Background

[PubMed]

[¹³N]Ammonia ([¹³N]NH₃) is a useful ¹³N-labeled compound that has been developed as a positron emission tomography (PET) imaging agent for assessing regional blood flow in tissues (1-3). The compound is labeled with ¹³N which is a positron emitter with a physical t_½ of 9.965 min. [¹³N]NH₃ was approved by the United States Food and Drug Administration in 2000 for PET imaging of the myocardium under rest or pharmacologic stress conditions to evaluate myocardial perfusion in patients with suspected or existing coronary artery disease.

Ammonia is important in many metabolic activities of various organs and is involved in biochemical pathways leading to the production of amino acids, purines, and urea (4). Ammonia is produced in the body from the deamination of amino acids and the deamidation of amides (5). About 20% of urea produced in the body is converted to ammonia and carbon dioxide in the gut. Ammonia is absorbed and converted back to urea in the liver. It also plays a significant role in glutamine synthesis. Ammonia is produced from glutamine and other amino acids in the kidney. NH₃, as a nonionic form, is freely permeable to all cell membranes (6). In an acidic environment, NH₃ accepts a proton and exists as NH₄⁺. With a dissociation constant (pK_a) of 9.3, NH₄⁺ constitutes about 99% of the total ammonia (NH₃ + NH₄⁺) concentration in the pH range of body fluids. As an ionized form, NH₄⁺ is a relatively impermeable cation to cell membranes. The mechanism of cellular localization of [¹³N]NH₃ is not entirely known. One known mechanism is cellular membrane diffusion of [¹³N]NH₃ and then metabolic trapping of radioactivity with the conversion of ammonia to glutamine, glutamic acid and carbamyl phosphate (6-8).

Ammonia labeled with ¹³N was first produced by Joliot and Curie (9, 10). The short t_½ of ¹³N requires on-site cyclotron production of ¹³N and a short synthesis time of [¹³N]NH₃. After i.v. intravenous injection, [¹³N]NH₃ rapidly clears from the circulation. It is taken up mainly by the myocardium, brain, liver, kidneys, and skeletal muscle (2, 6, 11, 12). In the myocardium and brain, [¹³N]NH₃ is removed from the blood and metabolically trapped within the tissues. The apparent linear relationship between distribution of [¹³N]NH₃ and the regional blood perfusion makes feasible the use of this radiotracer for imaging and measuring cerebral and myocardial blood flows.

Synthesis

[PubMed]

As early as 1934, Joliot and Curie (10) first produced [¹³N]NH₃ by alpha particle irradiation and heating of boron nitride in sodium hydroxide. Later, [¹³N]NH₃ was produced by deuteron irradiation of methane gas or metal carbides, which gave relatively low yields (4). A more efficient method is the proton irradiation of a natural water target [¹⁶O(p,α)¹³N] (1, 13). In this procedure, [¹³N]NH₃ is initially produced by hydrogen abstraction from the target water matrix. There is increasing production of oxo anions of nitrogen (¹³NO₃− and ¹³NO₂−) from radiolytic oxidation with increasing target dose. These oxo anions can be converted to [¹³N]NH₃ in aqueous solution by use of reducing reagents such as DeVarda’s alloy in sodium hydroxide or titanium(III) chloride/hydroxide_. Ido et al. (14) first described a fully automated synthesis method with titanium(III) chloride used as the reducing agent. The radiochemical purity was >99.9% and the radiochemical yield was 87-91% within 10 min from the end of bombardment (EOB). [¹³N]NH₃ also can be produced directly in the target water (in-target production) by adding free radical scavengers (ethanol, acetic acid, or hydrogen) to prevent the formation of the oxo anions. Wieland et al. (15) applied this method for [¹³N]NH₃ production with the use of pressurized, dilute aqueous solutions of acetic acid and ethanol. Berridge et al. (16) reported that the combination of hydrogen and ethanol was more effective than either alone at high beam doses. One limiting factor of the [¹⁶O(p,α)¹³N] nuclear reaction is that the relatively small cross-section of the reaction requires accelerators/ cyclotrons of energies >10 MeV for production.

Ferrieri et al. (17) introduced a novel solid ¹³C-enriched target for on-line [¹³N]NH₃ production with use of the ¹³C(p,n)¹³N reaction. This nuclear reaction has a higher cross-section which can be used in accelerators/cyclotrons with <10 MeV energies. In their method, [¹³N]nitrogen gas was converted to [¹³N]NH₃ by use of microwave radiation to dissociate the nitrogen gas in a hydrogen plasma. The reaction time was about 10 min, and radiochemical yield was 25% at 10 min after the EOB. Bida et al. (18) also used the method of ¹³C(p,n)¹³N reaction by placing enriched ¹³C carbon powder between two frits and irradiating with a 10 MeV proton beam while water was pumped through the bed to extract the ammonia. Welch et al. (19) successfully produced [¹³N]NH₃ with the use of ¹²C(d,n)¹³N reaction and a 7 MeV deuteron beam in aluminum carbide. Shefer et al. (20) and Dence et al. (21) showed that a windowless, solid target could be irradiated with relatively low-energy (0.8-3.2 MeV) deuterons to produce [¹³N]NH₃ by the ¹²C(d,n)¹³N reaction. This was accomplished by heating a graphite target in a stream of pure oxygen at 800ºC, and ¹³NO₂− (99% radioactivity produced) was eluted with water and converted to [¹³N]NH₃ with Raney nickel. The total extraction, trapping, and synthesis time was <17 min with a radiochemical purity of nearly 100%.

Mulholland et al. (22) reported a method of direct simultaneous production of [¹³N]NH₃ and [¹⁵O]H₂O with the use of 26 MeV proton irradiation and a double liquid chamber. Suzuki et al. (23) designed an automated system that used10 mM ethanol solution saturated with pure O₂ gas as the target and bombardedment with 18 MeV protons. Krasikova et al. (24) reported an alternative method that used low-pressure methane gas (150-550 kPa) in the natural water target irradiated with protons. The radiochemical purity of [¹³N]NH₃ was >99% even at very high beam currents. Firouzbakht et al. (25) used a cryogenic target containing frozen water for the ¹⁶O(p,α)¹³N reaction, and they found that this procedure (with beam currents from 1 µA to 20 µA) could produce [¹³N]NH₃ directly because low temperature decreased radiolysis. The radiochemical purity of the [¹³N]NH₃ produced was >95%. When frozen carbon dioxide was irradiated, >95% of ¹³N activity was in the form of nitrate and nitrite.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Isolated arterially perfused rabbit interventricular septa (n = 9 at 28ºC, n = 10 at 37ºC) were used to study the relationship between blood flow, myocardial uptake, and the metabolism and clearance of [¹³N]NH₃ (8). Analysis of the time-activity curves revealed that the [¹³N]NH₃ kinetics were best described by three components. Component 1, with a short clearance t_½ of <7 min, was consistent with a freely diffusible NH₃ compartment. A large metabolic compartment was primarily responsible for component 3, which had a long clearance t_½ of 30 to 500 min. At 37^oC and 15 min after injection, all radioactivity was essentially in component 3. In another study, glutamine synthetase appeared to be responsible for retention of [¹³N]NH₃ radioactivity in rabbit myocardium (26). In their study of [¹³N]NH₃ uptake in myocardial single cells, Rauch et al. (27) found that in addition to metabolic trapping, the extraction of [¹³N]NH₃ might also be influenced by cell membrane integrity, intracellular-extracellular pH gradient, and possibly an anion exchange system for bicarbonate.

Animal Studies

Human Studies

[PubMed]

Harper et al. (12) performed imaging with 370 MBq (10 mCi) at the time of administration, (TOA) of [¹³N]NH₃ in 2 healthy volunteers and showed that radioactivity localized in the myocardium, mediastinum, and liver. There was an initial moderate uptake in the lungs, and it was washed out after 5-10 min. In a metabolic study (33), 9 healthy male volunteers received iv. administration of 740 MBq (20 mCi at the TOA) of [¹³N]NH₃ in normal saline. About 93.1 ± 4.9% (n = 6) of the blood radioactivity remained as [¹³N]NH₃ within the first 2 min but was decreased to 50.2 ± 18.9% (n = 5) at 5 min after injection. At 1 min, the blood radioactivities associated with [¹³N]glutamine and [¹³N]urea were 3.0 ± 2.4% (n = 6) and 3.1 ± 4.6%, respectively. By 5 min, the radioactivities for [¹³N]glutamine and [¹³N]urea had increased to 16.2 ± 11.2% (n = 5) and 32.7 ± 10.0%, respectively. Lockwood et al. (11) reported a study of [¹³N]NH₃ metabolism in 5 normal subjects with a dose of 370 MBq (10 mCi at the TOA) that showed that the rates of both radioactivity clearance from the vascular compartment and brain ammonia utilization were linear functions of its arterial concentration. About 47 ± 3% of [¹³N]NH₃ radioactivity was taken up by the brain during a single pass from the arterial blood, and 7.4 ± 0.3% of [¹³N]NH₃ was metabolized by the brain. Approximately 50% of the arterial [¹³N]NH₃ was metabolized by the skeletal muscle. Changes in [¹³N]NH₃ kinetics were reported in patients with tumors, hypopituitarism, and liver diseases [PubMed].

In 1980, Lockwood (40) reported the radiation absorbed doses of i.v. injection of [¹³N]NH₃ with the use of body distribution data and the MIRD model. The urinary bladder wall was the critical organ with the highest total absorbed dose (0.014 mGy/MBq or 51 mrad/mCi). The whole body, liver, red marrow, ovaries, and testes received 0.0015 mGy/MBq (5.5 mrad/mCi), 0.0046 mGy/MBq (17 mrad/mCi), 0.0015 mGy/MBq (5.4 mrad/mCi), 0.0027 mGy/MBq (9.8 mrad/mCi), and 0.00027 mGy/Bq (1 mrad/mCi), respectively. The brain to brain-absorbed dose was 0.0043 mGy/MBq (16 mrad/mCi). Complete radiation dosimetry of [¹³N]NH₃ for adults has also been tabulated.

Hickey et al. (41) in a 2005 study compared the measurement of myocardial perfusion using [¹³N]NH₃ with that of [¹⁵O]H₂O in healthy volunteers, and they found that there was a discrepancy between the two measurements in a 2-compartment model analysis. The underestimation by [¹³N]NH₃ was most likely attributable to the regional myocardial uptake variation and metabolism of [¹³N]NH₃. This could be minimized by use of a 3-compartment model for data analysis. Other studies [PubMed] have shown the clinical utility of [¹³N]NH₃ for quantification of myocardial blood flow in humans. In a study of 27 patients, Khorsand et al. (42) reported that gated cardiac [¹³N]NH₃ imaging could be used for the estimation of LV function. In a study of 21 patients admitted for the assessment of myocardial perfusion with dynamic [¹³N]NH₃ PET, the absolute quantification of myocardial blood flow with 3-dimensional PET appeared to be in excellent agreement with those obtained with the 2-dimensional technique (43).

Supplemental Information

[Disclaimers]

Draft of Review and Evaluation of Pharmacology and Toxicology Data

Medical and Statistical Review

NIH Support

NIH HL-27841-01-03; NCI CA-18153-03, CA-08748-14; USPHS AM-16739, NS-00343; NIH DK-16739; NINCDS NS-05820, NS15665; NIH HL-11351-13, HL-27841-01; NCI 08748-11B; NIH RO1 HL-41047-01; USPHS GM-14889-06, 1 F11 NB-2169-01; NIH HL-29845, HL-33177, HL-29845-01; USPHS HE-06664, HL-15860, GM-16712, HV-71443; NCI CA-08748; NIH HL-43884; NINDS NS-15380; PHS NINCDS NS-15380; USPHS P01 HL-13851-11; NIH IR43HL48969, 2R44CA53953; NIH NS15655; NIH NS-02149; USPHS 1 P01 GM-18940-01, 43-NHL1-68-1334; NINCDS NS-05820, NS-14996, NIH 89-2359Ja, HL-27555, NIH HL-29845, HL-33177.

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