Uccelli L, Giganti M, Duatti A, Bolzati C, Pasqualini R, Cittanti C, Colamussi P, Piffanelli A
Nuclear Medicine Laboratory, University of Ferrara, Italy.
J Nucl Med. 1995 Nov;36(11):2075-9.
The aim of this study was to determine the subcellular distribution of bis(N-ethoxy N-ethyl)dithiocarbamato nitrido technetium(V) (99mTcN-NOEt) in rat heart by differential centrifugation techniques. Extraction of the activity from homogenized rat heart tissue was also performed to assess whether myocardial retention might induce changes in the chemical identity of the complex.
Anesthetized rats were intravenously injected with 99mTcN-NOEt, the heart tissue was extracted and homogenized and tissue fractions were obtained by differential centrifugation. The efficiency of organelle separation was determined by assay of each centrifugal fraction using enzyme markers. Lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (ALP) and 5'-nucleotidase (5'ND) activities were assayed using standard spectrophotometric methods. Succinic dehydrogenase (SDH) activity was determined using a p-iodo-nitrotetrazolium-linked assay. Severe cell membrane and organelle disruption were induced by prolonging the homogenization time and their effect on the subcellular distribution of 99mTcN-NOEt was studied. The activity from homogenized heart tissue was extracted using the Folch technique and analyzed by TLC and HPLC.
Most of the 99mTcN-NOEt activity was found to be associated with the hydrophobic components of the cell. No evidence of specific association of activity with the cytosolic and mitochondrial components was observed. Organelle and membrane cleavage did not cause release of activity into the cytosol. Approximately 90% of 99mTcN-NOEt activity was extracted from ventricular tissue and the chemical nature of 99mTcN-NOEt was not altered by uptake by myocardium.
Cell membranes are the most apparent site of localization of 99mTcN-NOEt in heart tissue.
本研究的目的是通过差速离心技术确定双（N - 乙氧基N - 乙基）二硫代氨基甲酰亚硝酰锝（V）（99mTcN - NOEt）在大鼠心脏中的亚细胞分布。还对匀浆后的大鼠心脏组织进行了活性提取，以评估心肌滞留是否会引起复合物化学特性的变化。
对麻醉的大鼠静脉注射99mTcN - NOEt，提取心脏组织并匀浆，通过差速离心获得组织级分。使用酶标志物对每个离心级分进行测定来确定细胞器分离的效率。采用标准分光光度法测定乳酸脱氢酶（LDH）、酸性磷酸酶（ACP）、碱性磷酸酶（ALP）和5'-核苷酸酶（5'ND）的活性。使用对碘硝基四氮唑连接法测定琥珀酸脱氢酶（SDH）的活性。通过延长匀浆时间诱导严重的细胞膜和细胞器破坏，并研究其对99mTcN - NOEt亚细胞分布的影响。使用Folch技术从匀浆的心脏组织中提取活性，并通过薄层层析（TLC）和高效液相色谱（HPLC）进行分析。
发现大部分99mTcN - NOEt活性与细胞的疏水成分相关。未观察到活性与胞质和线粒体成分有特异性结合的证据。细胞器和膜的裂解未导致活性释放到胞质溶胶中。约90%的99mTcN - NOEt活性从心室组织中提取出来，并且99mTcN - NOEt的化学性质不会因心肌摄取而改变。
细胞膜是99mTcN - NOEt在心脏组织中最明显的定位部位。
