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.

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.

Preparation and use of 123I-labeled highly iodinated fibrinogen for imaging deep-vein thrombi.

A method for producing protein-iodination-grade 123I suitable for use with a compact bio-medical cyclotron is reported. The preparation of highly iodinated fibrinogen (25 123I atoms per molecule) is described, and its successful use as a thrombus-imaging agent in experimental animals is reported. This new agent clears from the blood faster than conventional radioiodinated fibrinogen and gives higher thrombus-to-blood activity ratios. Thus, the detection of deep-vein thrombi in areas of large blood pool is enhanced, and images can be obtained sooner after administration of the radiopharmaceutical. Induced 4--8-hr-old femoral-vien thrombi in dogs can be well visualized with a scintillation camera as early as 4 hr and as late as 15 hr after administration of 1 mCi of 123I-labeled highly iodinated fibrinogen.

Detection of bacterial endocarditis with technetium-99m-labeled antistaphylococcal antibody.

The reliable diagnosis of bacterial endocarditis is an important but difficult clinical problem. The potential ability of technetium-99m-labeled antistaphylococcal antibody to detect infective endocarditis was investigated in a rabbit model. Radiolabeling of the purified antibody was effected by a mild electrolytic procedure, with full retention of immunologic activity. Infective endocarditis was induced in rabbits by placing a catheter through the carotid artery into the left ventricle, followed by i.v. injection of Staphylococcus aureus. The labeled antistaphylococcal antibody was subsequently injected, and its clearance and distribution were studied in the infected rabbits and in normal controls. The ratio of radioactivity on the aortic valve to that in the surrounding heart tissue or blood pool was significantly higher for the infected animals (> 10:1) than for the normals, and should permit visualization of the infection site. This radiolabeled antibody technique may provide a feasible approach to detection of infective endocardial lesions.

Radioiodinated plasminogen: an imaging agent for pre-existing thrombi.

We have reinvestigated radioiodinated plasminogen as an agent for localizing preformed thrombi. Canine plasminogen was isolated from fresh plasma by the affinity chromatography technique on a lysine-sepharose 4B column and tagged with I-123 or I-131, at less than one iodine atom per molecule of enzyme, by the conventional ICI method. When injected into dogs more than 2 days after thrombus induction, radioiodinated plasminogen produced thrombus-to-blood activity ratios of 7.8 +/- 2.4. Thrombi as old as 6 days can be visualized in 80% of the cases. Both the weight of the thrombus and the thrombus-to-blood ratio are more variable for 1-day-old thrombi; this may be associated with plasminogen release accompanying thrombus retraction. The results suggest that radioiodinated plasminogen has potential as an imaging agent for pre-existing thrombi.

In vivo behavior of 99mTc-fibrinogen and its potential as a thrombus-imaging agent.

We have investigated the in vivo behavior of 99mTc-fibrinogen, prepared by a mild and efficient electrolytic method employing tin electrodes. The clearance mechanisms of this agent were studied, and its efficacy for imaging deep-vein thrombi in dogs with an Anger camera was determined. The 99mTc-fibrinogen preparations, which are stable in vitro, undergo partial rapid exchange of the technetium with other plasma proteins and with anions of the blood buffer system in vivo, resulting in an early drop in the percent of radioactivity associated with clottable protein. However, very little or no oxidation to pertechnetate occurs. The nonclottable material is much more rapidly cleared from the blood than the remaining 99mTc-fibrinogen, and the proportion of clottable protein activity increases with time. The fraction of 99mTc-fibrinogen that remains intact in vivo is biologically active and will incorporate into thrombi. Higher thrombus-to-blood activity ratios are obtained with 99mTc-fibrinogen than with radioidinated fibrinogen when both agents are injected into dogs 4 hr after induction of femoral vein thrombosis. Clearly delineated images of the thrombi are obtained, beginning about 2.5 hr after injection. Thus, 99mTc-fibrinogen may be of clinical use as a thrombus-imaging agent in patients under-going active thrombosis, especially in regions of high blood pool.

Splenic imaging with 99mTc-labeled erythrocytes: a comparative study of cell-damaging methods.

Several methods of damaging red blood cells (RBCs) for splenic imaging were compared to determine the optimum approach. The RBCs from donor animals were labeled with 99mTcO4- and damaged by heat, excess acid citrate dextrose (ACD), excess Sn(II) ion, or the sulfhydryl inhibitors N-ethylmaleimide (NEM) or p-hydroxymercuribenzoate (PMB). The organ distributions of undamaged and damaged RBCs were determined in rats, and splenic imaging studies were performed in rabbits. Splenic deposition and spleen-to-liver ratios with heat- or sulfhydryl-damaged 99mTc-RBCs were significantly greater (p less than 0.001) than the values obtained using ACD or Sn(II) ion. Heat-damaging produces good splenic localization of 99mTc-RBCs but requires rigidly controlled incubation conditions. NEM-damaging provides an excellent and predictable alternative approach.

Fibrinogen uptake by thrombi: effect of thrombus age.

The uptake of radiolabeled fibrinogen in canine thrombi was determined at varying times after thrombus induction by electric current. The greatest thrombus/blood ratio was achieved when fibrinogen was administered 4 hr after thrombus induction but definite thrombus fibrinogen uptake was still observed when the tracer was administered up to 72 hr after thrombus induction. There was continued fibrinogen accumulation despite a decrease in weight of older thrombi suggesting that net thrombus propagation is not necessary for labeled fibrinogen uptake. Our results suggest that the fibrinogen uptake test may be useful for the diagnosis of deep vein thrombosis for several days after the onset of thrombosis.

Highly iodinated fibrinogen: a new thrombus-localizing agent.

We have examined radioiodinated fibrinogen prepared at high levels of iodination as an agent for improved in vivo thrombus detection. Fibrinogen containing 25, 50, and 100 atoms of iodine per molecule is prepared by an electrolytic procedure and is compared with conventional radiolabeled fibrinogen (less than 0.5 iodine atom per molecule) prepared by the iodine monochloride method. The level of iodination has little effect on the isotopic clottability of the product, but its degree of aggregation and its rate of blood clearance in experimental animals is strongly dependent on iodination level. Isotopic thrombus: blood ratios obtained in recently induced thrombi with the 25 atom per molecule preparation average about 50:1, twice as high as the ratios obtained with conventionally labeled fibrinogen.

Health risk assessment of Listeria monocytogenes in Canada.

In this review, the major steps used in the formulation of a health risk assessment for Listeria monocytogenes in foods are discussed. Data is given on the numbers of human listeriosis cases reported in Canada along with the current Canadian regulatory policy on L. monocytogenes. Four major steps in the health risk assessment of this organism in foods, namely, hazard identification, hazard characterization, exposure assessment and risk characterization, were examined. For hazard characterization, since it is known that no direct human dose response data is available for L.monocytogenes, a flexible dose response model called the Weibull-Gamma model was evaluated. For the exposure assessment, pâté and soft cheese, both high-risk foods in terms of listeriosis infection, were used as prototypes in some of the models that were used. Using disappearance data for cheese and 100 g as a typical serving, the data suggested an average of 102 servings per capita, per year in Canada. As a rough approximation, for L. monocytogenes, reference ID10 and ID90 dose levels of response for both normal and high risk populations were given as 10(7) and 10(9) for normal individuals, and 10(5) and 10(7) for high-risk people. The corresponding dose response models were graphically displayed. These models exhibited a higher degree of susceptibility and less host/pathogen heterogeneity for the higher risk group. The range of doses between the ID10 and ID90 reference values corresponded roughly to levels associated with cases of listeriosis. In the risk characterization stage, dose response data was combined with some predictive growth modeling data of L. monocytogenes on pâté, assuming an initial exposure of a single cell for food stored at 4 degrees and 8 degrees C. Storage of pâté at 4 degrees C for more than 35 days resulted in a rapidly increasing risk for the high risk population, while storage at 8 degrees C produced a similar risk after about 13 days. In addition, an equation, used to calculate the average probability of acquiring human listeriosis in Canada from soft and semi-soft cheese consumption, was formulated. Computations derived from this equation indicated a substantial level consistency between reported data and assumptions of the risk assessment model. An important part of risk characterization or possibly risk management is characterizing the economic and social consequences of estimated risks. The total annual estimated cost of listeriosis illnesses and deaths in Canada was estimated to be between 11.1 and 12.6 million dollars.

Prevention of foodborne listeriosis.

Listeria monocytogenes is a Gram-positive, rod-shaped bacterium which, although recognized in the medical literature as an opportunistic pathogen for the past 60 years, has only recently gained prominence as an important foodborne pathogen. Factors which make this organism unique among foodborne pathogens include its ability both to survive in foods under a variety of adverse conditions and to grow at low refrigeration temperatures. The organism is very widespread in the environment and can be found in a wide variety of foods. At least four major outbreaks definitively linked to the consumption of food containing L monocytogenes have occurred. In addition there have been a number of recent sporadic cases of listeriosis linked to the consumption of meat, fish and dairy products. The primary concern of the Health Protection Branch is contaminated foods in which L monocytogenes can grow well, and which would not normally be heated prior to consumption. Worldwide, the disease appears to be increasing in incidence, but definite links to foods are difficult to make. In most cases, individuals who come down with listeriosis include the immunocompromised, the elderly (older than 65 years) and pregnant women and their fetuses. Primary manifestations of the disease include meningitis, spontaneous abortion and septicemia. Mortality rates in foodborne listeriosis outbreaks are approximately 30%. Diagnosis of listeriosis usually requires isolation of the organism from sterile sites such as blood, cerebrospinal fluid, placenta and meconium and gastric aspirates from neonates. The recommended drug of choice is high dose intravenous ampicillin. Advice to physicians concerning measures to prevent foodborne listeriosis in high risk groups is reviewed. Included among these recommendations is avoidance of consumption of potentially hazardous foods such as soft cheese and raw products of animal origin.

Brine shrimp (Artemia salina L.) larvae as a screening system for fungal toxins.

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.

Screening Fusarium strains isolated from overwintered Canadian grains for trichothecenes.

A survey of 38 samples of Canadian overwintered grains showed that 14 (37%) contained viable Fusarium. Of a total of 38 Fusarium isolates, cultured on autoclaved corn, 20 (from 7 grain samples) showed toxicity to brine shrimp larvae and 12 (from 5 samples) produced levels of trichothecenes detectable by thin layer chromatography. The principal trichothecene found was T-2 toxin, produced by 10 strains and accompanied in half of these by neosolaniol; some of these strains were identified as F. sporotrichioides Sherbakoff. Two strains of F. poae (Peck) Wollenw. formed small amounts of diacetoxyscirpenol. T-2 toxin was the most toxic of 8 trichothecenes tested on brine shrimp larvae; the wide range of toxicities limits the usefulness of this bioassay as a general screening method for trichothecenes.

Methods for recovering poliovirus and rotavirus from oysters.

Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters.

Effect of pH on tumor cell uptake of radiogallium in vitro and in vivo.

When injected at tracer levels into the blood, radiogallium as 67Ga-citrate binds to, and it transported to the site of the tumor by, transferrin. The process by which transferrin-bound Ga is converted to tumor-bound Ga is not fully understood, but may involve the differential physiology of neoplasms compared with normal tissues. Based on the slight acidity known to be exhibited by the extracellular fluid of many animal and human tumors, we have studied the effect of pH on stability and dissociation of the Ga-transferrin complex and on the uptake of Ga by tumor cells in vitro and animal tumors in vivo. When plasma from rabbits injection 67Ga-citrate was dialyzed at pH 6.5-7.5, dissociation of Ga from transferrin showed an inverse pH-dependence. A similar inverse dependence on pH was observed for the uptake of Ga by L1210 leukemia cells and Ehrlich ascites cells incubated with Ga-transferrin complex. Tumor uptake of Ga in rats bearing Walker-256 carcinosarcoma or Murphystum lymphosarcoma whose tumor pH had been further lowered by administration of glucose showed a statistically significant increase over control rats receiving no glucose. These results demonstrate that the stability of the Ga-transferrin complex is pH-dependent and suggest that dissociation of this complex due to decreased pH at the tumor site may be one factor involved in tumor localization and binding of Ga.