responses of cerebral tissue to oxygen deficient conditions.

Phosphorus nuclear magnetic resonance and ocular metabolism

Phosphorus (31P) nuclear magnetic resonance (NMR) represents a noninvasive technique for the assessment of ocular metabolism. The measurement of a spectrum of phosphorus-containing metabolites (e.g., phosphorylated sugars and ATP), including a number of heretofore uncharacterized metabolites, can be made with a single analysis. In addition to quantitating phosphatic metabolites, 31P NMR can be employed to monitor (1) the rate of metabolic change in a specific biochemical reaction via T1 and T2 relaxation times, and (2) the rate of change in the concentration of a particular metabolite. Several calculations indicating tissue energy status (health) can be made using quantitative spectroscopic information including: the phosphorylation potential, the energy charge of the adenylate system, and the 31P spectral modulus. Tissue pH can be determined as a function of shift in 31P NMR signals. 31P NMR techniques have both research and diagnostic applications in ophthalmology since potentially it provides a noninvasive method to analyze ocular tissues metabolically and detect subtle biochemical changes that precede overt manifestation of disease states. Such detection may allow for early and more effective therapeutic intervention of disease. Furthermore, the noninvasive quality of NMR spectroscopy will permit continual evaluation of therapy.

Cardiac physiologic and tissue metabolic changes following chronic low-level cadmium and cadmium plus lead ingestion in the rat

Female Long-Evans hooded rats received Schroeders rye-based diet and 0 or 1 microgram/ml cadmium, or cadmium plus lead in mineral fortified drinking water from weaning to 18 months. The heavy metal-fed rats were normal with respect to control, including growth rates and final body weights. Rats receiving added cadmium and cadmium plus lead in the diet were characterized by a persistent hypertension which was evident after 2 months. Cardiac conduction system excitability was depressed preferentially in cadmium-(atrioventricular nodal region) and cadmium plus lead-(His-Purkinje system) fed rats. Although heart rates were comparable to control, myocardial contractile activity (peak active tension and dT/dt) was significantly decreased in intact perfused heart preparations from both heavy metal-treated groups. In conjunction with the observed physiologic changes, various tissue-specific metabolic alterations were detected in heart, kidney, and liver. Generally, prolonged heavy-metal ingestion at these levels resulted in impaired energy metabolism (e.g., decreased ATP, PCr; increased Pj, ADP concentrations) and altered essential mineral composition (e.g., calcium, magnesium, zinc, and to a lesser extent, sodium and potassium; copper levels were unaffected) that varied in severity according to the tissue. The addition of lead to the cadmium diet had little additive effect on the cardiovascular system; however, renal and hepatic tissues did exhibit apparent additive effects further suggesting that cadmium and lead actions and interactions may be tissue dependent. These experimental findings and the biologic inferences derived are consonant with the hypothesis that chronic, life-long cadmium exposure approximating environmental levels may have significant adverse effects on mammalian systems, that include effects on cardiovascular tissues.

Cardiotoxicity of lead at various perfusate calcium concentrations: Functional and metabolic responses of the perfused rat heart

Equilibrated rat hearts were perfused for 60 min with a standard crystalloid buffer containing either 0.9, 1.8, 3.5, or 5.0 mM Ca with or without added lead (0.3 and 30 microM). Contractile tension (T), rate of tension development (dT/dt), electrocardiographic (EKG), His bundle electrographic (HBE) indices, heart rate (HR), preejection period (PEP), and coronary flow rate (CFR) were recorded as a function of perfusion time. Endpoint analyses of myocardial phosphatic metabolites were performed on heart perchloric acid extracts by standard phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopic techniques. The contractile activity and glycerol 3-phosphorylcholine content of the myocardium were found to vary directly as a function of the perfusate calcium concentration; however, except for a prolonged atrioventricular-His bundle conduction time detected in hearts treated with 0.9 mM Ca, the variable perfusate calcium concentrations were devoid of any other significant physiologic and metabolic effects. In contrast, perfusion of control equilibrated hearts with 30 microM lead significantly attenuated the positive, and exacerbated the negative, inotropic responses to elevated, and low perfusate calcium concentrations, respectively. Moreover, a secondary, time-dependent decline in myocardial contractile strength was also observed in response to this lead concentration, which was progressively more pronounced with each increment in the perfusate calcium concentration. The preejection period of ventricular systole was also prolonged in response to 30 microM lead; however, this effect was less pronounced at higher perfusate calcium concentrations. Hearts perfused with 30 microM lead were also characterized by significant prolongation in atrioventricular node and His bundle conduction time, reduced coronary flow rate, and decreased heart rate, irrespective of the perfusate calcium concentration. Hearts treated with 0.3 microM lead exhibited functional properties that were diminished, but still comparable to control hearts. Analysis of myocardial phosphatic metabolite amounts following 60 min of perfusion revealed a significant lead-induced reduction in the energy status of the heart. The combination of 5.0 mM calcium with either 0.3 or 30 microM lead resulted in significant disturbances in phosphoglyceride, glycolytic, and high-energy phosphate pathways. These findings suggest that the cardiotoxic actions of lead are linked to complex mechanisms that are partially related to an interference with calcium-dependent cellular processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Altered metabolism and function of rat heart following chronic low level cadmium/lead feeding

Abstract Metabolic and functional (heart only) effects of long-term (15 months), low level (5 parts/10 6 in drinking water) cadmium and/or lead ingestion on myocardial, hepatic and renal tissues were assessed. Myocardial peak active tension was lower and tissue heavy metal levels were elevated significantly in cadmium and/or lead-exposed rats. The ATP ( P P P P P

The influence of chronic low-level cadmium and/or lead feeding on myocardial contractility related to phosphorylation of cardiac myofibrillar proteins

Rats fed 5 ppm cadmium and/or lead for 15 months duration were studied. Growth and food and water intakes were comparable to control. Depressed myocardial contractility and reduced positive inotropic responsiveness to isoproterenol (7 × 10−7 m) were detected in hearts from cadmium- and/or lead-fed groups. Hearts isolated from cadmium-fed rats generated significantly lower peak systolic tensions in response to isoproterenol than did control hearts, but in both cases the peak tension was well sustained during a 50-min isoproterenol perfusion. Hearts from both groups of lead-exposed rats also produced lower levels of tension than controls; however, the peak systolic tension decreased progressively during the isoproterenol challenge. Since the positive chronotropic responses to the β-adrenergic stimulation were comparable among the groups studied, altered β-receptor function or β-receptor desensitization were not suspected as contributory factors responsible for the altered positive inotropic responses. In contrast these altered contractile events were associated with depressed phosphorylation of the cardiac myofibrillar proteins: the myosin light chain-2 (all heavy-metal-treated groups) and the troponin inhibitory subunit (lead + cadmium group only). The phosphorylation process involving these cardiac myofibrillar proteins purportedly contributes to the regulation of cardiac contractility; consequently, the depressed phosphorylation of primarily the myosin light chain-2 may be implicated as a biochemical defect which contributes to the measured physiological dysfunction of hearts from chronic lead- and/or cadmium-exposed rats.

Simultaneous recording of His bundle electrogram, electrocardiogram, and systolic tension from intact modified Langendorff rat heart preparations. I: effects of perfusion time, cadmium, and lead.

Abstract A method for simultaneously recording His bundle electrograms (HBE), electrocardiograms (ECG), and systolic tensions from intact perfused rat heart preparations is described. This technique was developed for use in ionic, pharmacological, and toxicological studies to determine the effects of chemical agents on myocardial excitability and contractility under controlled conditions. The HBE recording technique using intact isolated perfused rat hearts represents a new method which may be used to study cardiac conduction disturbances and localize them to discrete sites within the cardiac conduction system. Corresponding changes in myocardial contractility also can be determined and correlated with observed electrophysiological alterations. A temporal control study of electrical and mechanical events of these hearts demonstrated the stability of hearts perfused with this method during a 3-hr postequilibrium period. The effects of cadmium (3 × 10 −2 m m Cd 2+ ) and lead (3 × 10 −2 m m Pb 2+ ) on myocardial systolic tension and excitability were compared. Both ions significantly depressed systolic tension and prolonged the PR interval of the ECG. Similarly, Cd 2+ and Pb 2+ significantly increased A-H and H-V conduction times of the HBE; however, conduction blocks occurred within the A-H interval (atrioventricular nodal depression) in Cd 2+ -treated hearts and within the H-V interval (His-Purkinje cell depression) of Pb 2+ -exposed hearts. By combining electrocardiographic, His bundle electrographic and peak systolic contractility analyses, this technique provides the necessary information for evaluating the cardiodepressive effects of toxicological and pharmacological agents, such as cadmium and lead, while localizing their sites of action to specific areas within the cardiac conduction system.