Modulation of iron transport proteins in human colorectal carcinogenesis.

BACKGROUND AND AIMS: Total body iron and high dietary iron intake are risk factors for colorectal cancer. To date there is no comprehensive characterisation of iron transport proteins in progression to colorectal carcinoma. In this study, we examined expression of iron import (duodenal cytochrome b (DCYTB), divalent metal transporter 1 (DMT1), and transferrin receptor 1 (TfR1)) and export (hephaestin (HEPH) and ferroportin (FPN)) proteins in colorectal carcinoma. METHODS: Perl's staining was used to examine colonocyte iron content. Real time polymerase chain reaction (PCR) and western blotting were used to examine mRNA and protein levels of the molecules of interest in 11 human colorectal cancers. Semiquantitative immunohistochemistry was used to verify protein levels and information on cellular localisation. The effect of iron loading on E-cadherin expression in SW480 and Caco-2 cell lines was examined by promoter assays, real time PCR and western blotting. RESULTS: Perl's staining showed increased iron in colorectal cancers, and there was a corresponding overexpression of components of the intracellular iron import machinery (DCYTB, DMT1, and TfR1). The iron exporter FPN was also overexpressed, but its intracellular location, combined with reduced HEPH levels, suggests reduced iron efflux in the majority of colorectal cancers examined. Loss of HEPH and FPN expression was associated with more advanced disease. Iron loading Caco-2 and SW480 cells caused cellular proliferation and E-cadherin repression. CONCLUSIONS: Progression to colorectal cancer is associated with increased expression in iron import proteins and a block in iron export due to decreased expression and aberrant localisation of HEPH and FPN, respectively. This results in increased intracellular iron which may induce proliferation and repress cell adhesion.

Coronary reoperation: results of adding an internal mammary artery graft to a stenotic vein graft.

Although it is desirable at coronary reoperation to replace a stenotic vein graft to the left anterior descending coronary artery (LAD) with an internal mammary artery (IMA) graft, previous reports have shown that if the stenotic vein graft is removed, that strategy can be complicated by severe hemodynamic deterioration and increased perioperative mortality. We report the results for 90 patients in whom an IMA was used to graft a completely obstructed LAD with the stenotic vein graft left intact. For 10 patients, reoperation involved only an IMA-LAD graft, and in 80 patients, a second IMA, veins, or both were used to graft other vessels. There were no hospital deaths. One patient had a perioperative myocardial infarction. Follow-up at a mean postoperative interval of 58 months documented 11 late deaths (eight cardiac related) and actuarial 5-year survival of 88%. Twenty-two patients underwent coronary angiography at a mean postoperative interval of 48 months. The IMA-LAD graft was found to be perfectly patent in 20 and obstructed in 2. The strategy of adding an IMA graft to the LAD and leaving a stenotic vein graft intact has been associated with a low risk of perioperative myocardial infarction, the late clinical results are favorable, and repeat angiography indicates that serious competitive flow from the stenotic vein graft is uncommon.

Evaluation of metaiodobenzylguanidine uptake by the norepinephrine, dopamine and serotonin transporters.

Metaiodobenzylguanidine (MIBG) is taken up by sympathetic neurons, but the precise mechanism of uptake has not been elucidated. Uptake of monoamines by presynaptic neurons is mediated by plasma membrane proteins, the monoamine transporters. The human norepinephrine transporter (hNET), the bovine dopamine transporter (bDAT) and the rat serotonin transporter (r5HTT) have been cloned, sequenced and expressed in various cell lines. This study involves the measurement of MIBG uptake by cell lines that have been transfected with complementary DNAs encoding these monoamine transporters. At 20 nM MIBG, hNET transfected cells demonstrate a ninefold greater uptake of MIBG than nontransfected cells. MIBG uptake in hNET transfected cells is inhibited by 3 x 10(-6) M norepinephrine (87% inhibition) and by hNET transport inhibitors: 10(-7) M desipramine (94% inhibition) and 10(-7) M mazindol (97% inhibition). hNET transfected cells exhibit a Km for MIBG transport of 264 nM. Percent nonspecific uptake rises with increasing concentrations of MIBG while specific uptake is saturable. There is no significant uptake by bDAT or r5HTT. The NET appears to be responsible for the specific uptake of MIBG.

Evaluation of metaiodobenzylguanidine heart and lung extraction fraction by first-pass analysis in pigs.

Metaiodobenzylguanidine (MIBG) is a norepinephrine analog that can be used to study cardiac sympathetic innervation. Most of the kinetic data on MIBG, however, have been obtained in vitro from adrenal chromaffin cells. To elucidate MIBG cardiac kinetics in vivo, we measured the first-pass extraction fraction (EF) of MIBG in pig heart and lungs and determined the relationship between the cardiac EF and myocardial blood flow (MBF) before and after dipyridamole, cocaine and imipramine. The first-pass lung EF was 24% +/- 0.80% (mean +/- s.e.). The baseline cardiac EF of MIBG was 79% +/- 1.6%. With dipyridamole, MBF increase significantly and the EF fell (82% +/- 2.5% to 71% +/- 3.5% baseline compared to 0.03 mg/kg/min dipyridamole, p less than 0.001), indicating that the cardiac EF of MIBG is dependent on MBF. Cocaine infusion had no effect on MBF or EF. Imipramine caused a significant increase in the EF (72% +/- 3.5% versus 77% +/- 2.5%, baseline versus imipramine p = 0.032) without a change in MBF. In adrenal chromaffin cells, cocaine and imipramine decrease MIBG uptake, suggesting that adrenal chromaffin cells may be an inappropriate model for studying MIBG kinetics in cardiac sympathetic neurons.

Effects of hypothyroidism and thyrotoxicosis on metaiodobenzylguanidine kinetics in the rat heart.

Metaiodobenzylguanidine (MIBG), a norepinephrine (NE) analogue, has been used to study cardiac sympathetic innervation. Although MIBG uptake and washout generally parallel the kinetics of cardiac NE (an accepted marker of sympathetic nerve function), quantitative differences exist between NE and MIBG. To determine the value of MIBG as a marker of cardiac sympathetic nerve function, cardiac MIBG uptake and washout were measured in rats with hypothyroidism and thyrotoxicosis which cause increased and decreased sympathetic nerve function, respectively. Rats were made hypothyroid by the ingestion of water containing 0.03% methimazole and thyrotoxic by the intraperitoneal injection of L-thyroxine. After 3 weeks of treatment, rats were injected with 25 mu Ci of 125I-MIBG. Rats were sacrificed at 1.5, 4, 8 and 24 h after injection and heart MIBG activity was determined. Initial uptake and cardiac washout (T1/2) were calculated in both treated groups and compared to controls. MIBG washout was similar in all three groups although it tended to be faster in the hypothyroid group. Uptake was highest in the hypothyroid group (130% of controls) and lowest in the hyperthyroid group (79% of controls). These results suggest that MIBG may be a marker of cardiac sympathetic nerve function although its kinetics may differ from NE.

Comparison of in vitro RBC labeling with the UltraTag RBC kit versus in vivo labeling.

This study compared cardiac-gated equilibrium blood-pool imaging studies using in vitro technetium-99m- (99mTc) labeled red blood cells (RBCs) prepared with the UltraTag RBC kit to in vivo labeling with stannous (pyro- and trimeta-) phosphates. The in vitro labeling procedure takes approximately 25 min and does not require centrifugation to separate free from bound 99mTc. Imaging studies were performed in 30 patients using the in vitro labeling procedure and in 30 patients with in vivo labeling. Regions of interest were placed over the center of the left ventricle, inferior and lateral to the left ventricle (background), and over the right midlung. The mean +/- s.e. in vitro RBC labeling efficiency was 98.5 +/- 0.2%. The heart-to-background ratios were significantly higher with in vitro labeling. The heart-to-background ratios, averaged among two blinded reviewers, were 4.6 and 3.4 for the in vitro and in vivo methods, respectively. The heart-to-lung ratio was generally higher with the in vitro procedure (3.6) than that observed with the in vivo method (3.2) but failed to attain statistical significance (p = 0.059). These results demonstrate the superiority of the in vitro labeling procedure over in vivo labeling for gated equilibrium blood-pool imaging.

Technetium-99m monoclonal antibody fragment (Fab) scintigraphy in the evaluation of small cell lung cancer: a preliminary report.

Small cell lung cancer (SCC) has the most rapid growth rate of the four cell types and metastasizes early. Present imaging modalities for staging include chest x-ray, CT, MRI and bone scans. In this preliminary study, we assessed the clinical role of 99mTc-monoclonal antibody (MOAB) scintigraphy in five patients with histologically proven SCC. Each patient was infused with 20-30 mCi of 99mTc labeled Fab fragment of MOAB (NR-LU-10, NeoRx, Seattle, Wash.). Total body simultaneous anterior and posterior images were obtained 14-16 h post injection. SPECT images of the chest were obtained through a 360 degrees rotation of the gamma camera and recorded on a 62 x 64 x 16 matrix. Images (1.2 cm thick) were generated in transaxial, sagittal and coronal views. Fourteen of fifteen chest lesions detected by CT were confirmed by 99mTc MOAB scintigraphy. Scintigraphy detected one additional chest lesion not seen by CT. Scintigraphy failed to detect a brain lesion (2 cm), a chest lesion, and two adrenal lesions, all of which were seen by CT. In one patient with multiple (more than 10) lesions in the liver, both scintigraphy and CT detected all lesions. Three spine lesions seen on 99mTc MDP scan and positive for metastasis on MRI concentrated 99mTc MOAB, but two rib lesions seen on 99mTc MDP bone scan did not concentrate 99mTc MOAB. It is concluded from these preliminary results that the potential usefulness of 99mTc MOAB scintigraphy as a complementary imaging modality in the staging of small cell lung cancer should be investigated further.

Pharmacokinetics and clinical application of technetium 99m-labeled hepatobiliary agents.

Hepatobiliary imaging, introduced first in late 1950s with iodine 131 rose bengal, has undergone major changes since the introduction of technetium 99m-labeled agents in the late 1970s. 99mTc-labeled iminodiacetic acid (IDA) agents, with their high hepatic uptake and fast biliary excretion, provide superior image resolution in addition to supplying simultaneous physiologic parameters quantitatively. The measurement of hepatic extraction fraction by deconvolutional analysis aids in separating hepatocyte from biliary disease. Excretion half-time calculation by nonlinear least square fit provides a quantitative method of analyzing hepatobiliary function and correlates directly with the severity of disease. The measurement of gallbladder ejection fraction, ejection rate, and common bile duct bile reflux index following cholecystokinin, enables quantification of the degree of obstruction to bile flow through the common bile duct. The combined application of morphologic and physiologic parameters enables diagnosis of various hepatobiliary disease, especially in early stages. Quantitative functional parameters not only provide criteria for diagnosis, but also assist in monitoring the therapeutic benefit from drugs and interventional techniques such as endoscopic sphincterotomy or balloon dilation of the obstruction. Biliary dynamic studies with cholecystokinin assist in distinguishing common bile duct dilation without obstruction from dilation with obstruction. Methods of application of 99mTc-IDA agents in quantitative physiologic studies are reviewed and future direction of their use is proposed.

Primary lung cancer: biodistribution and dosimetry of two In-111-labeled monoclonal antibodies.

This study was undertaken to measure the biokinetics and organ dosimetry of indium-111-labeled monoclonal antibodies (MoAbs) with a whole-body gamma camera imaging technique. Twenty patients with primary lung cancer were studied with two different MoAb agents (anti-carcinoembryonic antigen ZCEO25 and antiadenocarcinoma LA20207). Imaging was performed at 1, 24, 72, and 144 hours after injection. Scintigraphic whole-body retention was verified by means of comparison with the results from in vitro counting of excreta. Organ retention was verified in an abdominal phantom. The MoAb cleared slowly from the heart and lungs, the brain and spleen showed no clearance, and the liver showed increased activity over the 6-day period. Dosimetry for ZCE025 showed a dose to the liver of 1.3 rad/mCi (0.36 mGy/MBq); heart, 1.5 rad/mCi (0.40 mGy/MBq); spleen, 1.1 rad/mCi (0.29 mGy/MBq); total body, 0.49 rad/mCi (0.13 mGy/MBq); and testes, 0.39 rad/mCi (0.11 mGy/MBq). The dosimetry for LA20207 was similar.

Detection of experimental atherosclerosis with indium-111 radiolabelled hematoporphyrin derivative.

It has been observed that atherosclerotic lesions (AL) concentrate certain porphyrins. We evaluated the usefulness of 111In-labelled Photofrin II (PFII), a porphyrin derived from haematoporphyrin derivative, for detection of experimental AL in cholesterol fed rabbits. Three groups of rabbits were studied: non-atherosclerotic (n = 3), 6 and 12 week cholesterol fed (n = 4, 3). 111In-PFII was injected intravenously and gamma camera images were obtained at 24 and 48 h. At 48 h, explanted aortas were also imaged. Aortic arch (AA) to background (BKG) count ratios were calculated from images of the whole body and explanted aortas. AA/BKG ratios were significantly higher in the 12 week cholesterol fed rabbits (3.9 +/- 0.72 at 24 h) and (4.0 +/- 0.67 at 48 h) than in the non-atherosclerotic rabbits (2.2 +/- 0.07 at 24 h) and (2.3 +/- 0.18 at 48 h) (p less than 0.05). The AA/BKG ratio for the explanted aortas showed similar results. Additionally, in two of three 12 week cholesterol fed rabbits, focal count deposition was visible on the whole animal images at the site of aortic arch atherosclerosis. We conclude that 111In-PFII concentrates in AL as early as 24 h after injection and has the potential to be used as an imaging agent for experimental atherosclerosis.

Iodine-123 metaiodobenzylguanidine imaging of the heart in idiopathic congestive cardiomyopathy and cardiac transplants.

Iodine-123 metaiodobenzylguanidine ([123I]MIBG) is a norepinephrine analog which can be used to image the sympathetic innervation of the heart. In this study, cardiac imaging with [123I]MIBG was performed in patients with idiopathic congestive cardiomyopathy and compared to normal controls. Initial uptake, half-time of tracer within the heart, and heart to lung ratios were all significantly reduced in patients compared to normals. Uptake in lungs, liver, salivary glands, and spleen was similar in controls and patients with cardiomyopathy indicating that decreased MIBG uptake was not a generalized abnormality in these patients. Iodine-123 MIBG imaging was also performed in cardiac transplant patients to determine cardiac nonneuronal uptake. Uptake in transplants was less than 10% of normals in the first 2 hr and nearly undetectable after 16 hr. The decreased uptake of MIBG suggests cardiac sympathetic nerve dysfunction while the rapid washout of MIBG from the heart suggests increased cardiac sympathetic nerve activity in idiopathic congestive cardiomyopathy.

Gallbladder filling and post-ceruletide emptying in prairie dogs and rabbits.

The filling and emptying characteristics of the gallbladder in prairie dogs and rabbits were studied to assess the importance of the residual bile in the pathogenesis of gallstones. In prairie dogs under ketamine/xylazine anesthesia, a significantly larger fraction (p = 0.001) of hepatic bile entered the gallbladder (87 +/- 8%) than the intestine during fasting and very little bile emptied (0-3% ejection fraction) following ceruletide infusion. In rabbits under similar anesthesia, only a small fraction of hepatic bile entered the gallbladder (4 +/- 2%) during fasting, and the gallbladder emptied almost completely (85% ejection fraction) following ceruletide infusion. The resultant higher residual bile in the prairie dog gallbladder and lower residual bile in the rabbit gallbladder may explain why gallstones form so readily in prairie dogs but not in rabbits when fed a lithogenic diet. These similarities and differences in gallbladder function must be taken into account when considering any animal as a model for gallstone formation.

Ceruletide intravenous dose-response study by a simplified scintigraphic technique.

The intravenous dose response of a ceruletide diethylamine (ceruletide) was established by a simplified scintigraphic technique where multiple graded doses were given sequentially on a single occasion. The gallbladder volume was represented nongeometrically by 99mTc-IDA counts. The mean latent period, ejection period, and ejection rate were similar for all four groups of subjects given 1-20 ng/kg of ceruletide. The mean (+/- SD) ejection fractions after 1, 5, 10, and 15 ng/kg of ceruletide as the single dose were 19.4 +/- 11.9%, 59.6 +/- 26.0%, 55.2 +/- 23.3%, and 67.8 +/- 8.7%, respectively. These ejection fractions were similar to the values when the identical dose of ceruletide was administered sequentially either before or after another dose. A dose of 5 ng/kg produced the most physiologic type of emptying. Intravenous doses of 10 ng/kg and larger caused adverse reactions in 42% of the total doses in the form of abdominal pain, nausea, systolic and diastolic hypotension, or bradycardia. It is concluded that the dose response of a cholecystokininlike agent (ceruletide) can be established reliably by a scintigraphic technique where multiple graded doses are given on a single occasion.

A new scintigraphic technique for cholecystokinin gallbladder dose-response study: validation in rabbits.

An investigation was undertaken to test whether the dose-response curve of a cholecystokinin-like agent (ceruletide) could be established by administering it in graded doses sequentially on the same day. The results were compared to individual doses given on separate days. The gallbladder ejection fraction (EF) was calculated for each dose using the Tc-99m-IDA counts to represent the gallbladder bile volume. The mean ejection fraction following 1, 2.5, 5, and 10 ng/kg given sequentially on the same day was 10 +/- 8, 22 +/- 12, 53 +/- 13, and 85 +/- 3 per cent, respectively. The ejection fraction for 2.5, 5, and 10 ng/kg given on separate days was 29.7, 57.14, and 93.3 per cent, respectively, and was similar to the values obtained when the identical dose was given sequentially on the same day (P less than 0.05). It is concluded that the sequential method is as accurate as the single-dose regimen and carries the advantage of simplicity in the establishment of dose-response curve for any future CCK-like agent.

Gallbladder dynamics induced by a fatty meal in normal subjects and patients with gallstones: concise communication.

A study was undertaken to establish the pattern of gallbladder emptying in normal subjects and in patients with gallstones, using a fatty meal as stimulus to release endogenous cholecystokinin. The time from meal ingestion to beginning of gallbladder emptying (latent period), the total duration of emptying (ejection period), degree of emptying (ejection fraction), and the rate of emptying (ejection fraction/ejection period) were measured noninvasively by a nongeometric scintigraphic technique. The mean latent period and ejection rate were similar in normal subjects and patients with gallstones, but the mean ejection period and ejection fraction were significantly reduced in the patients. This study suggests that for an identical stimulus, the gallbladder in cholelithiasis begins to empty at the normal time but empties for a shorter duration; the result is a reduction of ejection fraction but not of ejection rate.

Comparison of biokinetics and biliary imaging parameters of four Tc-99m iminodiacetic acid derivatives in normal subjects.

The biokinetics (blood clearance, urinary excretion, hepatic peak time, uptake, and excretion t-1/2) and the imaging parameters (the time of appearance of the common bile duct, gallbladder, and duodenum) were determined in 34 normal subjects using Tc-99m diethyl (EIDA), Tc-99m dimethyl (HIDA), Tc-99m paraisopropyl (PIPIDA), and Tc-99m parabutyl (PBIDA) iminodiacetic acid derivatives. The blood and hepatic clearance of the four agents were significantly different (P less than 0.05) from each other. The 24-hour urinary excretion of PBIDA was significantly lower (P less than 0.05) than the urinary excretion of the other three agents. There was no difference among the four agents in the time of appearance of the gallbladder and duodenum. The time of appearance of the common bile duct was significantly delayed with PBIDA. The maximum intensity of the common bile duct usually occurred between 20 to 40 minutes with all four agents. However, gallbladder intensity continued to increase up to 3 hours. It is concluded that in the presence of normal liver function, all four Tc-99m IDA agents show definite differences in biokinetics but these differences do not have a major effect on biliary imaging parameters. If imaging alone is the primary goal, the selection of any one of the four agents will meet the clinican's need satisfactorily.

Radiation-dose calculation for five Tc-99m IDA hepatobiliary agents.

The radiation absorbed doses from five commercially available hepatobiliary agents--Tc-99m-tagged analogs of IDA (EIDA, PIPIDA, HIDA, PBIDA, DISIDA) have been calculated from biokinetic data in 41 normal subjects. Serial gamma images, with blood and urine samples, were obtained to calculate cumulated radioactivity in the source organs: blood, kidney, bladder, liver, gallbladder, and intestines. The critical organ was the gallbladder, with an absorbed-dose range of 690 to 780 mrad/mCl. Absorbed doses for other target organs were: upper large intestine 320 to 370 mrad/mCi, lower large intestine 210 to 240, small intestine 170 to 200, liver 65 (DISIDA) to 130 (PBIDA), ovaries 63 to 72, and urinary bladder wall 23 (PBIDA) to 36 (EIDA). The radiation absorbed dose was largely independent of changes in chemical structure except in (a) the liver, where absorbed dose varied by a factor of two in proportion to the rate of excretion of the IDA agent from the liver, and (b) the urinary bladder, where absorbed dose varied by a factor of 1.6 because of differences in rate of excretion. When the stimulus for gallbladder emptying is changed from whole-meal ingestion to cholecystokinin injection, the absorbed dose to the gallbladder increases to approximately 1 rad/mCi; if no gallbladder emptying is assumed, its absorbed dose increases to approximately 1.9 rad/mCi. In the absence of contraindication, the gallbladder absorbed dose may thus be decreased by inducing gallbladder emptying at the end of the imaging study.