Mary Jane Izzo

High Specific Activity Labeling of Protein with I131 by the Iodine Monochloride Method.

Summary A modified iodine monochloride method suitable for preparing I131-labeled proteins of a high degree of radioactivity is described, and results are given. Hydrogen peroxide present in high level I131 preparations is destroyed with catalase. Then IC1 is added to a mixture of the I131 as iodide and the protein to be iodinated. Total iodine content of I131 preparations sets a limit on the specific activity of I131-labeled proteins that can be achieved with a low degree of iodination. In the two commonly used methods for producing I131 (fission of U235 and thermal neutron irradiation of natural tellurium) stable I127 and long-lived I129 are also formed. Analysis showed the total iodine content of fission product I131, as received from Oak Ridge National Laboratory soon after processing, to average 2.4 μg per 100 mc, 3 times the amount present as I131 (0.8 μg/100 mc). For I131 produced from tellurium it was substantially greater. Since the ratio of total iodine to I131 increases with time after processing, freshly produced I131 is necessary to make very high level labeled preparations. Precautions to prevent protein damage as a result of high level labeling procedures are described.

I131-labeling of proteins at high activity level with I131Cl produced by oxidation of total iodine in NaI131 preparations☆

A procedure is described for oxidizing the total iodine in “carrier-free” NaI131 preparations to ICl under conditions such that the resulting solution can be used directly for production of I131-labeled proteins of high specific activity and a low degree of iodination. When I131Cl is formed for this purpose by an exchange reaction between inactive ICl and NaI131, the ratio of total iodine in a very radioactive NaI131 preparation to that in the ICl may be so high that only a small percentage of the radioactivity is introduced into the protein. This inefficient utilization of I131 is avoided in the described procedure. The efficiency of incorporation of radioactivity in the protein is comparable to that which is achieved when inactive ICl is used in the presence of a tracer quantity of NaI131 to iodinate the protein.

Subcellular distribution of intraportally injected 125I-labeled insulin in rat liver☆

Abstract Time course studies revealed that at 30 s after intraportal injection of 200 μU of 125I-labeled insulin per 100 g rat 47.9 ± 2.8% of the injected radioactivity was recovered from the liver homogenate by precipitation with trichloroacetic acid. Trichloroacetic acid precipitable radioactivity declined to very low levels during the next 30 min whereas trichloroacetic acid soluble radioactivity reached a peak value of 9.56 ± 1.9% at 5 min and declined gradually thereafter. At 30 s mean peak accumulations ±SE of 6.83 ± 0.42, 5.06 ± 0.27, 14.90 ± 1.85, and 3.58 ± 0.58% of injected radioactivity were recovered in trichloroacetic acid precipitates from the 700g (nuclei + debris), 10,000g (mitochondria + lysosome), 105,000g (microsomes), and supernatant (cytosol) subfractions, respectively. Mean peak values of 0.72 ± 0.08, 0.12 ± 0.02, and 1.11 ± 0.16% of injected radioactivity were recovered in the partially purified mitochondrial fraction, purified nuclei, and plasma membranes, respectively, as trichloroacetic acid precipitable material. Most of the trichloroacetic acid precipitable activities in the subfractions were immunoprecipitable. Trichloroacetic acid soluble radioactivity was found mainly in the cytosol and microsomal fractions. Peak specific activity (percentage of injected dose/mg protein × 10−3) was highest in the microsomes, intermediate in the plasma membranes, and very low in the purified nuclei and partially purified mitochondrial fraction. The specific activity of the microsomes remained at or near peak levels for 5 min after 125I-labeled insulin injection and then declined, whereas specific activity of the plasma membranes dropped precipitously to 25% of peak values at 5 min. Sephadex gel filtration of the radioactivity in the deoxycholate soluble fraction of microsomes at 5 min after 125I-labeled insulin injection resulted in the elution of a major peak (Peak I) in the region of 125I-labeled insulin and a minor peak (Peak II) in the region of the labeled A and B chains. Incubation of the fraction for 30 min at 37 °C with 3 m m reduced glutathione and 15 m m EDTA resulted in a reciprocal fall in Peak I and rise in Peak II. The data suggest that intraportally injected 125I-labeled insulin is rapidly internalized and concentrated in the rat liver microsomes. The time courses of appearance and disappearance of trichloroacetic acid precipitable radioactivity in plasma membrane and microsomes further suggest, although do not prove, that insulin binds to plasma membranes before it is internalized. They also provide presumptive evidence suggesting that the sequential degradative pathway is operative in vivo.

Localization of an 125I-Labeled Rat Transplantation Antibody in Tumors Carrying the Corresponding Antigen

Summary A method was developed for preparing antibody to Fischer-344 rat transplantation antigen, using as an antibody source serum from Buffalo rats immunized by repeated transplants of a carcinogen-induced Fischer tumor. Immune y-globulin was labeled with 125I and radioactive antibody isolated by twice repeated adsorption onto Fischer red blood cells and elution from stroma prepared from these cells. In vitro such antibody bound specifically to Fischer red blood cells. In vivo this antibody localized preferentially in the Fischer tumor grown in immunologically suppressed Buffalo rats.

Detection of left atrial thrombi

Abstract Scintillation scanning to detect atrial thrombi after intravenous administration of 131 I labeled antibodies to human fibrinogen was performed on 30 patients who had mitral valve surgery under cardiopulmonary bypass. Twenty-six patients had negative scans and at operation no thrombus was found in 25. Of the 4 patients with positive scans, 3 had left atrial thrombi at surgery and 1 had a highly calcified valve. 131 I determinations of blood, surgically removed cardiac tissue, and thrombi demonstrated that the thrombi could have as much as 25 times more 131 I than that in an equivalent amount of blood. Little 131 I accumulation was found in organized, avascular portions of the thrombus or atrial appendage, mitral valve, and tricuspid valve. Because of the high concentration of 131 I in the thrombus compared to that in the cardiac tissue and blood, this procedure seems promising as a means of detecting atrial thrombi with little discomfort to the patient. It is suggested that the use of an antiserum to rabbit gamma globulin to remove immunologically the major portion of the blood-borne 131 I rabbit antiserum to human fibrinogen may appreciably increase the ratio of thrombus to blood and this would decrease the possibility of falsenegative scans and increase the resolution of positive scans.

Disposition of 131I Proinsulin in the Rat Comparisons with 131I Insulin

The patterns of disposition of intravenously injected doses of 131I proinsulin and 131I insulin were compared in the rat with respect to plasma clearance, uptake, and degradation in selected organs and tissues, and rates of accumulation of degraded products in plasma and urine. Small doses of 131I insulin (4 mμg.per 100 gm. rat body weight) were cleared from plasma approximately twice as rapidly as large doses (4 μg. per 100 gm. rat body weight) and three times as rapidly as equi molar small doses (6 mμg. per 100 gm. rat body weight) or large doses (6μg.per 100 gm. rat body weight) of 131I proinsulin. Conversely, the relative rate of accumulation of 13lI-labeled degradation products in plasma after injection of low doses of 13lI insulin was about twice as rapid as the rate of accumulation after large doses and about three times as rapid as the rate of accumulation after either small or large doses of 13lI proinsulin. Peak uptake and degradation in the liver at one minute was greatest after injection of low doses of 13lI insulin (28.8 ± 2.5 per cent of injected radioactivity), less after injection of large doses (15.1 ±1.5 per cent), and least after injection of low doses (5.5 ± 0.9 per cent) or high doses (4.6 ±0.6 per cent) of 131I proinsulin. In contrast, peak uptake and degradation in the kidneys at seven to eleven minutes was greatest after injection of low doses (24.7 ± 2.7 per cent) or high doses (27.5 ± 1.2 per cent of 13lI proinsulin), least after injection of low doses (9.6 ±0.6 per cent) of 13lI insulin, and intermediate after injection of high doses (17.5 ±2.6 per cent). No large differences were noted in patterns of uptake of radioactivity in muscle, fat, or skin compartments. Excretion of degraded products in urine was more delayed after 13lI proinsulin injections. An inverse relationship was noted between initial concentration of hormone in plasma and uptake by the liver. Compared to 131I insulin, the hypoglycemie effect of 13lI proinsulin was weaker (58 per cent) and more delayed in onset. No evidence of conversion of l3lI proinsulin to 131I insulin was noted. The studies indicate that the differences in dose dependency in plasma clearance and degradation of 131I insulin and 131I proinsulin can be attributed to differences in relative uptakes and degradation by the liver and kidneys. The liver appeared to be the major organ involved in the removal and degradation of insulin, whereas the kidney appeared to be the major organ involved in the case of proinsulin. The hepatic process was rapid but relatively saturable, whereas the kidney process was slower but relatively unsaturable. The inverse relationship between initial concentration of hormone in plasma and uptake by liver suggests that the ratio may provide a sensitive index of the role of the liver in plasma hormone clearance.

Persistent Grossly Elevated Plasma Immunoglobulin A Levels in Untreated Streptozocin-Induced Diabetic Rats

Experimental diabetes mellitus was induced in adult male and female rats by injecting streptozocin (STZ; 60 mg/kg i.p.) in preparation for a screening survey of changes in the pattern of undenatured plasma proteins, as revealed by two-dimensional (2-D) gel electrophoresis followed by silver staining. As early as 8–12 days later, the 2-D gels revealed three high-molecular-weight plasma protein spots, which persisted for 150 days in the blood of untreated diabetic rats. Such spots were not seen in plasma of normal control rats. Evidence is presented for the presumptive characterization of these proteins as oligomers of immunoglobulin A (IgA). Specific measurement of total IgA content of diabetic plasma samples by single-radial immunodiffusion, after reduction with dithiothreitol and alkylation with iodoacetamide, reveals that IgA content increases linearly from control values of 11.1 ± 4.6 to 358 ± 249 mg/dl (means ± SE) 21 days after STZ and persists at these high levels for as long as 150 days. Diabetic rats injected daily with insulin showed IgA levels only two to four times higher than normal. Neither experiments designed to quantitate the rates of clearance (catabolism plus excretion) of 125I-labeled secretory IgA from the circulation of normal and diabetic rats nor measurement of total IgA in the bile from diabetic and normal bile fistula rats supports the view that slowed clearance from the circulation or impaired biliary excretion in the diabetic rat causes observed gross hyperimmunoglobulinemia A.

Localization in Rat Skin Transplants of Purified 125l-Labeled Xenogeneic Histocompatibility Antibody

Summary It is shown that appropriate xenogeneic rabbit and horse antiserum, as well as allogeneic rat serum, can serve as sources from which 125I-labeled antibody to a strong histocompatibility antigen expressed on Fischer-344 strain rat cells can be prepared. After iv administration all three types of antibody would localize preferentially in F-344 skin grafts made 4 days earlier on Buffalo strain rats. After 24 hr, 125I localization ranged from 10 to 20% of the injected dose on F-344 skin grafts averaging 0.3 g weight on 150-190-g Buffalo rats. The horse serum used as an antibody source was a commercially available anti-rat-lymphocyte serum. Prior ip administration of two or three doses of 0.5 ml of this serum largely blocked uptake by F-344 skin grafts of both the 125I-labeled alloantibody and the 125I-labeled xenogeneic histocompatibility antibody prepared from this horse antiserum. This work was supported by USPHS Research Grant CA-16749 from the National Cancer Institute and under contract with the U.S. Energy Research and Development Administration with the University of Rochester and has been assigned Report No. UR-3492-913