Structural changes in fibrin clot associated with the proteolytic activity induced by tissue type plasminogen activator. An NMR study.

The kinetic behavior of fibrin clot lysis as induced by tissue-type plasminogen activator (t-PA) was studied using proton magnetic resonance (PMR) and a release assay of fibrin labeled with technetium-99m isotope (99mTc). The lysis pattern of the preformed clot was examined as a function of gradual changes in the amounts of added t-PA and deactivated t-PA. The behavior of fibrinolysis was found to depend strongly on the amount of t-PA in the assay, which markedly affects the lysis rate of fibrin. The changes induced by the lysis were reflected in pronounced prolongation of the transverse relaxation time. The PMR and the radioisotope release measurements point to the possibility that at least two steps are involved in the mechanism of lysis. The PMR seems to be associated with structural features of the clot and reflects the liberation of compartmentalized water from the clot, while the 99mTc analysis reflects the further fragmentation of fibrin.

Clot uptake of labeled active and inhibited tissue plasminogen activator.

The clot uptake of labeled active and inhibited t-PA was compared. The most efficient inhibition was obtained with diisopropyl fluorophosphate (DFP) after 4 h incubation at room temperature. Enzyme activity was followed by fibrin-plate assay, radioactivity-release technique and proton magnetic resonance (PMR). The novel PMR method developed by us is sensitive to the effect of as low as nanogram amounts of t-PA on the interaction between the fibrin and the compartmentalized water trapped in the clot. Binding of labeled enzyme to fibrin-coated plates showed that the deactivation by DFP did not impair the affinity of t-PA for fibrin. A rapid binding of 125I-labeled t-PA to the clot occurred, which reached a maximum in 30 min and declined with time. This pattern was explained by consecutive clot binding and lysis. The binding of DFP-t-PA to the clot differed markedly from that of the active protein; 2 h post-incubation the uptake of DFP-t-PA was more than double that of the untreated t-PA. Parallel measurements in clots prepared from human blood showed a qualitatively similar trend. The biodistribution of radiolabeled t-PA in mice was similar for the active and inhibited forms. Blood activity reached 10% of the injected dose within 10 min. DFP-t-PA may prove to be a useful reagent for in-vivo localization of thrombi.

Osmium-191/iridium-191m generator based on silica gel impregnated with tridodecylmethylammonium chloride.

A new separation system for an 191Os/191mIr generator is described. The system is composed of two columns in a series: a main column, packed with silica gel impregnated with tridodecylmethylammonium chloride (loaded with high activity 191Os as an osmyl chloride); and a scavenger column, packed with activated charcoal. Iridium-191m is eluted from the generator by pH 1 saline. For clinical use the eluate is buffered by succinate solution before injection. This new system is characterized by high performance (approximately 25% 191mIr recovery with 5 X 10(-4)% 191Os breakthrough) and long shelf-life (3 wk). The buffered eluate is sterile, pyrogen-free, and nontoxic, and contains no 192Ir. It is suitable for first-pass radionuclide angiocardiography with a very low radiation dose to the patient.

Evaluation of ventricular function with first-pass iridium-191m radionuclide angiocardiography.

Iridium-191m would appear to be a highly useful agent for first-pass radionuclide angiocardiography (FPNA), with its very short half-life (4.96 sec), dual photopeaks (65 and 129 keV), and high injectable activity levels (greater than 100 mc). In order to compare 191mIr FPNA to current methods used to define cardiac function, 20 patients referred for cardiac catheterization were studied. Count rate data, right ventricular (RV), and left ventricular ejection fraction (LVEF), LV and diastolic volume (EDV), and end diastolic long axis (AXIS) were evaluated. Count rate data using 191mIr FPNA was consistently better than similar data obtained by 99mTc FPNA. There were acceptable correlations between 191mIr and 99mTc FPNA RVEF (r = 0.848), 191mIr FPNA and contrast angiography LVEF (r = 0.944), LVEDV (r = 0.917), and LV AXIS (r = 0.866). The data thus suggests that 191mIr FPNA has great potential in the evaluation of cardiac function.

Paramagnetic enhanced proton magnetic resonance measurement in rats: correlates with renal function.

Renal cortical, medullar and papillary T1 and T2 relaxation times were measured in rats with normal (n = 13) and impaired renal function (n = 11) with a Bruker Multispec, 20 MHz at 37 degrees C. In one group of seven rats, decreased renal function was obtained by 50% glycerol solution administration (10 ml/kg-body weight) 24 hours before the experiment, while in another group of four rats the renal function was decreased, by ureteral ligation for 72 hours. Immediately after the excision of one kidney, Gadolinium-DTPA (70 mumole/kg body weight) was injected intravenously. The second kidney was excised 5 min later. From the T1 and T2 relaxation times measured in the cortex, medulla, and papilla, their respective ratios before and after GdDTPA administration were calculated and correlated with GFR determined by creatinine clearance (Ccr range was between 0 and 850 microliters/min/g kidney weight). For T1: the ratios in the cortex, medulla, and papilla the correlation coefficients were r = 0.81 (p less than 0.001), r = 0.85 (p less than 0.001), and r = 0.87 (p less than 0.0001), respectively. The respective correlation coefficient r values for T2 were r = 0.38 (NS), r = 0.76 (p less than 0.001), and r = 0.73 (p less than 0.001). The present study indicates that a combination of MR measurements, with and without GdDTPA paramagnetic enhancement, can offer a new possibility for obtaining information on renal function and suggest the possibility of concomitant anatomo functional magnetic resonance imaging.

Proton MR study of different types of experimental acute renal failure in rats.

Kidney cortical and medullary spin-lattice (T1) and spin-spin (T2) relaxation times were measured in several types of experimental acute renal failure in rats with a Bruker PC "Multispec." Gentamicin ARF was obtained after one i.p. injection of 100mg Gentamicin/kg BW/day for 8 days. Glycerol ARF: 24 hours after one i.m. injection of 10 ml 50% Glycerol/kg BW. Obstruction ARF: 3 days after complete ureteral ligation. Renal tissue total water content, hydration fraction, fraction bound, blood urea and creatinine were measured at the end of the experiments. Shortened T1 and prolonged T2 were found in both cortex and medulla in the Glycerol ARF group. Gentamicin renal toxicity and the non-functioning kidney with ureteral obstruction are characterized by significant prolongation of T1 and T2 in cortex, while the medullary T1 and T2 were prolonged only in obstruction ARF. The highest T1 and T2 were found in the obstructed non-functioning kidney. The total water content decreased in the Glycerol ARF, increased in the obstruction and remain unchanged in Gentamicin ARF. The hydration fraction and the fraction bound changed significantly in the opposite direction with the total water content. Different profiles of renal cortical and medullary magnetic resonance properties found in several models of experimental ARF in rats indicate that MR properties may provide etiopathogenetic diagnostic possibilities.

Proton MR properties of lyophilized urine samples from normal and stone former individuals.

Proton MR measurements were performed in lyophilized urine samples collected from 5 normals (N) and 5 idiopathic hypercalciuric recurrent stone formers (SF). T1 and T2 relaxation times were measured with a Bruker PC Multispec at 20 MHz and 37 degrees C in the lyophilized samples and in samples gradually rehydrated. Significantly (p less than 0.01) prolonged T1 and T2 relaxation times were measured after addition of water to the lyophilized samples. The relaxation time prolongation patterns were significantly different (p less than 0.01) for the two groups; the rehydration curves of the lyophilized urine samples from the SF group had relatively shorter lag than that of N group. In calculations of water compartmentalization for similar water content, significant (p less than 0.01) differences in the fraction of bound water (FB) were found between the two groups. These results may reflect differences in the macromolecular properties, contents, in the amount of water binding sites and/or in the water multilayer thickness between the two groups. These differences, expressed as changes of the relaxation times values may provide new diagnostic possibilities of different renal pathologies.

Renal proton magnetic resonance in experimental acute renal failure in rats.

Cortical, medullary and papillary T1 and T2 water proton relaxation times were measured at 37 degrees C, 20 MHz. The measurements were made using kidneys from rats affected by many forms of experimental acute renal failure (ARF), namely acute hemorrhagic hypovolemia, angiotensin II administration, antidiuretic hormone (ADH) administration, glycerol, and other nephrotoxins (gentamicin, cisplatinum, cyclosporine), renal artery occlusion for different periods of time, and ureteral ligation. From the T1 and PW (percent tissue water content) the bound water (FB) and HF (percent water bound/g solid) were calculated according to a fast proton diffusion model. In most experimental models studied, the experiments were repeated following paramagnetic enhancement with GdDTPA administration (70 mmol/kg BW). By profiling the deviations from normal, it was possible to differentiate the ischemic (shortened T1, prolonged T2), obstructive (very high T1 and T2 in both cortex and medulla) and nephrotoxic (prolonged T2) forms of ARF. Significant changes in free/bound water compartments occurred, though their biological significance is unknown. T1 and T2 ratios before and after paramagnetic enhancement correlated well with estimates of glomerular filtration rate. In the first minutes following acute hemorrhagic hypovolemia, the intrarenal water distribution remained unchanged. After GdDTPA significant water proton T1 and T2 changes characterized the immediate posthemorrhagic state similar to the effect of ADH.

Renal proton magnetic resonance and bound and free water distribution in the normal, angiotensin II-and ADH-infused rats, before and after Gd DTPA paramagnetic enhancement.

Female Charles River rats, 250-300 g, were used in all experiments. The rats were divided into the following experimental groups. Group A: Normal rats were injected with 1 ml saline intravenously. Exactly after 8 min the kidneys were excised as rapidly as possible. The papilla was dissected and cortical and medullary slices were prepared. Immediately following the excision T1 and T2 were measured with a Bruker PC-20 Minispec spectrometer operating at 20 MHz at 37 + 1 degree C. Water content (PW) of the samples was calculated as a percentage tissue weight, after 24 h drying to constant weight. The fraction bound (FB = % water bound) and hydration fraction (% water bound/g solid) were computed from T1 relaxation time, and the PW, according to a fast proton diffusion model. Group B: As group A but 3 min after the saline administration bolus of 1 ml solution of Gd DTPA. 70 mumol/kg body weight, was given intravenously. Group C: As group A but instead of saline the rats were given a solution of ADH (5 mU/kg body weight i.v.). Group D: As group C plus Gd DTPA as in group B. Group E: As group A but instead of saline the rats were given a solution of angiotensin II (Ag II) (5 micrograms/kg body weight). Group F: As group E plus Gd DTPA as in group B. In groups B-F the magnetic resonance and water distribution were determined and calculated as in group A. In the normal rats there is a prolongation of T1, T2 and an increased total water content from the cortex to the papilla.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydration properties of urinary compounds obtained from normal and stone former individuals. An NMR study.

Proton magnetic resonance measurements were performed on lyophilized urine samples collected from 10 recurrent calcium oxalate stone former (SF) patients, 10 thiazide treated (TSF) patients and 14 normal (N) individuals. T1 and T2 relaxation times were measured with a Bruker PC Multispec at 20 MHz and 37C on the lyophilized sample and thereafter during gradual controlled rehydration. The prolongation of the relaxation times was found to be significantly different (p less than 0.001) for the SF and N groups, while it was similar for the SF and TSF groups. Water compartmentalization was then calculated according to the Fast Proton Infusion model. After the addition of similar amounts of water, significantly (p less than 0.01) different hydration fraction (HF) and fraction bound (FB) values were calculated for the N and SF groups, while similar values were obtained for the SF and TSF groups. The results reflect differences in the urinary contents and/or properties of N and SF groups. It seems that there are more hydrophilic compounds in the urine of N individuals than in that of SFs. It can be speculated that the urinary macromolecules of SFs differ from those of Ns in the amount of water binding sites and in the water multilayer thickness surrounding them. The present results point to possible pathogenic differences between SF and N individuals.

Proton magnetic resonance in experimental acute and chronic renal failure in rats.

Kidney cortical and medullary "spin-lattice" (T1) and "spin-spin" (T2) relaxation times were measured by spectroscopy in several types of experimental renal failure in rats. The T1 and the measured tissue water content were used to calculate the fraction bound (FB) and hydration fraction (HF) according to a fast proton diffusion model. The present study demonstrated the possibility to differentiate between normal and pathological renal tissue resulting from renal artery clamping (RAC), renal pedicle clamping (RPC) with or without reflow, glycerol-induced acute renal failure with or without previous dehydration, and chronic hypertensive renal failure induced by 5/6 nephrectomy and saline loading, with low (6%) or normal (21%) protein diet. Shortened T1 and prolonged T2 found in both cortex and medulla of the glycerol-induced ARF in dehydrated rats seem to represent a MR ischemic pattern. The prolongation of T1 and T2 and the increase in water content in the other groups seem to relate to different amounts of tubular obstruction and renal congestion. In summary, characteristic MR properties of different types of renal failure may provide etiological and pathogenetic diagnostic possibilities.

Hydration feature of urinary compounds. Evidence for molecular abnormality in calcium oxalate urolithiasis.

Proton-relaxation-time measurements were performed on lyophilized urine samples collected from 11 recurrent calcium oxalate stone-formers, 9 uric acid stone-formers, 9 patients with various urological disorders, and 20 normal individuals. The T1 and T2 relaxation times were determined using a Bruker PC Multispec at 20 MHz at 37 degrees C for measurements of lyophilized sample and thereafter during gradual controlled rehydration. The prolongation of the relaxation times as a function of rehydration was found to differ significantly (P less than 0.005) between, on the one hand, the calcium oxalate stone-formers and, on the other hand the normal, uric-acid stone-formers, and patients with other urological disorders. Water compartmentalization was then calculated according to the fast proton diffusion model. At most of the experimental points during rehydration process, significantly (P less than 0.001) less water was bound to the compounds of urine from calcium oxalate stone-formers than that obtained from normal individuals. The variations in the bound hydration water may have been associated with possible changes in the structure or configuration of the compounds present in the urine of the different groups. The results reflect differences in the urinary content and/or properties of normal subjects and calcium oxalate stone-formers, and indicate that the mechanism of bound water relaxation is in some way specific to the pathophysiological state of urine.