Radiosynthesis of 99mTc(CO)3(NTA)
Nitrilotriacetic acid (NTA) was purchased from Aldrich and labeled as previously described [14]. 99mTc-pertechnetate (99mTcO4−) in 0.9% saline was received from Triad Isotopes (Norcross, GA, USA). The “CRS Isolink kit” (Center for Radiopharmaceutical Science, Paul Scherrer Institute, Villigen, Switzerland) was used to prepare the [99mTc(CO)3(H2O)3]+ precursor according to the manufacturer’s insert. 99mTc(CO)3(NTA) was separated from unlabeled ligand by high-performance liquid chromatography (HPLC) instrument (System Gold Nouveau, Beckman Coulter, Brea, CA) equipped with a model 170 radiometric detector and a model 166 ultraviolet light-visible light detector, 32 Karat chromatography software (Beckman Coulter), and an octyldecyl silane column (C18 RP Ultrasphere; 5-µm, 4.6 x 250 mm; Beckman Coulter). The solvent system was 0.05 M TEAP buffer pH 2.5 (solvent A) and ethanol (solvent B) and the flow rate was 1 mL/min; the radiochemical purity was > 99%. The gradient method was the same as reported previously [15].
Ethanol was partially removed by N2 gas, and the collected solution of 99mTc(CO)3(NTA) was buffered in a physiological phosphate buffer (PBS) at pH 7.4. The HPLC-purified complex in PBS (pH 7.4) was passed through a sterile Millex-GS 22 µm filter (Millipore Co., St Louis, MO) (primed with 4 mL of saline) into a sterile, pyrogen-free empty vial. The final concentration was 74–148 MBq/mL (2–4 mCi/mL) and the final pH was 7.4. Test samples were sent for analysis and determined to be sterile and pyrogen free.
Patient Studies
An eIND was obtained from the FDA and all studies were performed with the approval of the Radiation Safety Committee for Human Use of Radiation (RSC1) and the Institutional Review Board. Signed consent was obtained from each participant. Adult patients referred by urologists for diuretic renography were asked to participate. Nineteen subjects entered the protocol but one withdrew after signing the consent due to lack of adequate venous access. Each of the remaining 18 subjects (mean age 51.3 ± 16.8 years, 11 females, 7 males) was monitored with measurements of blood pressure, heart rate, and temperature obtained before and after the study; they were under constant observation during the study and were further monitored via a follow-up phone call 24 hours later. Each patient drank 200 mL of water on arrival; an intravenous line was connected to a one-liter bag of normal saline for fluid replacement during the diuretic portion of the study.
Both studies were performed on the same day using a protocol similar to that of a two-stage ACE inhibition study [16]; a low activity acquisition was followed by a high activity acquisition to minimize the effect of residual activity on calculations derived from the second acquisition. Our intention was to alternate the order of 99mTc(CO)3(NTA) and 99mTc-MAG3 administration but the HPLC preparation of 99mTc(CO)3(NTA) rarely yielded enough activity to allow 99mTc(CO)3(NTA) to be administered as the second administration; consequently, 99mTc(CO)3(NTA) was the first tracer administered in 16/18 subjects. The mean activity of the initial tracer was 47 ± 4.4 MBq. The majority of patients received an intravenous injection of 40 mg of furosemide 15 min prior to the administration of first tracer and 14 mg of furosemide prior to administration of the second tracer to maintain a comparable serum concentration of the diuretic [17]. Exceptions were patients 10 (60mg and 30 mg), patient 16 (30 mg and 10 mg) and patient 17 (80 mg and 27 mg).
Imaging was performed for 24 minutes following tracer injection using a General Electric Infinia camera (Milwaukee, WI) with a 20% window centered over the 140 keV photopeak of 99mTc. Data were acquired in a 128 x 128 matrix by using a three-phase dynamic acquisition and processed on a General Electric Xeleris computer using non-commercial, in house update of the QuantEM™ renal software. Kidney regions of interestwere automatically assigned and could be modified by the operator; background was automatically assigned as a peri-renal region of interest 2 pixels wide and one pixel outside of the renal ROI [18].
The acquisition was followed by an anterior image of the liver and gall bladder, and a measurement of voided volume. Patients drank an additional 200 mL of water before receiving the second injection of furosemide. Fifteen minutes following furosemide, the alternate tracer was administered, mean activity of 320 ± 34 MBq, and the same acquisition protocol was utilized. Intravenous hydration was continued throughout both acquisitions. Renogram curves were generated using whole kidney regions of interest (ROIs). Clearances were measured using a camera-based technique and the kidney to background ratios were calculated from the same summed frames (1-2.5 minutes after injection) used to calculate relative uptake and the camera-based clearance [19].
Two subjects had only one kidney. Six of 18 subjects had to void prior to completing the first 24 minute acquisition; all of these patients received 99mTc(CO)3(NTA). Two patients had to void prior to completing the second acquisition, one following 99mTc-MAG3 and one following 99mTc(CO)3(NTA), and two studies demonstrated marked 99mTc(CO)3(NTA) tracer infiltration at the injection site. Comparisons impacted by dose infiltration or incomplete 24 minute acquisitions were not included in the data analysis.
The camera-based clearance method in this study utilized a regression equation developed for 99mTc-MAG3 that converts the percent injected dose in the kidney at 1-2.5 minutes post injection to a clearance expressed in mL/min/1.73m2 [19]. To test the applicability of this regression equation for 99mTc(CO)3(NTA), we used the 99mTc-MAG3 regression equation to calculate the camera-based clearance of 99mTc(CO)3(NTA) from two previous studies in which the 99mTc(CO)3(NTA) clearance was calculated determined using the single-injection, 2-compartment model of Sapirstein et al. [8, 9, 20]. These two studies contained 17 subjects, 9 volunteers with normal renal function and 8 with a diagnosis of chronic kidney disease [8, 9].
STATISTICAL ANALYSIS