John F. Harwig

Differences in the sites of iodination of proteins following four methods of radioiodination.

The rate of deiodination of radioiodinated proteins varies with the method of iodination. To elucidate differences in the iodinated protein labeled by various methods, we have hydrolyzed fibrinogen and several small peptides iodinated by the iodine monochloride, chloramine-T, electrolytic and enzymatic methods. Under conditions of either acidic or basic proteolysis, extensive deiodination occurred and the major product was I-. When a protease of Streptomyces griseus was used, radio-iodinated fibrinogen and other polypeptides were degraded to single iodinated amino acid residues and only a small yield of I-. The iodinated amino acids resulting from proteolysis were separated by ion-exchange chromatography. The iodine monochloride and enzymatic methods yielded largely iodotyrosine with small amounts of other iodinated amino acids. The chloramine-T product spectrum varied with the chloramine-T:protein ratio, whereas the electrolytic method yield was a complex function of the reaction conditions. The different methods of iodination lead to some differences in the site of iodination which correlate with stability of the protein-iodine bond.

Effect of iodination level on the properties of radioiodinated fibrinogen

Abstract The effect of iodination level on the biological clearance of radioiodinated fibrinogen was investigated. Canine, human and rabbit fibrinogen were included in the study to also test for possible differences among various species. Iodination of fibrinogen with 3 to 9 iodine atoms/molecule resulted in essentially unchanged isotopic clottability as compared to preparations at the conventional level of 0.5 iodine atom/molecule. No changes in molecular size due to covalent aggregation were observed by SDS gel electrophoresis with radioiodinated human fibrinogen preparations. Canine and human fibrinogen iodinated with up to 4.5 iodine atoms/molecule exhibited little change in biological clearance behavior relative to the conventional iodination level. However, rabbit fibrinogen iodinated with 3.5 iodine atoms/molecule displayed a difference in clearance when compared to the 0.5 iodine atom/molecule preparations. Thus, canine and human fibrinogen can be iodinated with 4.5 iodine atoms/molecule without altering their biological clearance behavior while rabbit fibrinogen is more sensitive to this iodination level.

Preparation and in vitro properties of 99mTc-fibrinogen

Abstract We have developed a simple, mild and efficient electrolytic labeling technique for preparing 99mTc-fibrinogen for use in the radioisotopic detection of deep vein thrombosis. The method employs tin electrodes with in situ generation of Sn2+ as intermediate reducing agent for 99mTcO4−. Wide differences in labeling behavior are observed as the reaction conditions are varied. The optimum labeling system consists of 2 mg fibrinogen and the desired level of Na 99mTcO4 in 2 ml 0.01 M phosphate-0.15 M NaCl buffer, pH 6.0. The 99mTc-fibrinogen labeling efficiency is 70–80%, with a total preparation time of ca. 25 min. Sepharose-4B gel chromatography indicates that the non-protein bound radioactivity is in the form of a chelate derived from anions of the buffer system, with no free unreduced 99mTcO4− present. The labeled fibrinogen is easily separated and purified by ammonium sulfate precipitation and shows good in vitro stability. The isotopic clottability of the product is 50–65%, depending on the species of fibrinogen. When prepared by the method reported here, 99mTc-fibrinogen appears to be a potentially useful radiopharmaceutical for external scintillation imaging of deep vein thrombi in patients.

Serial Radionuclide Determinations of the Ejection Fraction with 99mTc-Labeled Red Blood Cells

The reproducibility of area/length ejection fraction determinations was evaluated in 21 subjects using 99mTc-labeled red blood cells (99mTc-RBCs). The ejection fraction was determined within 30 minutes and 3-5 hours after a single injection of 99mTc-RBCs. The mean absolute difference between the initial and subsequent ejection fractions was 7% (maximum, 15%) (r = 0.90). 99mTc-RBCs offer a reproducible method of determining the ejection fraction which is suitable for serial noninvasive evaluations of left ventricular function.

Development and Evaluation of a Rapid and Efficient Electrolytic Preparation of 99mTc-Labeled Red Blood Cells*

Abstract This paper reports a new, simple and reproducible method for routine preparation of 99 m Tc-labeled red blood cells (RBC). This method employs electrolytic generation of minute quantities of stannous ion to serve as the reducing agent for 99 m Tc-pertechnetate. The RBC are incubated with Sn(II), washed with saline, incubated with Na 99 m TcO 4 , and washed again with saline. The entire procedure requires about 45 min and consistently provides labeling efficiencies above 90%. The half-clearance time in the blood in humans is approximately 24 hr. Activity released from the labeled RBC in vivo is largely in chelate form which is rapidly excreted in the urine. High quality blood pool images can be obtained for many hours after injection. In a damaged form, 99 m Tc-RBC provide spleen images of diagnostic quality. This 99 m Tc-RBC preparation appears to have good potential for routine use.

123I-labeled soluble fibrin: preparation and comparison with other thrombus imaging agents.

Abstract Radioiodinated soluble fibrin, a derivative of radioiodinated fibrinogen, has been investigated for thrombus localization. Soluble fibrin can be labeled with 123 I as the radionuclide for imaging thrombi with a scintillation camera. Protein iodination grade 123 I is produced by a distillation and extraction procedure from the product of the 121 Sb ( α , 2 n ) 123 I reaction in the Washington University 54-in. cyclotron. Labeling with these 123 I preparation is effected by the iodine monochloride method. Induced 4–6-hr-old femoral vein thrombi in dogs can be visualized beginning 2 hr after injection of 1 mCi of 123 I-labeled soluble fibrin. This new radiopharmaceutical may be useful for imaging actively-propagating thrombi in patients, especially in areas of large blood pool. Its potential appears to be similar to that of 123 I-labeled highly iodinated fibrinogen, another new thrombus imaging agent.

Solid phase bovine thrombin. Preparation and properties.

A new solid-phase thrombin (EC 3.4.21.5) was prepared through conjugation of the enzyme under mild conditions to a glass support bearing an active ester of N-hydroxysuccinimide. The immobilized enzyme retained 50 +/- 10% of the specific esterase activity of the parent soluble enzyme. The Km (apparent) for the esterase activity of the immobilized enzyme has a value of 5 mM, identical of the Km value of the parent-soluble enzyme. Only 6 +/- 1% of the specific proteolytic activity was retained and a higher Km (apparent) value of 67 muM was obtained for the insoluble enzyme compared to Km value of 12.5 muM for the parent soluble thrombin. Solid-phase thrombin prepared by the diazocoupling technique was previously reported to retain only 3% of the specific proteolytic activity. The observed loss of specific proteolytic activity can be attributed to steric interference, a change in charge characteristics, or both. Nevertheless, the present method of preparation has the advantages of rapidity and simplicity. It can readily be adapted to use for studying the fate of various complexes of fibrinogen, fibrin and their degradation products. It should also be useful for preparing radiolabeled autologous soluble fibrin for thrombus detection in patients undergoing active thrombosis.