versão impressa ISSN 1516-8913
Braz. arch. biol. technol. v.51 n.spe Curitiba dez. 2008
DRUG INTERACTION AND SURGERY, AND THE BIODISTRIBUTION OF RADIOPHARMACEUTICAL
Kércia Regina Santos Gomes PereiraI; Maria Kadja Meneses Torres AçucenaI; Arthur Villarim NetoI; Amália Cínthia Meneses RêgoI; Mário Bernardo-FilhoII; Ítalo Medeiros AzevedoIII; Irami Araújo FilhoIII; Aldo Cunha MedeirosIII,*
ICentro de Ciências da Saúde; Universidade Federal do Rio Grande do Norte; Natal - RN - Brasil
IIDepartamento de Biofísica e Biometria; Instituto de Biologia Roberto Alcantara Gomes; Universidade do Estado do Rio de Janeiro; Av. 28 de Setembro, 87; 20551030; Rio de Janeiro - RJ - Brasil
IIIDepartamento de Cirurgia; Universidade Federal do Rio Grande do Norte; Av. Nilo Peçanha, s/n; email@example.com; 59012300; Natal - RN - Brasil
Drugs and surgery can interfere with the biodistribution of radiopharmaceuticals and data about the effect of splenectomy on the metabolism of phytate-Tc-99m are scarce. This study aimed at evaluating the interference of splenectomy on phytate-Tc-99m biodistribution and liver function in rats. The SP group rats (n=6) underwent splenectomy. In group C (control) the animals were not operated on. After 15 days, all rats were injected with 0.1mL of Tc-99m-phytate via orbital plexus (0.66MBq). After 30 minutes, liver samples were harvested, weighed and the percentage of radioactivity per gram (%ATI/g) was determined by a Wizard Perkin-Elme gama counter. The ATI%/g in splenectomized rats (0.99±0.02) was significantly higher than in controls (0.4±0.02), (p=0.034). ALT, AST and HDL were significantly lower in SP rats (p= 0.001) and leukocytosis was observed in SP rats. In conclusion, splenectomy in rats changed the hepatic biodistribution of Tc-99m-phytate and liver enzimatic activity.
Keywords: splenectomy, sodium phytate, biodistribution, liver
O radiofármaco fitato-Tc-99m é usado no diagnóstico através de exames de imagem, na dependência de sua biodistribuição. O objetivo do trabalho foi avaliar efeito da esplenectomia na biodistribuição do fitato-Tc-99m e função hepática em ratos Wistar. Sob anestesia e técnica asséptica, os animais do grupo SP (n=6) foram esplenectomizados. Grupo C(controle; n=6) não operado. Após 15 dias, injeção de 0,1ml de fitato-Tc-99m via plexo orbital (0,66MBq). Após 30 minutos, retiradas biópsias hepáticas para determinação do percentual de radioatividade/grama (% ATI/g), usando-se contador gama WizardPerkin-Elmer®. Realizada dosagem de ALT, AST e HDL, e leucometria. Estatística pelo teste t, significância 0,05. O %ATI/g nos ratos esplenectomizados foi 0,99 ± 0,2 e nos controles 0,40 ± 0,2 (p=0,034). ALT, AST e HDL tiveram dosagens significativamente menores nos esplenectomizados (p=0,01), com leucocitose, comparando com controles. Em conclusão, em ratos a esplenectomia provocou alteração na captação de fitato-Tc-99m pelo fígado, coincidindo com alteração da função hepática.
Palavras-Chave: esplenectomia, fitato de sódio, biodistribuição, fígado
The spleen is the largest lymphoid organ in the body. Its functions are many, but they are generally related to 1 of 4 categories: filtration, immunologic, reservoir, and hematopoietic. Many of the spleen's immunologic functions, therefore, are common to other immunologic organs. For example, the spleen is efficient at removing nonopsonized bacteria, mostly encapsulated organisms (Müftüoğlu et al., 2000). Splenectomy is frequently performed for a multitude of reasons, including trauma and various pathologic processes. Blunt abdominal trauma remains the most common indication for splenectomy, but patients with a variety of hematologic disorders also benefit from this procedure. Loss of the spleen, however, leads to a lifelong higher risk of sepsis and severe infection (Bader-Meunier et al., 2001, Waghorn et al., 1997) and may be associated with an increased rate of thromboembolic complications, enhanced arteriosclerosis, and late coronary heart disease (Schilling, 1997). It has been reported that splenectomy might promote hepatic regeneration, prevent liver fibrosis (Akahoshi et al., 2002, Murata et al., 2001), reduce serum bilirubin concentration and improve liver function (Shimada et al., 2000, Lin et al., 1999). However, considering the relationship between the spleen and liver physiology, nuclear medicine may have an important role in studying the diagnosis and physiology of the liver after splenectomy.
Since its introduction a number of years ago, Tc-99m-phytate colloid has been used as an imaging agent for the liver and spleen (Huet et al., 1980). Subjective assessment of parameters such as liver dimension, colloid shift, and radiopharmaceutical uptake in the bone marrow have been used for both liver disease diagnosis and evaluation of its progression. Subjective assessment of radiocolloid distribution has been shown to be unreliable, and quantitative techniques have been used to evaluate liver function (Jago et al., 1987). The distribution of radiocolloid uptake in the liver and bone marrow has been shown to correlate well with the severity of chronic liver disease, the severity of histologic fibrosis, prognosis, and hepatic function (Hoefs et al., 1997). Thus, quantitation of liver uptake of 99mTc-phytate colloid provides a practical index of hepatic function, by using planar and single photon emission computed tomography (SPECT) techniques (Strauss et al., 1984). The ability to quantitate individual organ volumes and radiopharmaceutical concentrations with SPECT (Front et al., 1987, Iosilevsky et al., 1989) stimulates the use of Tc-99m-phytate colloid scintigraphy of the liver as a quantitative test of hepatic function. Radiopharceutical biodistribution may provide important information about its uptake to some target organs but post -surgery data are scarce (Chacon et al., 2007, Araújo-Filho et al., 2007).
This study aimed at evaluating if splenectomy interferes with liver function and the biodistribution of Tc-99m-fytate in the liver of Wistar rats.
MATERIAL AND METHODS
Male Wistar rats weighing 274±21g were obtained from the Center for Health Sciences vivarium, Universidade Federal do Rio Grande do Norte (UFRN), Brazil. The animals were housed in polypropylene cages and fed standard rat chow and water ad libitum. Prior to surgery, the rats were fasted overnight in separate cages. After 7 days of acclimatization, they were allocated to two groups of 6 animals each. The groups were denominated splenectomy (SP) and control (C). The protocol for this study was approved by the Institutional Animal Care Committee, and the research was performed in accordance with Brazilian College of Animal Experimentation guidelines
Surgical procedure for splenectomy
The rats were anesthetized with ketamine 50 mg/kg IM and thiopental (20 mg/kg IP), shaved and placed on an operating board and secured with tape. Midline laparotomy (3cm) was performed after skin sterilization with 70% ethanol. The animals were then covered with a small sterile drape with a 6cm circular opening. In group SP (n=6) the spleen was identified and resected after ligature of the splenic vessels with vicryl 5-0 (Ethicon, São Paulo, Brazil). The peritoneal cavity was irrigated with warm (37 ºC) normal saline. The laparotomy wound was closed with 4-0 nylon in layers and individual rats were placed in separate cages. They were allowed water ad libitum and rat chow 24 h postoperatively. The C rats (n=6) were neither anesthetized nor operated on. Hydration was done with normal saline (10 mL/100g weight) injected subcutaneously in the back of the rats for the first two postoperative days. Postoperative pain was treated with tenoxicam (Roche Pharm., Brazil); 10 mg/kg was given i.m. to the rats once a day for three days.
Body weight and clinical observation
Body weight was monitored weekly throughout the entire 15-day experimental period. Activity, mucosa and skin color were observed daily.
Sodium pertechnetate (Na99mTcO4) was obtained by elution of a 99Mo/99mTc generator (Instituto de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, Brazil), and 99mTc-phytate was prepared. On the 15th day all the animals were anesthetized again, and injected with 0.lmL of 99mTc-phytate in the orbital plexus, corresponding to radioactivity of 0.66MBq. After 30 minutes, blood was collected by cardiac puncture for dosages and the animals were killed by lethal dose of anesthetic. Samples of the liver were harvested. The samples were washed in 0.9% saline, weighed on a high-precision digital scale (Bel-Mark 160-II Itália®) and subjected to radioactivity detection using a 1470 WizardTM Gamma Counter -Perkin-Elmer, Finland, with automatic correction of radiation decline. The percentage of radioactivity/g (%ATI/g) of each organ was calculated by dividing the activity/g of the hepatic tissue by the total activity administered to each animal.
A blood sample was used for hematological analysis of white blood cells (Abbot Cell Dyn 3500 automatic analyzer) and for the serum measure of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactic desydrogenase activity, we used the Konelab 60i spectrophotometer (assay kit from Weiner, São Paulo, Brazil).
The data were expressed as mean±standard deviation (SD). The groups were compared using ANOVA and the post-hoc Student test, considering a significance level of p<0.05.
Postsplenectomy symptoms such as hematuria, pale mucosa and lethargy were observed in 3 animals but not in control rats. We observed no significant body weight loss in splenectomized rats, compared to controls. Radioactivity per gram of liver tissue ((%ATI/g) was increased by prior splenectomy in the SP group rats (0.99±0.2), when compared to the control (C) group (0.4±0.2). The difference between the two groups was significant (p=0.034; Table 1).
To examine the effects of prior splenectomy on liver function, we assessed the levels of ALT, AST and LDH activity, as a marker of liver injury, at 15 days postsplenectomy (Table 1). In splenectomized rats, ALT, AST and LDH values were markedly reduced, compared with the control group rats (p=0.001; Table 1). In addition, splenectomy resulted in increased white blood cell counts in the SP rats (13.8± 2.7 k/L), when compared to the C rats (2.5±0.9 k/L; Table 1).
Based on current knowledge, there is no doubt that the spleen is a lymphoid organ. Filtration, immunology, reservoir, and hematopoiesis are such important functions, that partial splenectomy or the preservation of the organ by suture is indicated in cases of traumatic injuries (Resende and Petroianu, 2003). Various complications and beneficial effects of the operation are described in postoperative splenectomy (Cadili and Gara., 2008). Splenectomy might promote hepatic regeneration to a certain extent and prevent liver fibrosis (Murata et al., 2001, Chen et al., 1998).
In this study we demonstrated that in splenectomized rats the biodistribution of 99mTcphytate to the liver was higher than in controls, suggesting that the operation favored the hepatic uptake of the radiopharmaceutical. This result coincided with the improvement in liver function, confirmed by the better alanine aminotransferase, aspartate aminotransferase and lactic dehydrogenase activities in splenectomized rats, compared with controls. In some studies splenectomy promoted hepatic regeneration (Akahoshi et al., 2002, Murata et al., 2001), prevented liver fibrosis (Chen et al., 1998), reduced serum bilirubin concentration and, consequently, reflected the effect of splenectomy on reducing the burden of hepatocyte bilirubin metabolism, improving liver function (Shimada et al, 2000, Lin et al., 1999).
Tomikawa et al (1996) reported that splenectomy increased hepatocyte growth factor (HGF) activities in plasma, suggesting that the spleen played an inhibitory role in hepatic regeneration. HGF, first identified as the most potent mitogen for primary hepatocytes, not only stimulates hepatic regeneration but also accelerates liver function. Moreover, the fact that a large amount of splenic tissue connects to liver tissue through the portal vein system, suggests the existence of a humoral factor originating in the spleen, which thus inhibits hepatic regeneration and promotes liver fibrosis. In rats submitted do hepatic ischemia/reperfusion, prior splenectomy ameliorated acute multiple organ damage (Jiang et al., 2007). These findings, associated to the favorable serum activity of ALT, AST and LDH in our splenectomized rats, partially explain the high hepatic biodistribution of 99mTc-phytate in the operated animals.
The postsplenectomy diagnosis of sepsis based on elevated white blood cell (WBC) counts is confounded by the fact that leukocytosis is considered a physiologic response to splenectomy (Horowitz et al., 1992). Some reports suggest that postsplenectomy WBC counts in patients with sepsis are greater and more persistent than the WBC in patients without sepsis (Weng et al., 2005, Rutherford et al., 1994).
In this study we were not able to diagnose sepsis in the splenectomized rats. Nevertheless, all the operated animals had leukocytosis (WBC ranging from 11 to 18 k/L) in the 15th postoperative day. It has been reported that by the fifth day following post-traumatic splenectomy, the WBC count may help the physician to confirm the development of sepsis and the need for treatment. If the WBC is greater than 15x103/L on that day, the physician should seriously consider treatment by empiric antibiotics and further diagnostic follow-up to prevent the untoward sequelae of postsplenectomy sepsis (Toutouzas et al., 2002). In conclusion, splenectomy improved the liver uptake of Tc-99m-phyate in rats, coinciding with changes in hepatic enzymatic activity.
The authors thank Michael Germain, from Canada, for his help in revision of English language.
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Received: August 18, 2008;
Revised: September 16, 2008;
Accepted: September 18, 2008
* Author for correspondence