Kinetic Analysis

The area under the concentration-time curves (AUC, %ID/mL x min) was calculated for each region of interest using Prism 8 Software (GraphPad, La Jolla, CA, USA). The kidney and liver uptake rate constants (k_{uptake,kidney} and k_uptake,liver, respectively - mL/min/mL tissue) of radioactivity were estimated from 0.4 to 4 min after [¹¹C]metoclopramide injection by integration plot analysis (34):

where X_t,tissue is the amount of radioactivity per mL tissue in the kidney or liver at time t, C_t,blood is the radioactivity concentration in the blood (image-derived curve from the region of interest placed in the left ventricle of the heart) at time t, and AUC_0-t,blood is the area under the concentration-time curve of the left ventricle of the heart from time 0 to time t. V_E corresponds to the y-intercept of the integration plot. k_{uptake,tissue} is obtained by performing linear regression analysis of a plot of X_t,tissue/C_t,blood versus AUC_0-t,blood/C_t,blood and calculating the slope of the regression line. The intrinsic urinary excretion clearance of total radioactivity (CL_int,urine, mL/min) was calculated for the animals which underwent 90-min PET scans from 30 to 70 min after radiotracer injection using integration plot analysis (34) as follows:

where X_t,urine is the total amount of radioactivity excreted in urine at time t, and AUC_0-t,kidney is the area under the concentration-time curve of the kidney from time 0 to time t. V_E is the y-intercept of the integration plot. CL_int,urine was obtained by performing linear regression analysis of a plot of X_int,urine versus AUC_0-t,kidney and calculating the slope of the regression line.

The total radioactivity concentration ratios (kidney/plasma, liver/plasma and urine/kidney) were calculated using the following equation:

where Concentration_{total PET,tissue1} and Concentration_{total PET,tissue2} are the %ID/mL values of total radioactivity obtained at 15 min after radiotracer injection from the PET images. The plasma concentration was obtained by multiplying the concentration in the left ventricle of the heart by the plasma/blood radioactivity ratio, determined from the blood sample collected at the end of the PET scan.

Since [¹¹C]metoclopramide is highly metabolized in mice, total radioactivity was corrected for radiolabeled metabolites in order to calculate the concentration ratios of unchanged [¹¹C]metoclopramide. The correction for metabolites of the total radioactivity concentration in each organ was performed using the following equation:

where Concentration_{total PET} is the %ID/mL value of total radioactivity obtained at 15 min after radiotracer injection from the PET images and P_unchanged is the average percentage of unchanged [¹¹C]metoclopramide in each sampled organ or fluid (Table ​(TableII).II). No metabolite data at 15 min after radiotracer injection were available for the PET scanned animals with C57BL/6 genetic background, since the PET scan lasted 90 min; thus, P_unchanged was used from the separate cohort of C57BL/6 animals which did not undergo a PET scan (Table ​(TableI).I). For the PET scanned animals with FVB genetic background, P_unchanged was measured at the end of the 15-min PET scans for all animals.

Percentage of Unchanged [¹¹C]Metoclopramide in Different Organs or Fluids Determined with Radio-TLC Analysis at 15 Min After Radiotracer Injection

Cimetidine

(150 mg/kg)

Sulpiride

(25 mg/kg)

Percentage of unchanged radiotracer is given as mean ± SD. ** p < 0.01, *** p < 0.001, Kruskal-Wallis followed by a Dunn’s multiple comparison test against a reference group (baseline)