A data sheet for the simultaneous assessment of dual radioactive tracer uptake in the heart

Graphical abstract

When 18 FDG images were acquired (A), graded standards of both 18 FDG which consisted 18 FDG only and 125 I-9MPA which consisted 125 I-9MPA olny as described in second method in the section 2 in Measurement of radioisotope activity were placed on the sheet. When 125 I-9MPA images were acquired 9 days after the first experiment (B), the same sheet was exposed; note that the 18 FDG standards had completely decayed. The respective 18 FDG and 125 I-9MPA doses per rat were 436 mCi (16.1 MBq) and 11.6 mCi (0.43 MBq). The arrow indicates graded 18 FDG standards. Arrowheads indicate graded 125 I-9MPA standards.

Difficulties with in vivo analysis
Complex metabolic networks can be analyzed Expensive tracers and equipment Do not decay or emit radiation Natural abundance of a given isotope (and presence of multiple other isotopes) must be low Amounts should be sufficiently large to account for when calculating a metabolic rate after injection. Hearts were removed from the rats and washed in cold saline. A portion comprising one third of the apical side was frozen in liquid nitrogen, and radioisotopic activity was measured using a scintillation counter. Mid-ventricle specimens were embedded in methylcellulose, cut into serial 20-mm-thick transverse sections, and analyzed using a computer-assisted imaging-processing system. The analyzed portion of the heart is provided as an analytical example. To measure 125 I-labeled fatty acid tracer uptake, radioisotopic activity was measured at least 48 h after the initial measurements. The amount of incorporated radioisotope was reported as a percentage of the administered dose, corrected by heart weight in grams, or as a standard uptake value (SUV) of the tissue concentration (mCi/g)/injected dose (mCi)/body weight (g). Cross-talk between the two tracers was negligible ( Fig. 1A and B).

Radioisotope preparation
1. Determine the total radioisotopic activity of 18 FDG and 125 I-labeled fatty acid tracer 2. Mix 18 FDG and 125 I-labeled fatty acid tracer with saline to a final injection volume (100-200 ml Â number of rats to inject plus 5) containing 500 mCi-1 mCi of 18 FDG and 10-20 mCi of 125 I-labeled fatty acid tracer. Note that the prepared volume should be greater than the precise volume [100-200 ml Â (number of rats plus 5)]. If mice are used, an injection volume of 20-50 ml per mouse is appropriate [5,6].
3. Fill in the date, measured dose, measured time, and numbers used for preparation, as well as the time at the start of the experiment in the data sheet (supplemental materials). This will allow automatic calculation of the radioisotope activity at the start of the experiment.

Animal methods
Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Kyoto University and conducted following the Guide for Care and Use of Laboratory Animals published by the United States National Institutes of Health.
1. Assess the body weight and cardiac function in advance. Monitor blood glucose using a selfmonitoring blood glucose kit to determine whether 18 FDG uptake is influenced by blood glucose levels. 2. In rats fasted overnight, insert a plastic needle (24-gauge) attached to a thin tube containing saline into the tail vein. Inject a 100-200-ml volume containing 1 mCi of 18 FDG and 20 mCi of 125 I-labeled fatty acid tracer simultaneously, followed by saline to wash the tube. 3. Euthanize animals via decapitation or deep sedation 45 min after injection; remove the hearts and place them in cold saline. 4. Collect blood, wash hearts quickly in saline, and weigh each heart 5. Embed mid-ventricle specimens in methylcellulose, and freeze them on dry ice. Collect and weigh a one-third portion of the apical side and freeze this tissue in liquid nitrogen. This will be used to measure radioisotopic activity with a scintillation counter. The analyzed portion of the heart is provided as an analytical example.
Measurement of radioisotope activity using a scintillation counter 1 Count the levels of 18 FDG or 125 I-labeled fatty acid tracer in the above-described apical portion via direct measurement with a scintillation counter. Window levels should be set in advance at 15-75 KeV for 125 I and 250-750 KeV for 18 F. The energy peak of 18 FDG is 511 KeV; that of 125 I-9MPA is 35 KeV. The counting efficiency specific to the scintillation counter should also be known before the experiment. For this manuscript, a 12-s counting period was appropriate for the proscribed settings and doses. The first count is acquired to determine the 18 F uptake in the one-third apical portion of the heart. This data should be filled in the yellow-colored cells of the second Excel sheet. In the first experiment, the 18 FDG activity was not influenced by the 125 I-labeled fatty acid tracer activity due to the narrow energy peak and difference in the injected dose; however, the 125 I-labeled fatty acid tracer activity measured during the first analysis exceeded the actual values; 18 FDG has a relatively scattered energy peak and these values are not usually used in the analyses. Fill in the values in the second Excel sheet attached as a supplemental Excel file. 2 Two methods exist for the preparation of graded standards -The first involves a count of the mixed radioisotopes for injection. Prepare duplicates of the following: 5-10 ml of the mixed radioisotopes (A), 5-10 ml of a 2Â volume of the mixed radioisotopes diluted in saline (B), 5-10 ml of a 4Â volume of the mixed radioisotopes diluted in saline (C), 5-10 ml of an 8Â volume of the mixed radioisotopes diluted in saline (D), and 5-10 ml of a 16Â volume of the mixed radioisotopes diluted in saline (E). These will be used to calculate standard curves of known injected doses and actual counts. Count, seal, and preserve these standard samples. Note: do not discard these standard tubes because they will be needed for the second count and 125 I-labeled fatty acid tracer calculation. Fill in the time of graded standard measurement, the volumes of graded standards (usually the same volume), and radioisotope activity in the first Excel datasheet (Supplemental materials) -The second method is the count of each of the used radioisotopes respectively, for which two separate graded standards will be prepared. Prepare duplicates of the following: 5-10 ml of the prepared 18 FDG (pre-mixed) (A), 5-10 ml of a 2Â volume of the prepared 18 FDG (pre-mixed) diluted in saline (B), 5-10 ml of a 4Â volume of the prepared 18 FDG (pre-mixed) diluted in saline (C), 5-10 ml of an 8Â volume of the prepared 18 FDG (pre-mixed) diluted in saline (D), and 5-10 ml of a 16Â volume of the prepared 18 FDG (pre-mixed) diluted in saline (E). Again, the same procedure is performed for 125 I-labeled fatty acid tracer. Prepare duplicates of the following: 5-10 ml of the prepared 125 I-labeled fatty acid tracer (pre-mixed) (A), 5-10 ml of a 2Â volume of the prepared 125 I-labeled fatty acid tracer (pre-mixed) diluted in saline (B), 5-10 ml of a 4Â volume of the prepared 125 I-labeled fatty acid tracer (pre-mixed) diluted in saline (C), 5-10 ml of an 8Â volume of the prepared 125 I-labeled fatty acid tracer (pre-mixed) diluted in saline (D), and 5-10 ml of a 16Â volume of the prepared 125 I-labeled fatty acid tracer (pre-mixed) diluted in saline (E).
3 Count the actual administered radioisotope activities. Note that 5 ml of the injected isotope mixture (triplicate) should be counted. Fill in the time of measurement and radioisotope activity on the first Excel sheet (see a Supplemental Excel file). Note: do not discard these tubes because they will be needed for the second count. 4 After 2 or more days, perform a second 125 I count (half-life = 60 days) of both the sample and standard tubes. Fill in the yellow-colored cells on the second Excel sheet (see also a Supplemental Excel file). The activity of 18 FDG during the second analysis is almost identical to the background activity; hence, the count of 125 I activity was not influenced by 18 FDG activity. paper is then sealed in a thin plastic wrap and placed on a sheet sized to fit the imaging plate or X-ray film (arrow in Fig. 1A). In Fig. 1A, graded standards that contained only 18 FDG were used following preparation as described above (second method) in the section 2 in Measurement of radioisotope activity using a scintillation counter. For 125 I-labeled graded standards, previously used graded standards (for example, an arrowhead in Fig. 1A and 1B) are prepared and placed on the sheet to account for the longer half-life. 2. Place serial 20-mm-thick transverse sections of methylcellulose-embedded mid-ventricle specimens on a prepared slide with a glass coverslip (6 slices per heart). These prepared slides should be attached to a paper sized to fit the imaging plate along with the graded standards. 3. After preparing the paper sheet with attached slides and graded standards, place the sheet upsidedown on the imaging plate such that the covered glass or plastic membrane touches the imaging plate or X-ray film directly for 1 h. Next, analyze the imaging plate using a computer-assisted imaging-processing system (BAS 1000, Fuji Photo Film Co., Ltd., Tokyo, Japan) to acquire photostimulated luminescence or X-ray film to acquire an image. If using the BAS system, a linear correlation should be fitted between the relative activities (Photo-Stimulated Luminescence; PSLs) of the graded scales and actual radioisotopic activities (see detailed in explanation of the data sheet and a Supplemental material). If using X-ray film, densitometry should be performed, and the results should be fitted between the densities of the graded standards and actual radioisotopic activities. The dose and energy of 18 FDG is enough large that the activity of the 125 I-labeled fatty acid tracer might be negligible in the first exposure (arrowhead in Fig. 1A). 4. After obtaining the 18 FDG image (Fig. 1A), the imaging plate should be removed, and the next exposure prepared. 5. After 2 or more days, perform the next exposure in the same way (Fig. 1B), using the abovementioned paper sheet. As the 18 F will have completely decayed, all radioisotopic activity will be derived solely from 125 I. The exposure time is longer than that used for 18 F; for example, an exposure of 3-4 days is appropriate, due to the low injected dose and weak energy peak of 125 I.   where t is defined as the elapsed time between the time of the start of the experiment and the measurement (cell E13), and 110 is the half-decay time of 18 F. Calculate the radioisotopic counts of the injected isotope (cells N4 and N5 or N7 and N8) according to the gamma camera counting efficiency (P4 and P5). The counting efficiency differs for each camera; accordingly, each camera uses a different calculation formula. In general, however, the radioisotopic counts of the injected isotope (cells N4 and N5 or N7 and N8) should = Correcting coefficient (the counting efficiency) x (actual values; L4 and L5 or L7 and L8) (Eq. (D)). 2. Open the second sheet. Fill in the cells indicating the identification number of each animal (cell D6), animal group (cell C6), animal body weight (cell E6), date of the experiment (cell F6), the time of the start of the experiment (cells W4 and X4), and blood sugar concentration (cell G6) before injection. 3. After euthanization, record the weight of the whole heart into the appropriate cell (cell H6) in the second Excel sheet. The mid-ventricle and apical one-third portions would be excised; the latter is then weighed, and this value is filled into the cell (cell I6) in the second sheet. The weight of the collected blood is also determined and entered into the second Excel sheet (cell J6); note that the weight of the empty collection tube should be measured previously, and the weight of blood should thus be corrected. 4. Measure the radioisotope activity of the heart and blood fill in the yellow-colored Excel cells (K6 to K11 and M6 to M11, Q6 to Q11 and S6 to S11, respectively) and the time of measurement on the second Excel sheet (cells O6 and P6 where t is the elapsed time (cells W6 or L18) between the start of the experiment and the time of the measurement of the sample.
(Activity per gram) = (Actual values at the start of the experiment)/(tissue or blood weight [g]) (the% dose/g) = (Activity per tissue gram)/(injected radioisotope activity) (SUV) = (the% dose/g) Â (body weight) 9. During the second count for 125 I activity, the 125 I activity values should be filled in where appropriate. Count the actual radioisotope activities of 5 ml of the injected (mixed) radioisotope (triplicate) and fill in the first Excel sheet (cells B62 to B64). In addition, fill in the time of measurement (cell B57) and calculation of days form the first experiment (cell B58). The 125 I at the start of the experiments are calculated (cell H65) according to the Eq. (F), and copy and paste these data to the second sheet (cell Q37).
(Activity at the experiment) = (measured activity) Â (0.5) (Àt/60) (F) where t is defined as the days from the first experiment. 60 (days) is the half decay time of 125 I. 10. Measure the 125 I activities of the heart and blood, and fill in these values (cells AD6 to AD11 and AF6 to AF11, or directly into cells AE6 to AE11 and AF6 to AF11 if a gamma camera yields actual values) and the date of measurement (cell AF3) on the second Excel sheet. On the second Excel sheet, calculate the activity per gram of tissue, according to Eq. (F). 11. For both 18 FDG and 125 I-labeled fatty acid tracer autoradiography, acquire the PSL and calculate PSL/mm 2 . For X-ray film, acquire the image and calculate the image density instead of the PSL values. Calculate the PSL of the heart minus background PSL. This calculation is performed by fitting values to a standard curve generated using graded standards and their known radioisotope activity levels. An example of the third and the fourth Excel sheets is provided in Fig. 1A and B.

Discussions
Recent technical advantages have enabled system-wide measurements of metabolites in various organisms [9][10][11]. Two tracers, one radioactive and one stable, are used in these experiments. The advantages and disadvantages of these tracers are briefly summarized in Table 1. Dual-tracer methods do not limit the use of radioactive tracers, and both tracers can be used in a dual-tracer method. In this methodological paper, a dual-tracer method based on 2 radioactive tracers is introduced.

Conflicts of interest
No declared.

Source of funding
The funding source had no role in this study.

Acknowledgement
This work was supported by Tobe-Maki Foundation. MethodsX thanks the reviewers (anonymous) of this article for taking the time to provide valuable feedback.

Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi. org/10.1016/j.mex.2016.03.015.