Washout Profile Research Articles

AN EXAMINATION OF THE INTERPLAY BETWEEN ENTEROCYTE-BASED METABOLISM AND LYMPHATIC DRUG TRANSPORT IN THE RAT

The current study has examined whether drugs that are transported to the systemic circulation via the intestinal lymph (and therefore associate with lipoproteins within the enterocyte) are accessible to enterocyte-based metabolic processes. The impact of changes to the mass of lipid present within the enterocyte-based lymph lipid precursor pool (LLPP) on the extent of enterocyte-based drug metabolism has also been addressed. Low (5 mg oleic acid/h) or high [20 mg oleic acid/5.2 mg lyso-phosphatidylcholine/h] lipid dose formulations containing halofantrine (which is lymphatically transported and metabolized) or dichlorodiphenyltrichloroethane (DDT) (which is lymphatically transported and relatively metabolically inert) and radiolabeled oleic acid were infused into the duodenum of lymph duct-cannulated rats. After 5 h, drug and radiolabeled oleic acid were removed from the infusions, allowing calculation of the first order turnover rate constants describing drug and oleic acid transport from the LLPP into lymph from the washout profiles. In one group of animals, bolus doses of ketoconazole were also administered to inhibit cytochrome P450-based metabolism. The rate constant describing halofantrine transport from the LLPP into the lymph was lower than that of oleic acid, whereas these differences were abolished in the presence of ketoconazole. DDT and oleic acid exhibited similar turnover rate constants. The data therefore suggest that enterocyte-based metabolism removes halofantrine from the LLPP before transport into the lymph. Furthermore, enhancing the lymphatic transport of halofantrine by coadministration of larger quantities of lipid reduced the difference between the turnover rate constant for halofantrine and oleic acid and seemed to reduce the extent of enterocyte-based metabolism.

Distinguishing Benign from Malignant Adrenal Masses: Multi–Detector Row CT Protocol with 10-Minute Delay

To retrospectively evaluate the accuracy of precontrast attenuation, relative percentage washout (RPW), and absolute percentage washout (APW) in distinguishing benign from malignant adrenal masses at multi-detector row computed tomography (CT). This HIPAA-compliant retrospective study had institutional review board approval; the need for informed consent was waived. One hundred twenty-two adrenal masses were evaluated in 99 patients (51 men, 48 women; age range, 37-86 years) who had undergone CT performed according to the study protocol and who either were given a pathologic diagnosis or underwent follow-up imaging. Unenhanced images were obtained before administration of 120 mL of an intravenous contrast agent with a 75-second scan delay. Delayed images were obtained after 10 minutes. RPW and APW were computed. Receiver operating characteristic (ROC) analysis was performed to compare mean attenuation and both RPW and APW. Analysis was first performed with the exclusion of pheochromocytomas, myelolipomas, and cysts. Precontrast attenuation criteria specific for benignity or malignancy were determined, and ROC analysis of results for the entire nonpheochromocytoma group was then performed. By using an RPW of 37.5% and excluding cysts and myelolipomas, all malignant lesions were detected with a sensitivity of 100% (17 of 17 lesions) and a specificity of 95% (90 of 95 lesions). Area under the binomial ROC curve (A(z)) values were 0.912, 0.985, and 0.892 for precontrast attenuation, RPW, and APW, respectively. Precontrast attenuation of less than 0 or more than 43 HU indicated benign and malignant entities, respectively. Incorporation of these criteria into the APW analysis yielded a sensitivity of 100% (17 of 17 lesions) and a specificity of 98% (93 of 95 lesions) for a threshold washout value of 52.0%. This attenuation-corrected APW generated the greatest A(z) value (ie, 0.988). Combining all the information available from the protocol yielded a sensitivity of 100% (17 of 17 lesions) and a specificity of 98% (98 of 100 lesions) for differentiating benign from malignant masses. Precontrast attenuation of less than 0 HU supercedes the washout profile in the evaluation of an individual adrenal mass. Noncalcified, nonhemorrhagic adrenal lesions with precontrast attenuation of more than 43 HU should be considered suspicious for malignancy.

Screening Cascade and Development of Potential Positron Emission Tomography Radiotracers for mGluR5: In vitro and In vivo Characterization

Use of mGluR5 receptor radiotracers to determine whether an in vitro binding assay is able to predict how good a radiotracer is likely to be in imaging receptor in the central nervous system (CNS) via positron emission tomography (PET). Saturation and equilibrium competition studies in rat and rhesus membranes were used to determine receptor concentrations and tracer affinities. In addition, specific binding of metabotropic receptor subtype 5 (mGluR5) radioligands in rhesus and rat brain sections was determined using a "no-wash protocol," and the in vivo binding signal in rats was determined using micro-PET. Affinity values were determined for a series of mGluR5 antagonists (1-5) and ranged from 0.1 to 11 nM in rat. A previously reported "no-wash protocol" was then employed to determine specific binding in tissue sections following a 20-min incubation, and the regional distribution of these mGluR5 radiotracers determined in rat brain via autoradiography. The analogs 1b, 2b, 3b, and 4b, but not 5b, displayed good signal-to-noise ratios under these conditions with high density of binding in caudate, cortex, and hippocampus and lower density in cerebellum. With this information it was predicted that 1c, 2c, 3b, and 4b would display measurable signal-to-noise ratios in vivo, and that the larger in vitro signals for 3b and 4b would translate to 3b and 4b yielding the best in vivo signals. These predictions were investigated using micro-PET imaging in rat. Compound 1c showed a rapid wash-in and rapid wash-out profile in rat brain. Compound 2c showed similar signal-to-noise ratio as 1b, but slower washout. Compounds 3b and 4b showed the best signal-to-noise ratio in vivo, while 5b did not provide a significant signal, as predicted. In vivo occupancy estimates for 2-methyl-6-(phenylethynyl)-pyridine (MPEP) following intravenous administration were determined using radiolabeled compounds 1c, 2c, and 3b; they were essentially the same and were on the order of 1 mg kg(-1) (ID(50)). An in vitro screen of several mGluR5 tracers was used to rapidly predict whether radiolabeled mGluR5 analogs would be useful as PET radiotracers. Results provided an extension to previously reported data. Two of the four radiotracers with the best in vitro "no-wash" results also showed the best potential as measured noninvasively using micro-PET.

Low-Density Pheochromocytoma on CT:A Mimicker of Adrenal Adenoma

Attenuation values on CT of less than 10 H are considered characteristic of adrenal adenomas. Adrenal pheochromocytomas can infrequently contain fat that could result in low attenuation on CT. The purpose of our study was to determine if pheochromocytomas could be confused with adenomas by virtue of their attenuation values on unenhanced CT. CT attenuation and size of nine adrenal nodules producing pheochromocytoma syndrome were measured on unenhanced CT in nine patients. For five patients who received IV contrast material, washout profiles were also calculated. Two of the nine patients had adrenal lesions with attenuation values of less than 10 H; one had a pheochromocytoma with an attentuation of 9.0 H, and the other had a medullary hyperplasia with an attenuation of 1.8 H. These two nodules showed evidence of microscopic fat at histologic examination. No macroscopic fat was seen on the CT scans. The remaining seven patients had lesions with attenuation values exceeding 10 H (mean value, 25.6 H; range, 1.8-41 H). Mean diameter of the nine tumors (including the hyperplastic nodule) was 3.2 cm (range, 0.8-6.7 cm; SD, +/- 2.3 cm). The two low-attenuation lesions also mimicked adenomas by displaying more than 60% contrast washout on 10-min-delayed contrast-enhanced scans, unlike the other three pheochromocytomas for which we had washout data. On CT, pheochromocytomas may have attenuation values less than 10 H and also may display more than 60% washout of contrast agents on delayed scanning. Adrenal pheochromocytomas should be included with adenomas in the differential diagnosis both for masses with low attenuation on unenhanced CT and for lesions exhibiting a high percentage of contrast washout.

Method for measuring luminal efflux of fluorescent organic compounds in isolated, perfused renal tubules.

To examine directly in real time the efflux of organic compounds [e. g., organic anions (OAs) such as fluorescein (FL)] across the luminal membrane of isolated, perfused renal tubules during net secretion, we devised an approach utilizing a recently developed epifluorescence microscopy system for continuous monitoring of fluorescence in the collected perfusate. To illustrate this approach, we measured the luminal efflux rate of FL in mineral oil-covered, isolated, perfused S2 segments of rabbit renal proximal tubules. The washout profile of FL showed a deviation from linearity at time 0 when plotted on a semilog scale, indicating that the luminal efflux of FL was a saturable process. We were able for the first time to determine the kinetic parameters of luminal efflux [FL concentration at one-half maximal FL efflux (K(t)(lumen)) of approximately 560 microM and maximal rate of FL efflux across the luminal membrane (J(max)(lumen)) of approximately 635 fmol. min(-1). mm(-1)]. From the present study, we conclude that the transport step for OAs across the luminal membrane of OAs is a carrier-mediated process. This approach will work to measure luminal transport in real time for any secreted organic compound that is sufficiently fluorescent to be measured with commonly available, highly sensitive optical equipment.

Improved washout profiles with a new exercise/adenosine Tl-201 myocardial perfusion imaging protocol

Improved washout profiles with a new exercise/adenosine Tl-201 myocardial perfusion imaging protocol

Particle motion within in vitro models of stenosed internal carotid and left anterior descending coronary arteries.

Asymmetric 75% and 95% area reduction, transparent Sylgard stenotic models were operated under internal carotid artery (ICA) [Womersley parameter, alpha=5.36, Re(mean) =213 and 180, respectively, and Re(peak)=734 and 410, respectively] and left anterior descending coronary artery (LAD) flow wave forms (alpha=2.65, Re(mean)=59 and 57, respectively, and Re(peak)= 137 and 94, respectively) to evaluate the effect of these conditions on particle residence times downstream of the stenoses. Amberlite particles (1.05 g/cm3, 400 microm) were added to the fluid to simulate platelets and their motion through the stenotic region and were traced using a laser light sheet flow visualization method with pseudo-color display. Two-dimensional (2D) particle motions were recorded and particle washout in the stenotic throat and downstream section were computed for all cases. All four model cases demonstrated jetting through the stenosis which followed an arching pattern around a large separation zone downstream. Considerable mixing was observed within these vortex regions during high flow phases. Particle washout profiles showed no clear trend between the degrees of stenosis although particles downstream of the stenoses tended to remain longer for LAD conditions. The critical washout cycle (1% of particles remaining downstream of the stenosis), however, was longer for the 95% stenoses cases under each flow condition due to the larger protected region immediately downstream and maximal for the LAD 95% case. Results of this study suggest that particle residence times downstream of 75% and 95% stenoses (approximately 3-6 s for ICA and approximately 8-10 s for LAD) exceed the minimum time for platelet adhesion (approximately 1 s) for at least 1% of cells and, thus, may be sufficient to initiate thrombus formation under resting conditions.

Biophysical factors and contractile function during coronary bolus perfusion: an experimental study in the isolated guinea pig heart.

The purpose of this study was to evaluate the role of biophysical factors (viscosity, temperature, osmalality, perfusion pressure, and ionic washout) on cardiac contractility during coronary bolus perfusions. Guinea pig hearts were perfused in the osmolality mode at constant and high flow (30 mL/min) and were subjected to boluses (injected in 1-8 seconds) of purely nonionic media: mannitol and iohexol (in hyposmolal, isosmolal, and twice isosmolal forms) and iodixanol (in a hyposmolal form). All contrast media elicited a dose-dependent increase in aortic pressure and contractile variables that was attenuated by an increased osmolality. Elevation of aortic pressure was followed by an increase in left ventricular systolic pressure and, with iodixamol and iohexol, a subsequent elevation of left ventricular end-diastolic pressure. Moderate hypothermic solutions (29 degrees C) did not alter the responses. The ionic washout profile was the same for the three substances, as were the individual washout profiles for sodium, potassium, chloride, magnesium, and calcium. An increase in osmolality attenuates the increased contractility induced by a purely nonionic medium. A viscosity-induced rise in perfusion pressure may increase contractility. A moderate reduction in bolus temperature does not alter the contractile responses. The ionic washout profile is the same for the main cardioactive electrolytes.

The use of computer-assisted diagnosis in cardiac-perfusion nuclear medicine studies: a review.

The use of computer-assisted diagnosis has become widespread in cardiac nuclear medicine. Quantitative programs are commercially available for ventriculography, phase analysis, and thallium 201 perfusion studies. The goal of these programs is to eliminate interobserver variability by objectively analyzing the studies without causing a loss of accuracy. In addition, by using quantitative data not apparent on visual inspection, some programs attempt to increase sensitivity for disease above that possible by the visual reading of images. Programs that analyze perfusion studies to detect coronary artery disease have received the most interest. Results have varied, but sensitivities as high as 95% have been reported. This review discusses the techniques of computer-assisted diagnosis for thallium 201 myocardial-perfusion studies. The circumferential and washout profile methods are discussed in detail.

Technical note validity and limitations of the closed-vessel analytical solution to the axial dispersion model

The validity and limitations of the pseudo-analytical solutions of the axial dispersion model, for the “closed-closed” boundary conditions, are examined in conjunction with the determination of the Peclet number and the corresponding axial dispersion coefficient; the first ten roots of the transcendental equation related to them are calculated and tabulated. The generation of theoretical RTDs by differentiation of the washout profile is shown to be superior to the use of the analytical equation available for a Dirac delta impulse, for values of the Peclet number < 10. For Pe > 16, the analytical solution for the “open-open” boundary conditions may be used, instead. For the intermediate region (10 < Pe < 16), the Pe corresponding to the “open-open” boundary conditions may be determined and then corrected using the correlation of Ityokumbul et al.

Regulation of liver cell volume and proteolysis by glucagon and insulin

The effects of insulin and glucagon on liver cell volume and proteolysis were studied in isolated perfused rat liver. The rate of proteolysis was assessed as [3H]leucine release from single-pass-perfused livers from rats which had been prelabelled in vivo by intraperitoneal injection of [3H]leucine. The intracellular water space was determined from the wash-out profiles of simultaneously added [3H]inulin and [14C]urea. In normo-osmotic (305 mosM) control perfusions the intracellular water space was 548 +/- 10 microliters/g wet mass (n = 44) and was increased by 16.5 +/- 2.6% (n = 6), i.e. by 85 +/- 14 microliters/g, after hypoosmotic exposure (225 mosM). Glucagon (0.1 microM) decreased the intracellular water space by 17 +/- 4% (n = 4), whereas insulin (35 nM) increased the intracellular water space by 9.3 +/- 1.4% (n = 15). Also, in isolated rat hepatocyte suspensions insulin (100 nM) caused cell swelling by 10.7 +/- 1.8% (n = 16), which was fully reversed by glucagon. In perfused liver, insulin-induced cell swelling was accompanied by a hepatic net K+ uptake (4.5 +/- 0.2 mumol/g) and an inhibition of proteolysis by 21 +/- 2% (n = 12); further addition of glucagon led to a net K+ release of 3.8 +/- 0.2 mumol/g (n = 7) and fully reversed the insulin effects on both cell volume and proteolysis. Similarly, insulin-induced cell swelling and inhibition of proteolysis were completely antagonized by hyperosmotic (385 mosM) cell shrinkage. Furthermore, cell swelling and inhibition of proteolysis after hypo-osmotic exposure or amino acid addition were reversed by glucagon-induced cell shrinkage. There was a close relationship between the extent of cell swelling and the inhibition of proteolysis, regardless of whether cell volume was modified by insulin, glucagon or aniso-osmotic exposure. The data show that glucagon and insulin are potent modulators of liver cell volume, at least in part by alterations of cellular K+ balance, and that their opposing effects on hepatic proteolysis can largely be explained by opposing effects on cell volume. It is hypothesized that hormone-induced alterations of cell volume may represent an important, not yet recognized, mechanism mediating hormonal effects on metabolism.

A Non-Invasive Technique for Cell Volume Determination in Perfused Rat Liver

1) In isolated perfused rat liver, the intracellular ([14C]urea-accessible minus [3H]inulin accessible) water space was determined from the washout profiles of simultaneously infused [3H]inulin and [14C]urea. The washout profile of infused [14C]urea was indistinguishable from that of infused tritiated water. During normotonic perfusions and without hormones or amino acids in influent, the intracellular water space was 548 +/- 10 microliters/g liver wet weight (n = 44). Use of [3H]raffinose instead of [3H]inulin as marker for the extracellular space yielded almost identical values for the intracellular water space (i.e. 98.9 +/- 0.2% of that found with [3H]inulin/[14C]urea). When volume-regulatory K+ fluxes were completed following hypo- and hypertonic exposure of perfused rat livers and a steady state was reached, the intracellular water space was found to be increased and decreased, respectively. The extent of anisotonic exposure was linearly related to the change of intracellular water space. 2) Anisotonicity-, glutamine- and glycine-induced liver mass changes were almost fully explained by the simultaneously occurring alterations of the intracellular water space, indicating that cell volume changes in perfused rat liver under these conditions are not accompanied by significant changes of the extracellular space. Volume-regulatory K+ (plus accompanying anion) efflux following hypotonic perfusion accounted for about 70-85% of regulatory cell volume decrease, which occurred during the first 10 min of hypotonic exposure. 3) Cell volume of isolated hepatocytes was determined as the "hepatocrit" after gentle centrifugation of the cell suspension.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of glucose on enzyme activities and phenol utilization in Trichosporon cutaneum grown in continuous culture

Summary: The soil yeast Trichosporon cutaneum was grown in continuous culture on a minimal medium containing phenol or glucose, or both. The utilization rate of the carbon substrate and the specific activities of catechol 1,2-dioxygenase (EC 1.13.11.1), pyruvate kinase (EC 2.7.1.40), alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1) were determined. Both substrates were utilized simultaneously, the rate of glucose utilization being much higher than that of phenol. Basal activity of catechol 1,2-dioxygenase [3-5 mU (mg protein)−1] was found in glucose-grown cells. High glucose concentrations (20 g I−1) did not prevent the induction of phenol metabolism as reflected in the derepression of catechol 1,2-dioxygenase and the consumption of phenol. However, glucose partially repressed the overall phenol metabolism when T. cutaneum was grown on phenol together with glucose: phenol utilization and catechol 1,2-dioxygenase activity were reduced by 60% and 75% respectively. Complete derepression of catechol 1,2-dioxygenase did not occur until the glucose was depleted. Catechol 1,2-dioxygenase was repressed, but not inactivated, by glucose, as judged from the wash-out profile during transition from growth on phenol to growth on glucose. Pyruvate kinase activity was about twice as high in glucose-grown cells as in phenol-grown cells, and the level of the enzyme during growth on glucose was unaffected by the presence of phenol. Aspartate aminotransferase activity was higher in cells grown with phenol, or phenol together with glucose, than in cells grown on glucose alone. Alanine aminotransferase activity increased only when both substrates were present.

Preparation and characterization of adsorbents for use in high-performance liquid affinity chromatography

Adsorbents suitable for use in high-performance liquid chromatography were produced by covalently immobilizing Procion Blue MX-R to microporous silica. The adsorption capacities of the resulting materials were investigated. It was found that a Langmuir isotherm gave the best fit to data obtained for the adsorption of lactate dehydrogenase, while a Freundlich isotherm best described adsorpion of lysozyme. A 12-μm diameter silica with 28-nm diameter pores and a bound ligand concentration of 7 μmol (Procion Blue MX-R)/g (silica) had a capacity of 8.7 mg (lactate dehydrogenase)/g and 40.0 mg (lysozyme)/g. The rate of the overall adsorption process was found to be rapid. A model which assumes instantaneous equilibrium between solid and liquid phases was used to describe chromatography performed on these adsorbents. This gave a good approximation to the breakthrough and washout profiles.

Purine efflux after cardiac ischemia: relevance to allopurinol cardioprotection.

Allopurinol is thought to protect hearts against damage due to hypoxia or ischemia by inhibiting xanthine oxidase and oxygen radical generation. We subjected isolated rabbit hearts, equilibrated by perfusion at 37 degrees C, to 1 h of global ischemia at 27 or 37 degrees C with or without brief pretreatment with 100 microM allopurinol. The total absence of xanthine or uric acid in the coronary effluent following ischemia, the presence of hypoxanthine (25 +/- 4 microM peak concentration), and the failure of allopurinol to alter purine washout profiles or postischemic cardiac function suggest that rabbit myocardium lacks xanthine oxidase or dehydrogenase. Data obtained with a similar rat heart preparation showed appreciable formation of xanthine (12 +/- 2 microM peak) and uric acid (10 +/- 3 microM). Allopurinol pretreatment inhibited xanthine and uric acid formation and significantly improved key indicators of postischemic left ventricular function. We conclude that there is species dependency in the myocardial activity of xanthine oxidase or dehydrogenase, that when present it can be inhibited by acute allopurinol pretreatment, and that xanthine oxidase activity and its ability to generate oxygen radicals are not universal contributors to cardiac ischemic damage.

Continuous culture of RPMI 8226 human cells

Continuous culture of RPMI 8226 human hematopoietic cells was performed. The viable cell number and glucose, lactate and ammonium concentrations became constant within 3–4 days at a constant dilution rate. The viable cell number decreased at low and high dilution rates. The growth and product yields slightly depended on the dilution rate, except for product yield for lactate based on cell number. Growth characteristics of these cells at various dilution rates could be expressed by equations considering the maintenance energy in growth yield. Maximum specific growth rate could be evaluated from the wash-out profile and the known inhibition constants.

Tracer Elimination in I-123-Heptadecanoic Acid: Half-Life, Component Ratio and Circumferential Washout Profiles in Patients with Cardiac Disease

Sixty patients with different cardiac diseases and healthy volunteers were given omega-I-123-heptadecanoic acid (HDA) intravenously. Tracer kinetics were followed for 90 min, and tracer elimination curves were obtained regionally. In addition, circumferential washout profiles were evaluated for 26 patients and interpolative as well as constant background subtraction was performed for comparison in selected patients. Rest and stress radionuclide ventriculography allowed formation of a group with normal ventricular function (control group); the remaining patients had an abnormal ventricular function at rest or under stress. Regions of patients in the control group were significantly different (P less than 0.005) from regions of patients with CHD or CMP with regard to the initial half-life or the component ratio between a fast and a slow component (Ca/Cb). Regions of patients after MI without exercise-induced angina did not differ strikingly from control regions. Circumferential washout analysis showed homogeneous tracer kinetics in healthy subjects, bus some individuals showed increasing regional activity, mainly by late activity uptake of the stomach. Dynamic heart scintigraphy with HDA is an additional nuclear cardiologic tool that makes possible the classification of patients with myocardial disease and abnormal ventricular function already under resting conditions. Initial half-life allows reasonable discrimination between different severely diseased patient groups; expansion of acquisition time to 90 min refines biexponential tracer analysis which, by means of an altered component ratio Ca/Cb, may allow better clinical judgement of the individual patient. Circumferential washout analysis and interpolative background correction lead to a better specificity of examination.

Clinical pharmacology of netilmicin in preterm and term newborn infants

Sixty-four neonates, with gestational age ranging from 27 1/2 to 40 weeks, postnatal age from 1 to 15 days, and birth weight from 800 to 3400 gm, were given netilmicin 2.5 mg/kg intramuscularly two or three times per day according to postnatal age, for 5 to 14 days. Serum concentrations were measured before and 1 hour after a dose at least twice during treatment. The serum washout profile of the drug was observed in 22 neonates after discontinuation of therapy. Renal function was studied in 37 infants by measuring serum creatinine concentrations and in 27 by urinary excretion of N-acetyl-glucosaminidase during and up to 15 days after therapy. Behavioral and impedance audiometry, and in infants failing those, auditory brainstem evoked response tests, were performed between 6 and 12 months of age. In 23.5% of the neonates, trough serum levels were greater than 3 micrograms/ml. The serum washout followed a multiexponential decay, accounting for distributional, rapid (initial), and slow (tissue) elimination phases. Linear regression analysis performed between each kinetic parameter and gestational age or birth weight showed that initial elimination half-life, steady-state volume of distribution, and total body clearance were significantly correlated with both variables. Netilmicin did not cause detectable renal or auditory damage.

NETILMICIN IN PREMATURE AND FULL-TERM INFANTS: ITS EFFECTS ON THE RENAL AND AUDITORY FUNCTIONS AND ITS KINETICS ACCORDING TO MULTICOMPARTMENT MODEL

68 neonates with gestational age ranging from 27 to 40 weeks, 6-15 days postnatal age and 800-3400 g birth weight were treated for 5-14 days with Netilmicin (N), 2.5 mg/kg/b.i.d. or t.i.d. according to postconceptional age. Their renal function was studied by measuring serum creatinine concentrations during and up to 15 days after therapy. The urinary excretion of N-acetyl-glucosaminidase (NAG) was measured as a marker of tubular injury. Valley N serum levels were monitored during the treatment. Serum and urinary washout profile of the drug were followed for up to 10-15 days after discontinuation. Behavioral and impedance audiometry are being performed between 6 to 12 months of age. Treatment with N (always in association with a β-lactam antibiotic) did not represent for the newborns a source of toxicity as far as either renal or auditory (38 infants so far examined) function are concerned. Small preterm neonates (≤34 weeks G.A.) more frequently had elevated valley serum N concentration (≥3 ug/ml). The serum and urinary washout followed a multiexponential decay with prolonged terminal T 1/2 indicating tissue accumulation.

The intracellular localization of amiloride in frog skin.

The diuretic compound amiloride is often used as a specific inhibitor of the passive Na+ entry step in the transepithelial transport of Na+ across frog skin. We have utilized the fluorescence properties of amiloride to study the distribution of this transport inhibitor in the ventral skin of Rana pipiens. After a 30 s exposure of 1-100 microM amiloride to the external surface of frog skin, amiloride fluorescence was evident in the cytoplasm of all cell layers of the epidermis and alveolar gland epithelium. Changes in the conditions of incubation which alter the pharmacological activity of amiloride did not affect the intracellular distribution of amiloride or the washout profile of [14C]amiloride. The presence of amiloride fluorescence in the cytoplasm prevented our examination of changes in the amiloride fluorescence at the cell surface with various conditions of incubation. Four derivatives of amiloride that differed in their ability to inhibit short-circuit current were also localized intracellularly but varied in their relative distribution among the cell layers of the epidermis. Our results indicate that when incubated at concentrations from 1 to 100 microM, a large fraction of the amiloride taken up by frog skin is not directly involved with the inhibition of passive Na+ transport at the apical surface of the stratum granulosum. The mechanism of intracellular uptake of amiloride is not clear. However, the cytoplasmic localization of amiloride could explain the action of the drug on intracellular enzymes and may account for the large proportion of non-displaceable [14C]amiloride that has been observed in frog skin.