Effect of glucose on the distribution of iodine-123-IHA-16 between esterification and oxidation in canine myocardium

To evaluate the effect of glucose perfusion on the myocardial metabolism of [123I]-16-iodo-9-hexadecenoic acid (IHA), the latter was injected intravenously into six fasting dogs perfused with a solution lacking glucose (controls) and seven fasting dogs perfused with glucose and insulin. The distribution of myocardial 123I among iodides, free IHA, and esterified IHA was measured in myocardial biopsy specimens. The increase in esterification and decrease in oxidation of IHA due to glucose were quantified using a compartmental mathematical model of myocardial IHA metabolism. Subsequently, in six control and six glucose-perfused dogs, cardiac radioactivity was measured with a scintillation camera for 1 hr following i.v. injection of IHA. Four different methods were used to analyze the myocardial time-activity curves and to calculate the distribution of IHA between oxidation and esterification. Results comparable to those provided by analysis of biopsy specimens can be obtained by considering the curve to be the sum of an exponential and a constant, or by analyzing it with a compartmental mathematical model.     

Effect of myocardial perfusion and metabolic interventions on cardiac kinetics of phenylpentadecanoic acid (IPPA) I 123

The effect of regional myocardial perfusion and flow-independent adrenergic stimulation, as well as lactatemediated inhibition of cardiac lipolysis, on cardiac IPPA uptake and metabolism was examined in canine hearts (flow studies) and in the isolated perfused Langendorff rat heart (metabolic interventions). In both normal and ischaemic myocardium, local perfusion is a major determinant of cardiac IPPA uptake. In pacing-induced hyperaemia, the strict flow-dependence of cardiac IPPA uptake is not preserved. Adrenergic stimulation raises the rate of oxidation of both palmitic acid ¹⁴C and IPPA. This change is reflected by increased metabolite production released into the perfusate and radioactivity clearance recorded externally. Lactate in high concentrations exerts the opposite effect on cardiac free fatty acid oxidation. IPPA is stored in this condition preferentially in tissue phospholipids and triglycerides.

     

Omega-halofatty acids: a probe for mitochondrial membrane integrity. In vitro investigations in normal and ischaemic myocardium

Long-chain omega-halofatty acids, especially omega-123I-iodoheptadecanoic acid (IHA), are widely used clinically as radiopharmaceuticals for functional heart imaging. The metabolic interpretation of the various elimination rates, however, remains in dispute. It has been previously shown (Kloster and St?cklin 1982) that in isolated perfused guinea-pig hearts halide diffusion from the mitochondrion to the blood is the rate-determining step of IHA pharmacokinetics in normal myocardium. We have now extended these in vitro experiments to normal and globally ischaemic isolated perfused rabbit hearts. Again, in normal hearts a single phase iodide elimination half-time (14.3 +/- 2.1 min) was observed. In hearts made globally ischaemic for 90 min, the iodide elimination was biphasic with a first fast phase (T 1/2 = 3.8 +/- 0.49 min) and a late slow phase (T 1/2 = 60.5 +/- 14.0 min). The first fast phase is attributed to iodide ion released by residual beta-oxidation (more rapid than in normal hearts due to damaged membranes in ischaemia), while the late slow phase is explained by beta-oxidation of IHA slowly released by hydrolysis of intracellular lipid stores. These data were compared with published data from investigations in patients which seem to support our interpretation.     

Myocardial metabolism of radioiodinated methyl-branched fatty acids

Methylated fatty acids labeled with radioactive iodine have been proposed as a means of studying regional myocardial uptake of fatty acids in man. To investigate the methylated fatty acid that is best adapted for an assessment of uptake, we have studied the influence of the number and the position of the methyl groups of IFA intracellular metabolism; 16-iodo-2-methyl-hexadecanoic (mono-alpha), 16-iodo-2,2-methyl hexadecanoic (di-alpha), 16-iodo-3-methyl-hexadecanoic (mono-beta), and 16-iodo-3,3-methyl-hexadecanoic (di-beta) acids were injected into the coronary arteries of isolated rat hearts. Intracellular analysis shows that the degradation of mono-alpha was always lower than that of IHA and the storage was always much higher. The differences between mono-beta and IHA were similar to those observed with mono-alpha, but were much more pronounced. With the two dimethylated IFAs there was an inhibition of both oxidation and esterification which led to an accumulation of free FAs in myocardial cells. In conclusion, mono-beta, di-alpha, and di-beta are potentially suitable for studying the cellular uptake of IFA since all of them, and particularly the dimethylated IFAs, have a low oxidation rate.     

ω-Halofatty acids: A probe for mitochondrial membrane integrity

Long-chain -halofatty acids, especially -¹²³I-iodoheptadecanoic acid (IHA), are widely used clinically as radiopharmaceuticals for functional heart imaging. The metabolic interpretation of the various elimination rates, however, remains in dispute. It has been previously shown (Kloster and Stöcklin 1982) that in isolated perfused guinea-pig hearts halide diffusion from the mitochondrion to the blood is the rate-determining step of IHA pharmacokinetics in normal myocardium. We have now extended these in vitro experiments to normal and globally ischaemic isolated perfused rabbit hearts. Again, in normal hearts a single phase iodide elimination half-time (14.3±2.1 min) was observed. In hearts made globally ischaemic for 90 min, the iodide elimination was biphasic with a first fast phase (T_1/2=3.8±0.49 min) and a late slow phase (T _1/2=60.5±14.0 min). The first fast phase is attributed to iodide ion released by residual -oxidation (more rapid than in normal hearts due to damaged membranes in ischaemia), while the late slow phase is explained by -oxidation of IHA slowly released by hydrolysis of intracellular lipid stores. These data were compared with published data from investigations in patients which seem to support our interpretation.     

Measurement of myocardial fatty acid metabolism: kinetics of iodine-123 heptadecanoic acid in normal dog hearts

To define the potential of iodine-123 heptadecanoic acid (IHA) for the noninvasive assessment of myocardial fatty acid metabolism with gamma camera imaging, the influence of myocardial oxygen consumption (MVO2) and blood flow (MBF) on extraction and half-times of IHA were investigated in dogs. Following IHA injection into the left circumflex coronary artery, extraction fraction and half-times were derived from the peak and slope of the IHA time activity curve, which consisted of a vascular, early, and late phase. Single-pass extraction fraction of IHA averaged 0.53 +/- 0.11 s.d. at control and was not influenced by MVO2 and MBF. The half-time of the early phase (T = 9.3 min +/- 2.8 s.d. in controls) as well as the ratio between the size of the early and late phase increased with MVO2 (r = 0.82, r = 0.87, respectively). Thus, early phase intracellular turnover of IHA increased, yet clearance of 123I activity was slowed by augmented cardiac work. Preliminary data of HPLC and electrophoretic analysis of myocardial arterial and venous blood samples over time indicate that the early phase is characterized by a decreasing washout of IHA and a relative increase of radioiodine washout. The half-time of the late phase (T = 245 min +/- 156 s.d. at control) was not related to MVO2 and MBF. In conclusion, myocardial fatty acid metabolism cannot be measured from the half-time of the early phase but might be analyzed from the ratio between the size of the early and late phase when using IHA.     

Assessment of myocardial metabolism with iodine-123 heptadecanoic acid: effect of decreased fatty acid oxidation on deiodination

Terminally radioiodinated fatty acid analogs are of potential use for the noninvasive delineation of regional alterations of fatty acid metabolism by gamma imaging. Since radioactivity from extracted iodine-123 heptadecanoic acid [( 123I]HDA) is released from the myocardium in form of free radioiodide (123I-) the present study was performed to determine whether deiodination of [123I]HDA is related to free fatty acid metabolism. Myocardial production of free radioiodide was measured in rat hearts in vitro and in vivo both under control conditions and after inhibition of fatty acid oxidation. In isolated rat hearts perfused at constant flow with a medium containing [123I]HDA, release of 123I- was markedly reduced during cardioplegia and pharmacologic inhibition of mitochondrial fatty acid transfer with POCA by 67% (p less than 0.005) and 72% (p less than 0.005), respectively. In fasted rats in vivo, 1 min after i.v. injection of [123I]HDA, 51 +/- 5% of myocardial radioactivity was recovered in the aqueous phase, containing free iodide, of myocardial lipid extracts. Aqueous activity was significantly decreased in fed (20 +/- 2%; p less than 0.002) and POCA pretreated (30 +/- 3.7%; p less than 0.05) animals exhibiting reduced oxidation of [14C]palmitate. Thus, deiodination of [123I]HDA was consistently reduced during inhibition of fatty acid oxidation in vitro and in vivo. The results apply to the interpretation of myocardial clearance curves of terminally radioiodinated fatty acid analogs.     

31P NMR spectroscopy in chronic adriamycin cardiotoxicity

Abnormal cardiac energy metabolism has been postulated as a mechanism for adriamycin induced cardiotoxicity. This study was designed to determine high energy phosphate stores at rest and with hemodynamic stress in perfused rat hearts after animals had been chronically exposed to adriamycin (2 mg/kg weekly for 14 weeks). Morphologic and hemodynamic changes were mild in this model. Phosphorus-31 NMR determined intracellular pH and levels of inorganic phosphate (Pi) and ATP were comparable in treated and control hearts. Phosphocreatine (PCr) levels were markedly decreased in treated hearts (0.89 +/- 0.07 units/g versus 1.7 +/- 0.13 units/g, p less than 0.001). The PCr/Pi ratio decreased in both groups during hemodynamic stress. It recovered earlier in controls and there was a marked over-shoot after cessation of rapid pacing in this group which was not present in adriamycin treated hearts. These results suggest that metabolic regulation in response to hemodynamic stress is impaired after chronic adriamycin exposure. PCr depletion and delayed metabolic recovery after hemodynamic stress appear to be potentially useful markers for the effect of adriamycin on the heart.     

Quantitative analysis of myocardial kinetics of 15-p-[iodine-125] iodophenylpentadecanoic acid

Myocardial extraction and the characteristic tissue clearance of radioactivity following bolus injections of a radioiodinated (125I) long chain fatty acid (LCFA) analog 15-p-iodophenylpentadecanoic acid (IPPA) were examined in the isolated perfused working rat heart. Radioactivity remaining in the heart was monitored with external scintillation probes. A compartmental model which included nonesterified tracer, catabolite, and complex lipid compartments successfully fitted tissue time-radioactivity residue curves, and gave a value for the rate of IPPA oxidation 1.8 times that obtained from steady-state release of tritiated water from labeled palmitic acid. The technique was sensitive to the impairment of LCFA oxidation in hearts of animals treated with the carnitine palmitoyltransferase I inhibitor, 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA). IPPA or similar modified fatty acids may be better than 11C-labeled physiological fatty acids such as palmitate in this type of study, because efflux of unoxidized tracer and catabolite(s) from the heart are kinetically more distinct, and their contributions to the early data can be reliably separated. This technique may be suitable for extension to in vivo measurements with position tomography and appropriate modified fatty acids.     

Validation of Tc-labeled “4+1” fatty acids for myocardial metabolism and flow imaging: Part 2. Subcellular distribution

IntroductionOur group has synthesized technetium-labeled fatty acids (FA) that are extracted into the myocardium and sequestered due to heart-type fatty acid binding protein (H-FABP) binding. In this article, we further address the detailed subcellular distribution and potential myocardial metabolism of [^99mTc]“4+1” FA.MethodsExperiments were conducted using isolated hearts of Wistar rats, as well as of wild-type and H-FABP^?/? mice. Myocardium samples underwent subcellular fractionation [subsarcolemmal mitochondria (SM), intermyofibrillar mitochondria (IM), cytosol with microsomes, and nuclei and crude membranes] and analysis by thin-layer chromatography and high-performance liquid chromatography.ResultsThe largest fraction of tissue radioactivity was associated with cytosol [79.69±8.88% of infused dose]. About 9.07±0.95% and 3.43±1.38% of the infused dose were associated with SM and IM fractions, respectively. In the rat heart, etomoxir, an inhibitor of carnitin-palmitoyl transferase I, did not significantly decrease radioactivity associated with mitochondrial fractions, whereas myocardial extraction of [¹²³I]-labeled 15-(p-iodophenyl)-pentadecanoic acid (13.26% vs. 49.49% in controls) and the radioactivity associated with the SM and IM fractions were blunted. The percentage of the infused dose in the mitochondrial and crude fractions increased with the number of NH-amide groups of the FA derivative. Absence of H-FABP significantly decreased radioactivity count in the cytosolic fraction (P<.001). No metabolic product of [^99mTc]“4+1” FA could be detected in any isolated heart.ConclusionsMyocardial [^99mTc]“4+1” FA extraction reflects binding to H-FABP and membrane structures (including the mitochondrial membrane). However, the compounds do not undergo mitochondrial metabolism because they do not reach the mitochondrial matrix.     

Three-Dimensional gated EPR imaging of the beating heart: Time-resolved measurements of free radical distribution during the cardiac contractile cycle

In vivo or ex vivo EPR imaging, EPRI, has been established as a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. While instrumentation capable of performing EPR imaging of free radicals in whole tissues and isolated organs has been previously reported, it was not possible to image rapidly moving organs such as the beating heart Therefore instrumentation was developed to enable the performance of gated-spectroscopy and imaging on isolated beating rat hearts at L-band. A synchronized pulsing and timing system capable of gated acquisitions of up to 256 images per cycle, with rates of up to 16 Hz was developed. The temporal and spatial accuracy of this instrumentation was verified using a specially designed beating heart-shaped isovolumic phantom with electromechanically driven sinusoidal motion at a cycle rate of 5 Hz. Gated EPR imaging was performed on a series of isolated rat hearts perfused with nitroxide spin labels. These hearts were paced at a rate of 6 Hz with either 16 or 32 gated images acquired per cardiac contractile cycle. The images enabled visualization of the time-dependent alterations in the free radical distribution and anatomical structure of the heart that occur during the cardiac cycle.     

Do iodinated fatty acids undergo a nonspecific deiodination in the myocardium?

The intracellular and subcellular distribution of 16-(123I)-iodo-9-hexadecenoic acid were studied in isolated rat hearts, perfused with or without glucose. At various time intervals after injection, cardiac lipids were extracted and the activity was determined for all fractions and all lipid classes. The total cardiac activity was maximal within 1 min postinjection and most of the activity was in the aqueous phase. The presence of glucose in the perfusion medium induced an increase of total cardiac and organic fraction activities. In the latter fraction, activity was very low for FFA, but high for triglycerides (TG), and especially polar lipids. The presence of an exogenous substrate, led to a more active esterification of fatty acids. Coronary effluent analysis showed, in the hydrophilic phase, a lower activity spike in the presence than in the absence of glucose. In the mitochondrial fraction most activity occurred in the organic phase, especially as polar lipids. In the nonmitochondrial fraction, activity was much higher in the aqueous phase. At 90 s postinjection of 1-14C-palmitic acid, over 80% of the myocardial activity was found in the hydrophilic fraction, which indicates, as for the iodo-fatty acid (IFA), an immediate and important oxidation, especially without glucose. These data seem to prove that IFA is taken up by the myocardial cell, subsequently enters the mitochondria and, without an early deiodination, is oxidized with iodide release. Changes in IFA metabolism, consecutive to modifications of glucose concentration in the perfusion medium can be observed by external detection of the myocardial activity curve. Omega-Iodinated fatty acids do not undergo a nonspecific deiodination and are therefore well suited for an external study of myocardial metabolism.     

Myocardial imaging with radiolabeled free fatty acids: Applications and limitations

The assessment of myocardial fatty acid metabolism using radiolabeled substrates has recently become a new diagnostic modality in noninvasive cardiology. The development of metabolic tracers has been made possible largely due to a combined increase in the understanding of myocardial biochemistry and in nuclear-medicine technology. Initially, imaging and the exploration of myocardial metabolism appeared to be the exclusive domain of positron-emission tomography. However, investigators have been successful in applying radioiodine-labeled fatty acids that can be monitored using conventional gamma cameras. These metabolic substrates can be used not only for imaging purposes, but also for the evaluation of regional metabolic clearance rates, which may serve as a parameter for myocardial fatty acid metabolism. Although the initial results have been promising, the analysis and interpretation of clearance curves appears to be rather complicated and may produce a lot of unanswered questions. A great deal remains to be done due to the complex biological behavior of the tracers employed and the difficulties encountered in quantitatively delineating the distribution of radioactivity in the beating heart in vivo. Therefore, closer integration of myocardial biochemistry and the metabolic imaging technique seems to be necessary for enhancing our knowledge of myocardial fatty acid metabolism and to make metabolic imaging clinically useful.

     

In Vitro Assessment of Cardiac Performance after Irradiation Using an Isolated Working Rat Heart Preparation

Summary

The effect of irradiation on cardiac function was assessed using an isolated working rat heart preparation. The animals were given single doses of X-rays in the range 15–30 Gy to their hearts. Cardiac output (CO = aortic flow + coronary flow), heart weight and body weight were followed for a period of 10 months after treatment. Irradiation led to a decrease in cardiac function. This reduction was dose-dependent and progressive with time after treatment. The shape of the Frank-Starling curves constructed for irradiated hearts suggests a loss of contractile function of the myocardium. Coronary flow rates measured in ‘working’ hearts and in ‘Langendorff’ hearts were not significantly changed by the irradiation treatment. The isolated working rat heart preparation proved to be a simple and suitable animal model for the investigation of irradiation-induced cardiotoxicity.     

MR imaging of coronary artery flow in isolated and in vivo hearts.

Methods for imaging flow in coronary arteries with magnetic resonance (MR) imaging techniques are demonstrated in isolated heart preparations and live animal models. Coronary artery flow was first imaged with a flow-compensated gradient-echo pulse sequence in isovolumic and working perfused rat hearts and then in vivo. A bolus tracking technique was used to measure flow velocity in the coronary arteries. Ultrafast gradient-echo imaging techniques were then applied, with high resolution obtained by combining the information from several cardiac cycles. A stimulated-echo pulse sequence was demonstrated as a method for performing coronary angiography by flow tagging in isovolumic perfused hearts. This report describes the results of coronary flow MR imaging in isolated rat hearts and live mice and rats. The general approach has proved useful in evaluating new methods for coronary MR angiography and should permit well-controlled studies of pathologic conditions. This ability to image coronary flow in isolated hearts and in small animals should permit integrated MR studies of coronary flow, myocardial perfusion, myocardial metabolism, and cellular ionic status.     

I-123 heptadecanoic acid — value and limitations in comparison with C-11 palmitate

To define the potential of ¹²³I-labeled heptadecanoic acid (IHA) for the noninvasive assessment of myocardial free fatty acid (FFA) metabolism, the kinetics of IHA were compared to those of physiologic ¹¹C-palmitate (CPA). The single-pass myocardial extraction fraction of IHA was lower than that of CPA (0.53±0.11 vs 0.65±0.10 under control conditions). Following an intracoronary injection of IHA and CPA, the myocardial time-activity curves showed biphasic clearance of both tracers. While, for CPA, the half-time of the early phase of the time-activity curve was a function of myocardial oxygen consumption (MVO₂), this phase was not found to reflect the oxidative metabolism of IHA. However, for both tracers, the size of the early phase increased with augmented MVO₂, whereas the size of the late phase decreased. The late phase represents storage of both tracers in triglycerides and phospholipids. Hence, while quantitative measurement of CPA oxidation is possible from the early phase of the time-activity curve, only the ratio between the size of the early and late phase might be of value in assessing myocardial FFA metabolism using IHA.

     

In vitro assessment of cardiac performance after irradiation using an isolated working rat heart preparation.

The effect of irradiation on cardiac function was assessed using an isolated working rat heart preparation. The animals were given single doses of X-rays in the range 15-30 Gy to their hearts. Cardiac output (CO = aortic flow + coronary flow), heart weight and body weight were followed for a period of 10 months after treatment. Irradiation led to a decrease in cardiac function. This reduction was dose-dependent and progressive with time after treatment. The shape of the Frank-Starling curves constructed for irradiated hearts suggests a loss of contractile function of the myocardium. Coronary flow rates measured in 'working' hearts and in 'Langendorff' hearts were not significantly changed by the irradiation treatment. The isolated working rat heart preparation proved to be a simple and suitable animal model for the investigation of irradiation-induced cardiotoxicity.     

Simultaneous intracellular and extracelular pH measurement in the heart by 19F NMR of 6-fluoropyridoxol

6-Fluoropyridoxol (6-FPOL) was evaluated as a simultaneous indicator of intracellular and extracellular pH and, hence, pH gradient in perfused rat hearts. After infusion, ¹⁹F NMR spectra rapidly showed two well-resolved peaks assigned to the intracellular and extracellular compartments, and pH was calculated on the basis of chemical shift with respect to a sodium trifluoroacetate standard. To demonstrate use of this molecule, dynamic changes in myocardial pH were assessed with a time resolution of 2 min during respiratory and metabolic alkalosis or acidosis and ischemia. For a typical heart, intracellular pH (pHi) = 7.14 ± 0.01 and extracellular pH (pHe) = 7.52 ± 0.02. In response to metabolic alkalosis, pHi remained relatively constant and the pH gradient increased. In contrast, respiratory challenge caused a significant increase in pHi. Independent measurements using pH electrodes and ³¹P NMR confirmed validity of the ¹⁹F NMR results.     

Metabolic fate of radiolabeled palmitate in ischemic canine myocardium: implications for positron emission tomography

Interpretation of dynamic and integrated myocardial tomograms requires elucidation of the biochemical fate of the tracer and characterization of its tissue distribution and rate of efflux. The fate of [1-11C] and [1-14C]palmitate was studied in 13 open-chest dogs during control or ischemic extracorporeal perfusion of the left circumflex coronary artery. Residue detection of myocardial radioactivity, and radio-biochemical analyses of sequential transmural biopsies and arterial and coronary venous effluent were performed for 30 min after intracoronary bolus administration of tracer. In control hearts, 10.3% of initially extracted tracer was retained in tissue (2.9% in triglyceride, 3.5% in phospholipid, and 3.9% in other lipid and aqueous fractions), 73.7% was oxidized, and 16.1% back-diffused unaltered. With ischemia (pump flow 10% of normal), 28.1% was retained (18% in triglyceride, 6.0% in phospholipid, and 4.1% in other lipid and aqueous fractions), 27.2% was oxidized, and 44.4% back diffused (p less than 0.05 compared to control). Throughout the 30-min study interval, triglyceride, diglyceride, and nonesterified fatty acid comprised a significantly greater fraction of initially extracted radioactivity in ischemic than in control hearts. Thus, during ischemia externally detected clearance rates cannot be used as a direct measure of fatty acid metabolism because of marked influences on efflux of nonmetabolized radiolabeled palmitate and the distribution of tracer retained in tissue. Quantitative measurements of specific metabolic processes by tomography will require development and validation of tracers confined to individual metabolic pathways or pools.     

Reproducibility study for free‐breathing measurements of pyruvate metabolism using hyperpolarized 13C in the heart

Spatially resolved images of hyperpolarized ¹³C substrates and their downstream products provide insight into real‐time metabolic processes occurring in vivo. Recently, hyperpolarized ¹³C pyruvate has been used to characterize in vivo cardiac metabolism in the rat and pig, but accurate and reproducible measurements remain challenging due to the limited period available for imaging as well as physiological motion. In this article, time‐resolved cardiac‐ and respiratory‐gated images of [1‐¹³C] pyruvate, [1‐¹³C] lactate, and ¹³C bicarbonate in the heart are acquired without the need for a breathhold. The robustness of these free‐breathing measurements is demonstrated using the time‐resolved data to produce a normalized metric of pyruvate dehydrogenase and lactate dehydrogenase activity in the heart. The values obtained are reproducible in a controlled metabolic state. In a 60‐min ischemia/reperfusion model, significant differences in hyperpolarized bicarbonate and lactate, normalized using the left ventricular pyruvate signal, were detected between scans performed at baseline and 45 min after reperfusion. The sequence is anticipated to improve quantitative measurements of cardiac metabolism, leading to feasible validation studies using fewer subjects, and potentially improved diagnosis, serial monitoring, and treatment of cardiac disease in patients. Magn Reson Med 69:1063–1071, 2013. © 2012 Wiley Periodicals, Inc.