ORIGINAL ARTICLE 
Annals of Nuclear Medicine Vol. 13, No. 4, 235-239, 1999 
Pharmacokinetic analysis of 123I-labeled medium chain fatty acid as a radiopharmaceutical for hepatic function based on beta-oxidation 

Nono YAMAMURA Yasuhiro MAGATA,** Fumiyoshi YAMASHITA,*** Mitsuru HASHIDA*** and Hideo SAJI* 
*Department of Patho-Functional Bioanalysis, Greduate School of Pharmaceutical Sciences, * *Department of Nuclear Medicine, Graduate School of Medicine, ***Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University 

Beta-oxidation is the most important pathway to provide energy for the liver. Our recent findings indicated that radiolabeled medium chain fatty acid analogs could be used as radiopharmaceuticals in the liver, allowing us to monitor alterations in energy metabolism on the cellular level. In the present study, pharmacokinetical analysis of a radioiodinated medium chain fatty acid analog, p-[123I]iodophenylenanthic acid ([123I]IPEA), was carried out in normal and hepatitis model rats to investigate the index for the measurement of beta-oxidation activity in hepatocytes. The rate constant for metabolism of [ 123I]IPEA in the liver showed a strong correlation with the ATP level, which was determined as an indicator of beta-oxidation activity in hepatocytes. The radioactivity profile in the liver after [1231]IPEA administration provided important information regarding hepatic viability, and the metabolic rate constant of [123I]IPEA calculated by a pharmacokinetic method was a useful criterion for hepatic diagnosis based on hepatic cellular energy metabolism.
 
Key words: SPECT, medium chain fatty acid, beta-oxidation, pharrnacokinetic analysis, hepatic function
INTRODUCTION 
THE DEVELOPMENT of an in vivo method for quantitative and regional assessment of hepatic viability is an important goal of clinical nuclear medicine. For this purpose, we recently developed p-[123I]iodophenyienanthic acid ([123I]IPEA) as a radiopharmaceutical for use in single photon emission computed tomography (SPECT) (Fig. 1).1 [123I]IPEA showed high initial uptake in the liver immediately after the injection. [123I]IPEA incorporated into the liver was metabolized via beta-oxidation, and its radiometabolites were rapidly eliminated from the liver into the urine. In addition, the rate of elimination of radioactivity from the liver was significantly delayed in hepatitis model rats compared with that in normal rats. Thus, in the present study, the hepatic time-activity curves obtained by dynamic scanning with a SPECT instrument were analyzed by a pharmacokinetic method in normal and hepatitis model rats, and some parameters were evaluated as the criteria for determining liver function. 

Received February 18, 1999, revision accepted April 13, 1999. For reprint contact: Hideo Saji. Ph.D., Department of Patho-Functional Bioanalysis. Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501 , JAPAN. 

MATERIALS AND METHODS 
Materials 
Sodium [125I]iodide was purchased from Daiichi Pure Chemical Co. Ltd., Chiba, Japan. Ammonium [123I]iodide was kindly provided by Nihon Medi-Physics Co., Hyogo, Japan. All chemicals were of reagent grade and were used as received. 

Synthesis of Radioiodinated IPEA 
IPEA was synthesized by iodination of 7-phenylheptanoic acid which was synthesized by Friedel-Crafts reaction from benzene and pimelic acid followed by reduction. Details of the synthetic procedure will be published elsewhere. [123I]IPEA and [125I]IPEA were prepared by the 1271-1231 or 1271-1251 exchange reaction with non-radio-active IPEA as a precursor. The radiochemical purity of each radioiodinated IPEA was more than 96% as deter-mined by high performance liquid chromatography (HPLC) on a reverse-phase column (Cosmosil 5C18-AR-300, 250 mm x 10 mm, Nacalai Tesque. Co., Ltd.. Kyoto) with a mobile phase of acetonitrile/water (70/30, v/v) at 30dig. (Rt. = 32 min). 

Animals 
Animal experiments were conducted in accordance with our institutional guidelines and were approved by the Kyoto University Animal Care Committee. Male Wistar rats (200-250 g) were housed for 1 week under a 12-h light/12-h dark cycle and had free access to food and water. Hepatitis model rats were generated by administration of carbon tetrachloride (CCI4).2 Briefly, rats were orally administered 30% CCI4 (O.5-1.5 ml CCI4/ kg) in olive oil (v/v) after a 6-h fast. After fasting for a further 2 h, the animals were fed ad libitum. 

Metabolite Analysis Four normal rats anesthetized with pentobarbital were administered 74 kBq of [125I]IPEA via the tail vein. At 20 min post-injection, the rats were sacrificed by decapitation, and the liver was excised immediately. The total lipids in the liver samples were extracted with chloroform/methanol/water (4/2/2) as reported by Forch et al.3 The organic layer and water-soluble layer were separated, and radioactivity in each was quantified. Then the metabolites in the each layer were analyzed by TLC (reverse-phase TLC plate LKCl8F, Whatman International Ltd., Kent, U.K.) developed with acetonitrile/water/acetate (7/3/0.01).
 
Dynamic Scanning of [123I]IPEA 
Imaging studies were performed in 12 rats with a SPECT instrument (SPECT 2000H-40, Hitachi Medical Co., Japan). Normal rats and those treated with different doses of CCI4 were anesthetized with pentobarbital (50 mg/kg i.p.). Dynamic planar scanning (20 sec x 30 frames, 1 min x 10 frames, 2 min x 20 frames) was initiated at the time of [123I]IPEA (18 MBq) injection via the tail vein. Regions of interest (ROI) were chosen on the upper half of the liver images to eliminate the influence of radioactivity from the kidneys, and the time-activity curves were obtained after decay correction. After scanning, liver samples of about 100 mg were excised from each rat, and the ATP concentration in the 
liver was measured according to the procedure reported by Sellevold et al. with minor modifications.4 Briefly, the liver sample was immediately frozen and powdered in liquid nitrogen. Then the ATP in the powdered liver was extracted with 3 ml of perchloric acid (O.42 M). After precipitation of proteins and neutralization with potassium hydroxide, ATP was quantified by HPLC on a reverse-phase column (Cosmosil 5C18-AR-300, 150 mm x 4.6 mm, Nacalai Tesque, Co., Ltd., Kyoto) with a mobile phase containing potassium dihydrogen phos-phate 2 15 mM, tetrabutylammonium hydrogen sulfate 2.3 mM, acetonitrile 1O% and potassium hydroxide to adjust the pH to 6.0. The flow rate was maintained at 1.O ml/min, and the spectrophotometer was set at 254 nm. The 12 rats used in this study were divided into 3_ groups according to the ATP concentration in the liver as follows: severe injury group, ATP levels of less than 80 umol/1OO g wet liver; moderate injury group, from 80 to 11O ,umol/100 g wet liver; and normal group more than 11O ,umol/100 g wet liver. Each of these 3 groups consisted of 4 rats. 
Data Analysis (Pharmacokinetic Model) Pharmacokinetic models were constructed based on the following assumptions: (i) [123I]IPEA in the blood was transported into hepatocytes monoexponentially; (ii) [123I]IPEA in the hepatocytes was metabolized by one-way metabolism; (iii) the radiometabolites in the hepato-cytes were eliminated from the liver only into blood; and (iv) the radiometabolites in blood were not re-accumu-lated in hepatocytes. Two pharmacokinetic models are depicted schematically in Figure 2. Symbols and the mass-balance equation are given in Tables 1 and 2, respectively. Model 1 repre-sents clearance of [123I]IPEA from the blood of whole body. Model 2 represents hepatic uptake, metabolism and excretion. The constants ka and k, are the rate constants for liver uptake and elimination by the other organs, respectively. Eq. (1) and (2) were obtained from the mass-balance equation shown in Table 2, and Eq. (2) indicated that [123I]IPEA in the blood was transported into hepato-cytes monoexponentially. Thus, when ka ¥X and ka + ke were replaced by A and k1, respectively, Eq (2) was simplified by A ¥ e~kl 't as the input function of [1231]IPEA to the liver. 
The following first-order constants were used in Model 2 (Fig. 2): k2 for elimination of intact [123I]IPEA from hepatocytes; k3 for metabolism of [123I]IPEA in hepato-cytes; and k4 for elimination of radiometabolites from hepatocytes. Solving the differential equations for Model Since it was impossible to distinguish radioactivity from intact [ 123I]IPEA and that from radiometabolites in the hepatocytes by external imaging, the information obtained from the hepatic images on radioactivity represents the sum of them (X1 + X2). The equation for the amount of radioactivity in the liver (X1 + X2) (Eq. 5) was fitted to the radioactivity profiles obtained by dynamic scanning of [123I]IPEA by the non-linear least-squares method (MULTI),5 and constants k1, k2, k3, k4 and A were determined. 

RESULTS AND DISCUSSION 
Figure 3 shows the mean time-activity profiles of the livers obtained by dynamic scanning of each group. These results are shown by the relative radioactivity in the liver in order to evaluate the clearance rate. The normal group showed a higher initial uptake and faster clearance in the liver than the CCl4-treated group. Although about 44% of the maximum radioactivity uptake in the liver remained in the normal rats at 60 min after injection, this might be the result of the slower elimination from the liver of the radiometabolite rather than its production through beta-oxidation and/or of the relatively high background seen in the planar image. All of these time-activity profiles showed two phase clearance. Although the second phase clearance was not clearly different among these groups, the first phase clearance was delayed depending on the degree of injury due to CCl4. That is, at each clearance point in the severe injury group and after 5 min of moderate injury the portion of radioactivity remaining in the liver was significantly higher than that in the normal group. Administration of CCl4 causes peroxidation of fat, which causes the collapse of systems enclosed by mem-branes including mitochondria 6,7 The enzymes involved in beta-oxidation are located in the mitochondrial matrix.8 Therefore, the model animals used in the present study were expected to suffer damage to the beta-oxidation system, although other injuries also seemed to have occurred. Therefore, the delay in radioactivity clearance observed in the present study may have been responsible for the lack of normal beta-oxidation. The models shown in Figure 2 were therefore established and the time-activity profiles were fitted based on these models by using the MULTI program. The fitted values for parameters in each group are shown in Table 3. Metabolite analysis showed that 85.5% of radioactivity in the normal rat liver was derived from radiometabolites of [123I]IPEA. This percentage matched the calculated X2 /(X1+X2) x 100 value for normal rats based on Eq. 5(Table 4). Therefore, since the present pharmacokinetic model was considered appropriate, it was also applied to experimental data analyses for hepatitis model rats. For further investigation on the index of beta-oxidation activity in hepatocytes, the time-radioactivity profile of each animal was fitted individually. The rate constant k3 was considered to be related to beta-oxidation in hepatocytes based on Model 2. On the other hand, since beta-oxidation is the main pathway responsible for production of ATP in the hepatocytes 9,10 the level of ATP in the liver was regarded as an indicator of the beta-oxidation activity in hepatocytes. Therefore, the relationship between the first-order constant k3 for the metabolic rate of [123I]IPEA in hepatocytes and the ATP concentration in the liver was evaluated as shown in Figure 4. There was a good correlation between k3 and the ATP level (r = 0.769). This indicated that k3 is a potentially useful criterion which allows determination of the beta-oxidation activity in hepatocytes externally after [123I]IPEA administration. The metabolic rate via the beta-oxidation in the liver might be influenced by the anesthetic. But in order to eliminate the effect of the anesthetic, in all experiments in the present study the same anesthetic was used, and further study of the anesthetic may be required to elucidate its effect. In conclusion, when [123I]IPEA was administered to rats, the degree of injury in hepatocytes due to CCl4 could be detected as the delay in radioactivity clearance from the liver. Furthermore, pharmacokinetic analysis indicated that the rate constant k3 for metabolism of [123I]IPEA could be used to quantify hepatocyte viability based on beta-oxidation activity. 

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