Kinetics of 13N-ammonia uptake in myocardial single cells indicating potential limitations in its applicability as a marker of myocardial blood flow.

Abstract

To study kinetics and principles of cellular uptake of '3N-ammonia, a marker of coronary perfusion in myocardial scintigraphy, heart muscle cells of adult rats were isolated by perfusion with collagenase and hyaluronidase. Net uptake of '3N, measured by flow dialysis, reached equilibrium within 20 sec in the presence of sodium bicarbonate and carbon dioxide (pH 7.4, 370 C). Total extraction, 80 sec after the reaction start, was 786 ± 159 ,umol/ml cell volume. Cells destroyed by calcium overload were unable to extract 13N-ammonia. Omission of bicarbonate and carbon dioxide reduced total extraction to 36% of control. '3N-Ammonia uptake could also be reduced by 50 gM 4,4' diisothiocyanostilbene 2,2' disulfonic acid, by 100 ,ug/ml 1-methionine sulfoximine, and by preincubation with 5 gtM free oleic acid. These results indicate that in addition to metabolic trapping by glutamine synthetase, the extraction of 13N-ammonia by myocardial cells is influenced by cell membrane integrity, intracellular-extracellular pH gradient, and possibly an anion exchange system for bicarbonate. For this reason, the uptake of 13N-ammonia may not always provide a valid measurement of myocardial perfusion. Circulation 71, No. 2, 387-393, 1985. 13N-AMMONIA (13N),* a positron-emitting radionuclide, has been used as a marker of myocardial perfusion.' 8 Because cellular extraction of an ideal flow marker must be proportional to regional myocardial blood flow, extraction of such a marker should not be influenced by cell metabolism or cell membrane transport mechanisms. Since myocardial extraction fraction of '3N-ammonia can be significantly reduced when intracellular glutamine synthetase is inhibited,67 the usefulness of this compound as a flow marker has been questioned. This study provides additional data regarding the kinetics and possible role of the plasma membrane in myocardial '3N uptake and retention in isolated adult rat heart cells. Materials and methodsPreparation of myocardial single cells. Male Wistar rats 3 to 4 months old were heparinized and anesthetized by urethane (1.5 mg/g body weight, intraperitoneally). Hearts were extracted and perfused in a nonrecycling manner for 10 min with 35 mM NaCI, 4.75 mM KCl, 1.2 mM KH2PO4, 150 mM sucrose, 25 mM NaHCO3, 10 mM HEPES buffer, 10 mM glucose, and 5 g/liter albumin bovine fraction V (pH 7.4, 370 C, 95% 02/5% CO2).9 A recycling perfusion (25 min) was carried out with the same solution also containing 150 U/ml unpurified collagenase (E.C.3.4.4.19.) and 1000 U/ml hyaluronidase (E.C.3.2.1.36.).At the end of the perfusion the softened heart was sliced into small pieces and reincubated for 10 min in the same enzymecontaining solution; the suspension was filtered through a nylon mesh and centrifuged gently for 2 min at 50 g. The supernatant was removed and the pellet was washed three times in 130 mM NaCl, 4.0 mM KCI, 1.2 mM KH2PO4, 25 mM HEPES buffer, 10 mM glucose, and 20 g/liter albumin bovine fraction V (pH 7.4, room temperature, 100% 02). A small population of completely destr...

Treadmill Exercise Produces Larger Perfusion Defects Than Dipyridamole Stress N-13 Ammonia Positron Emission Tomography

“…Retention of N-13 ammonia is adenosine triphosphatedependant and requires the metabolism of ammonia to glutamine (13). Thus, uptake and retention might be altered by changes in metabolic state (14,15). Exercise-induced ischemia might cause metabolic stunning, reduce the metabolism of ammonia to glutamine, and attenuate N-13 ammonia retention in areas of ischemia.…”

Delineation of impaired regional myocardial perfusion by positron emission tomography with H2(15)O.

“…Because extraction and retention of both tracers are influenced by the metabolic states of myocardium, as well as by perfusion, 1,3,5,6,[8][9][10] these tracers can be used to evaluate myocardial meta-bolic activity, as well as flow,6 if enough tracer can be delivered to the myocardial segment in question.…”

“…However, the use of these tracers for quantification of flow entails some limitations. Single-pass extraction and myocardial retention of`3N-labeled ammonia are influenced by flow23,8 as well as by cell membrane integrity9 and the metabolic state of the myocardium.8 9 Extraction of 82Rb also varies with flow and metabolism. 5,6,10 An ideal perfusion tracer should be either freely diffusible or completely extractable and retainable by myocardium on a single pass through the coronary circulation without having its kinetics altered throughout the range of flow studied or by the metabolic status of the myocardium.…”

Novel and Simple Carbon-11-Labeled Ammonium Salts as PET Agents for Myocardial Perfusion Imaging

“…Its uptake is more in tune with flow rate in comparison to 82 Rb, and it generates good image quality [19]. However, the uptake of 13 N-ammonia may not always provide a valid measurement of myocardial perfusion since in addition to metabolic trapping by glutamine synthetase, the extraction of 13 N-ammonia by myocardial cells may be influenced by other parameters such as cell membrane integrity, intracellular-extracellular pH gradient, and an anion exchange process for bicarbonate [20]. Additional problems are faced with [ 13 N]ammonia's production and administration; despite high-yielding synthetic procedures [21], a maximum of two patients can be scanned per 30-min cyclotron irradiation, an expensive and time-consuming operation to any non-dual beam cyclotron unit.…”