Finite element model of antibody penetration in a prevascular tumor nodule embedded in normal tissue.

We have developed a pharmacokinetic model for monoclonal antibodies (mAb) to aid in investigating protocols for targeting small primary tumors or sites of metastatic disease. The model describes the uptake of systemically-administered antibody by a prevascular spherical tumor nodule embedded in normal tissue. The model incorporates plasma kinetics, transcapillary transport, interstitial diffusion, binding reactions, and lymphatic clearance. Antigen internalization can easily be incorporated. Simulations obtained from a three-dimensional finite element analysis are used to assess errors in predictions from earlier models in which the influence of the normal tissue was collapsed into a boundary condition at the tumor surface. The model employing a Dirichlet boundary condition substantially overpredicted the mean total tumor mAb concentration at all times. Although the model with a concentration-dependent flux (composite) boundary condition underpredicted mAb concentration, the discrepancy with finite element results is only notable at early times. Sensitivity analyses were performed on mAb dose and on the coefficients for mAb diffusion in the tissue regions, since reported antibody diffusivity values have varied over 30-fold. The results of the study suggest that mAb diffusivity and mAb binding site density in tumors should have major influences on optimizing doses and scheduling of mAb administration in tumor targeting protocols.

Probe calibration in transient microdialysis in vivo.

PURPOSE: We examine the theoretical basis for calibrating microdialysis probes in vivo for pharmacokinetic experiments in which the extracellular analyte concentrations vary in time. METHODS: A software package, MICRODIAL. was used to simulate microdialysis for illustrative transient situations with linear concentration dependence. RESULTS: For a constant distant extracellular analyte concentration. the calibration factor (extraction fraction, Ed) exhibits a mass transfer transient associated with the development of spatial concentration profiles within the tissue and the probe. Processes clearing the analyte from the extracellular fluid (ECF) strongly influence the rapidity of approach to steady-state and affect the magnitude of the steady-state calibration factor, Ess(d). For situations in which the distant ECF concentration varies in time as a result of exchange with the plasma compartment, different time profiles of the distant ECF and plasma concentrations yield different transient E(d). For the linear, transient cases examined, the area-under-the-curve (AUC 0-infinity) time integral of the distant ECF concentration was found to be proportional to the outflow dialysate concentration-time integral with Ess(d) being the proportionality constant. CONCLUSIONS: The options for calibrating microdialysis probes in solid tissues appear limited under non-steady state conditions; however, AUC integrals for linear systems may be determined by continuous microdialysis sampling and steady-state probe calibration approaches.

Computational models of antibody-based tumor imaging and treatment protocols.

We present improved computational models for investigating monoclonal antibody-based protocols for diagnostic imaging and therapy of solid tumors. Our earlier models used a boundary condition (Dirichlet) that specified concentrations of diffusing molecular species at the interface between a prevascular tumor nodule and surrounding normal tissue. Here we introduce a concentration-dependent flux boundary condition with finite rates of diffusion in the normal tissue. We then study the effects of this new condition on the tumor's temporal uptake and spatial distribution of radiolabeled targeting agents. We compare these results to ones obtained with the Dirichlet boundary condition and also conduct parameter sensitivity analyses. Introducing finite diffusivity for any molecular species in normal tissue retards its delivery to and removal from the tumor nodule. Effects are protocol- and dose regimen-dependent: generally, however, mean radionuclide concentration and tumor-to-blood ratio declined, whereas relative exposure and mean residence time increased. Finite diffusivity exacerbates the negative effects of antigen internalization. Also, the sensitivity analyses show that mean concentration and tumor-to-blood ratio are quite sensitive to transcapillary permeability and lymphatic efflux values, yet relatively insensitive to precise values of diffusion coefficients. Our analysis underscores that knowledge of antigen internalization rates and doses required to saturate antigen in the tumor will be important for exploiting antibody-based imaging and treatment approaches.

Effect of capillary efflux transport inhibition on the determination of probe recovery during in vivo microdialysis in the brain.

Intracerebral microdialysis probe recovery (extraction fraction) may be influenced by several mass transport processes in the brain, including efflux and uptake exchange between brain and blood. Therefore, changes in probe recovery under various experimental conditions can be useful to characterize fundamental drug transport processes. Accordingly, the effect of inhibiting transport on probe recovery was investigated for two capillary efflux transporters with potentially different membrane localization and transport mechanisms, P-glycoprotein and an organic anion transporter. Fluorescein/probenecid and quinidine/LY-335979 were chosen as the substrate/inhibitor combinations for organic anion transport and P-glycoprotein-medicated transport, respectively. Probenecid decreased the probe recovery of fluorescein in frontal cortex, from 0.21 +/- 0.017 to 0.17 +/- 0.020 (p < 0.01). Quantitative microdialysis calculations indicated that probenecid treatment reduced the total brain elimination rate constant by 3-fold from 0.37 to 0.12 (ml/min. ml of extracellular fluid). In contrast, the microdialysis recovery of quinidine, delivered locally to the brain via the probe perfusate, was not sensitive to P-glycoprotein inhibition by systemically administered LY-335979, a potent and specific inhibitor of P-glycoprotein. Recovery of difluorofluorescein, an analog of fluorescein, was also decreased by probenecid in the frontal cortex but not in the ventricle cerebrospinal fluid. These experimental observations are in qualitative agreement with microdialysis theory incorporating mathematical models of transporter kinetics. These studies suggest that only in certain circumstances will efflux inhibition at the blood-brain barrier and blood-cerebrospinal fluid barrier influence the microdialysis probe recovery, and this may depend upon the substrate and inhibitor examined and their routes of administration, the localization and mechanism of the membrane transporter, as well as the microenvironment surrounding the probe.

Evidence for the ability of hippocampal neurons to develop acute tolerance to ethanol in behaving rats.

BACKGROUND: The cellular mechanisms underlying acute tolerance to alcohol are unclear. This study aimed to determine whether hippocampal neurons have the ability to develop acute tolerance to alcohol in behaving rats. METHODS: Intrahippocampal microdialysis was performed in freely behaving rats, and the firing of single neurons in the dialysis area was recorded. The control microdialysis fluid, artificial cerebrospinal fluid (ACSF), was replaced with 1 M ethanol in ACSF for a 30 min period. One hour later, the ethanol perfusion was repeated. To test the functional integrity of the microdialysis probe in situ, each microdialysis session was completed with recording the effect of a 10-20 min perfusion of 500 microM N-methyl-D-aspartate (NMDA). The extracellular concentration profile of ethanol during intrahippocampal microdialysis with 1 M ethanol was estimated in a separate study in anesthetized rats. The ethanol content was measured in tissue slices surrounding the probe with gas chromatography (GC), and the generated data were analyzed with a mathematical model for microdialysis to estimate the concentration of ethanol at the recording site. RESULTS: The predominant effect of the first intrahippocampal microdialysis with ethanol was a decrease in firing rate in both pyramidal cells and interneurons. In contrast, such firing rate decrease did not develop during the second ethanol perfusion. Subsequent NMDA perfusion still induced robust changes in the electrical activity of the neurons. The estimated extracellular ethanol concentration at the recording site was 45-70 mM. CONCLUSION: This study revealed that hippocampal neurons have the ability to develop acute tolerance to a single exposure of clinically relevant concentrations of ethanol in behaving rats, without influences from the rest of the body.

Diffusion of small solutes in the lateral intercellular spaces of MDCK cell epithelium grown on permeable supports.

The diffusion coefficients of four solutes ranging in molecular weight from 238 to 10,000 in the lateral intercellular spaces (LIS) of cultured kidney cells (MDCK) grown on permeable supports were determined from the spread of fluorescence produced after the release of caged compounds by a pulse from a UV laser. Two types of experiments were performed: measurement of the rate of change of fluorescence after releasing a caged fluorophore, and measurement of the change in fluorescence of a relatively static fluorescent dye produced by the diffusion of an uncaged ligand for the dye. Fluorescence intensity was determined by photon-counting the outputs of a multichannel photomultiplier tube. Diffusion coefficients were determined in free solution as well as in the LIS of MDCK cells grown on permeable supports and the hindrance factor, theta, determined from the ratio of the free solution diffusivity to that in the LIS. The hindrance factors for 3000-MW dextran, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, MW 524) and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, MW 238) were not significantly different from 1. The diffusion of 10,000-MW dextran was substantially reduced in the LIS with a theta of 5.6 +/- 0.3. Enzymatic digestion by neuraminidase of the sialic acid residues of the glycosylation groups in the LIS increased the diffusivity of the 10,000-MW dextran 1.8-fold indicating hindrance by the glycocalyx. We conclude that small solutes, such as Na(+) and Cl(-), would not be significantly restricted in their diffusion in the LIS and that solute concentration gradients could not develop along the LIS under physiologic conditions.

Quantitative microdialysis of ethanol in rat striatum.

We have applied a steady-state theory of microdialysis to characterize the diffusion of ethanol through a microdialysis membrane and through rat striatum. Quantitative characterization required measurement of in vitro and in vivo extraction fractions for ethanol and determination of the clearance of ethanol from brain tissue during steady-state perfusion through a microdialysis probe. Extraction fraction of ethanol was determined in vitro by perfusing a known concentration of ethanol through probes immersed in water at 37 degrees C with stirring. The in vitro extraction fraction yielded a probe permeability value of 0.046 +/- 0.004 cm/min that is comparable with an estimate from published measurements for similar dialysis membranes. The in vivo extraction fraction was determined for probes placed in the striatum. Clearance of ethanol and a brain slice concentration profile of ethanol were determined by measurement of the amount of ethanol remaining in the brain tissue during steady-state perfusion of the probe. Steady state was achieved within 10 min after beginning the ethanol perfusion in vivo, and the extraction fraction was not altered by sedation of the rat with pentobarbital. The tissue concentration profile was symmetrical around the probe track, and ethanol was detected 1 mm from the probe. The experimental clearance rate constant value obtained for ethanol (2.0 +/- 0.3 min(-1)) was higher than that expected for removal solely by loss to the blood. The tissue diffusivity for ethanol, Dt, derived from the experimental measurements was 1.2 +/- 0.2 x 10(-5) cm2/sec. This value is greater than expected for interstitial diffusion, suggesting a substantial contribution by transcellular diffusion of ethanol as well. The predicted tissue concentration profile had a higher peak value and did not extend into the tissue (0.5 mm) as much as the experimental profile (1 mm), although there was reasonable agreement between experiment and theory. Our quantitative characterization of the microdialysis behavior of ethanol in brain provides a framework for interpretation of brain microdialysis experiments using ethanol by supplying, inter alia, a means for estimating the ethanol concentration achieved in the tissue volume being sampled by the probe.

Diffusion coefficients in the lateral intercellular spaces of Madin-Darby canine kidney cell epithelium determined with caged compounds.

The diffusion coefficients of two caged fluorescent dyes were measured in free solution and in the lateral intercellular spaces (LIS) of cultured Madin-Darby canine kidney (MDCK) cells after photoactivation by illumination with a continuous or pulsed UV laser. Both quantitative video imaging and a new photometric method were utilized to determine the rates of diffusion of the caged fluorescent dyes: 8-((4,5-dimethoxy-2-nitrobenzyl)oxy)pyrene-1,3,6-trisulfonic acid (DMNB-HPTS) and (4,5-dimethoxy-2-nitrobenzyl) fluorescein dextran (10,000 MW) (DMNB-caged fluorescein dextran). The diffusion coefficients at 37 degrees C in free solution were 3.3 x 10(-6) cm2/s (HPTS) and 0.98 x 10(-6) cm2/s (10,000 MW dextran). Diffusion of HPTS within nominally linear stretches of the LIS of MDCK cells grown on glass coverslips was indistinguishable from that in free solution, whereas dextran showed a 1.6 +/- 0.5-fold reduction in diffusivity. Measurements of HPTS diffusion within the LIS of multicellular regions also exhibited a diffusivity comparable to the free solution value. The restriction to diffusion of the dextran within the LIS may be due to molecular hindrance.

Effect of ethanol on extracellular dopamine in rat striatum by direct perfusion with microdialysis.

The concentration-related effects of ethanol on extracellular dopamine (DA) in rat striatum were studied by direct perfusion through microdialysis probes in freely moving rats. Two sets of three ethanol concentrations were separately tested using a Latin square experimental design. Potassium stimulation with high potassium (50 mM) in artificial CSF (ACSF) preceding ethanol treatment confirmed the neuronal function of dopaminergic cells by increasing DA concentrations to 200-1,500% of basal levels. The perfusion with calcium-free ACSF applied at the end of each experiment confirmed the calcium dependency of the basal levels of extracellular DA by decreasing basal DA levels by 70%. The striatal volume measurement to examine the possible brain damage by direct ethanol perfusion suggested that ethanol did not increase the damage caused by the probe implantation at any ethanol concentration tested in this study. The 30-min direct perfusion of 510 and 860 mM ethanol resulted in a significant concentration-related stimulatory effect on the extracellular DA concentration in rat striatum (510 mM, 29% increase, p < 0.05; 860 mM, 66% increase, p < 0.05). However, there was no significant effect of ethanol at low concentrations, < or = 170 mM. Considering the effective ethanol concentration in tissue areas in which DA is sampled, the data suggest that concentrations of ethanol associated with moderate intoxication do not directly affect the extracellular concentration of DA in the striatum. Therefore, the systemic effects of ethanol on striatal DA found in previous studies may be caused by the interaction with sites other than the striatum.

Cross-flow membrane plasmapheresis technique for continuous ex vivo plasma sampling.

A technique is described for plasma sampling by continuous membrane plasmapheresis performed on blood flowing through an extracorporeal arteriovenous shunt. The plasmapheresis sampler in the shunt employs replaceable commercial planar membranes 2.5 cm in diameter. Validation tests were conducted for 0.6-micron pore diameter microporous membranes with several low-molecular-weight, nonmetabolized solutes that either rapidly equilibrate between plasma and formed elements or remain extracellular. Ex vivo tests were performed for bolus intravenous administration to rabbits. The technique yielded values for time-averaged plasma concentrations comparable to those obtained with serial blood and continuous blood withdrawal methods. The new technique should be particularly advantageous when the distribution of the solute of interest between plasma and formed elements of the blood undergoes significant changes during the sampling interval as a result of binding, exchange, or metabolism in the formed element phase.

Estimation of blood sampling errors resulting from metabolism and solute exchange between plasma and formed elements.

The origin and magnitude of potential errors in whole-blood sampling are predicted on the basis of a mathematical model. The model describes the kinetics of solute metabolism, breakdown, and interphase distribution (i.e., partitioning and exchange between formed elements and plasma) within a blood sample during sample withdrawal and storage. The model is applied to the determination of the integral over time of solute concentration in the plasma (area-under-the-curve, or AUC) from a sample withdrawn through an arterial or venous catheter. Errors in AUC determination can be substantial and are strongly dependent on the duration of sampling (T), the rate constants for solute degradation processes, the rate constant for solute exchange between the formed elements and the plasma (ke), and the equilibrium ratio for distribution of the solute between formed elements and plasma (R). When the value of the dimensionless group keT/R is small, little solute exchanges between plasma water and formed elements before the two phases of the blood are separated. When keT/R is large, the solute distribution is close to equilibrium at all times. In these two keT/R limits, the contribution of solute redistribution to sampling error is small. Sizable errors resulting from redistribution are associated with intermediate values of keT/R, even in the absence of metabolism and despite rapid separation of the phases at the end of the withdrawal period. Chemical conversion within either of the blood phases introduces additional sampling error under most circumstances.

pH, morphology, and diffusion in lateral intercellular spaces of epithelial cell monolayers.

The lateral intercellular spaces (LIS) of reabsorptive epithelia are the site of the proposed local osmotic gradient responsible for transepithelial transport. We developed techniques for loading the LIS of living cultured renal cells (MDCK and LLC-PK1) with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), visualizing LIS geometry, measuring pH, and determining the BCECF diffusion coefficient within the LIS. The LIS pH was remarkably constant and differed substantially from that of the superfusate in both the presence and absence of HCO3 or CO2. The LIS of MDCK cells had a pH of 7.66 +/- 0.04 in bicarbonate-free solutions of pH 7.0, 7.4, or 7.8. In bicarbonate-containing solutions, MDCK LIS pH was acidic to the superfusate by 0.3-0.4 units. In the absence of bicarbonate, the LIS of LLC-PK1 cells was markedly acidic (6.83 +/- 0.05), becoming alkaline by approximately 0.25 units in the presence of bicarbonate. Gradients in pH or dye concentration were not detected within the LIS. The diffusion coefficient of BCECF within the LIS was approximately equal to that seen in free solution.

Microdialysis study of zidovudine (AZT) transport in rat brain.

The concentration profiles of [14C]3'-azido-3'-deoxythymidine (AZT) emanating from an acutely implanted microdialysis probe were measured in rat caudate putamen by quantitative autoradiography for infusions of 14 min and 1 and 2 h. A mathematical model which simulated diffusive solute transport, unaffected by the processes of microvascular exchange or tissue metabolism, did not fit the observed concentration profiles. Chromatographic analysis of brain homogenates for metabolites of AZT showed that the rate of metabolic transformation was not large enough to affect transport of the drug through the brain tissue. A model simulating the effect of microvascular exchange on the diffusion profiles fit the observed concentration profiles and the transient change in the dialysate extraction fraction. This analysis yielded an estimated tissue elimination rate constant for microvascular exchange of Kel = 0.013 ml/(g.min) and an intra- to extracellular partition coefficient of K pi = 1.04. Inclusion of probenecid in the dialysate, together with an i.p. injection, led to a substantial increase in the diffusion distance of the labeled AZT from the microdialysis probe, suggesting at least a 4-fold decrease in the microvascular exchange rate constant. These results imply that AZT is actively transported out of the brain parenchyma to the microvasculature and that this active transport mechanism is responsible for the limited central nervous system penetration of systemically administered AZT, in spite of its high lipid solubility.

Quantitative examination of tissue concentration profiles associated with microdialysis.

Spatial solute concentration profiles resulting from in vivo microdialysis were measured in rat caudate-putamen by quantitative autoradiography. Radiolabeled sucrose was included in the dialysate, and the tissue concentration profile measured after infusions of 14 min and 61.5 min in an acute preparation. In addition, the changes in sucrose extraction fraction over time were followed in vivo and in a simple in vitro system consisting of 0.5% agarose. These experimental results were then compared with mathematical simulations of microdialysis in vitro and in vivo. Simulations of in vitro microdialysis agreed well with experimental results. In vivo, the autoradiograms of the tissue concentration profiles showed clear evidence of substantial differences between 14 and 61.5 min, even though the change in extraction fraction was relatively small over that period. Comparison with simulated results showed that the model substantially underpredicted the observed extraction fraction and overall amount of sucrose in the tissue. A sensitivity analysis of the various model parameters suggested a tissue extracellular volume fraction of approximately 40% following probe implantation. We conclude that the injury from probe insertion initially causes disruption of the blood-brain barrier in the vicinity of the probe, and this disruption leads to an influx of water and plasma constituents, causing a vasogenic edema.

Flow rate measurements in isolated perfused kidney tubules by fluorescence photobleaching recovery.

We have developed a new application of the fluorescence photobleaching recovery (FPR) technique for instantaneous measurement of volume flow rates at any axial position along isolated perfused kidney tubules. The method requires fast data acquisition of emitted fluorescence through a photomultiplier (time resolution, 0.5 ms) coupled with differential interference contrast microscopy to measure luminal diameters accurately. While the tubule is perfused in vitro with an impermeant fluorophore (fluorescein sulfonate), a 20-ms bleach pulse reduces the fluorescence in the observation region by 20-25%. Fluorescence recovery is a direct function of perfusate velocity; diffusion plays no significant role in the early phase of recovery. A fluid dynamics approach to data analysis shows that fractional recovery increases linearly with time until t = L/2vm, where L is the length of the observation window and vm is the mean axial velocity. Practically, a linear regression analysis of the early recovery phase allows measurement of vm of up to 0.14 cm/s, i.e., a 40-nl/min flow rate in a 25-microns-diameter tubule. Calibration experiments in small glass tubes perfused at predetermined flow rates demonstrated good accuracy (within 10%) and reproducibility (coefficient of variation, 8.7%). In rat inner medullary collecting ducts microperfused at 4-40 nl/min, the correlation with a standard fluid collection method was excellent (r2 greater than 0.97). The method should also be suitable for the direct measurement of fluid flow rate in kidney tubules or blood vessels microperfused in vivo.

Quantitative microdialysis: analysis of transients and application to pharmacokinetics in brain.

The behavior of a microdialysis probe in vivo is mathematically described. A diffusion-reaction model is developed that not only accounts for transport of substances through tissues and probe membranes but also accounts for transport across the microvasculature and metabolism. Time-dependent equations are presented both for the effluent microdialysate concentration and for concentration profiles about the probe. The analysis applies either to measuring the tissue pharmacokinetics of drugs administered systemically, or for sampling of endogenously produced substances from tissue. In addition, an expression is developed for the transient concentration about the probe when it is used as an infusion device. All mathematical expressions are found to be a sum of an algebraic and an integral term. Theoretical prediction of time-dependent probe behavior in brain has been compared with experimental data for acetaminophen administered at 15 mg/kg to rats by intravenous bolus. Plasma and whole striatal tissue samples were used to describe plasma kinetics and to estimate a capillary permeability-area product of 0.07 min-1. Theoretical prediction of transient effluent dialysate concentrations exhibited close agreement with experimental data over 60 min. Terminal decline of the dialysate effluent concentration was slightly overestimated but theoretical concentrations still lay within the 95% confidence interval of the experimental data at 112 min. Microvasculature transport and metabolism play major roles in determining microdialysate transient responses. Extraction fraction (recovery) has been shown to be a declining function in time for five probe operating conditions. High rates of metabolism and/or capillary transport affect the time required to approach steady-state extraction, shortening the time as the rates increase. Conversely, for substances characterized by low permeabilities and negligible metabolism, experimental situations exist that are predicted to have very slow approaches to microdialysis steady state.

Effects of different semipermeable membranes on in vitro and in vivo performance of microdialysis probes.

The in vitro and in vivo performance of three different semipermeable microdialysis membranes was compared: a proprietary polycarbonate-ether membrane made by Carnegie Medecin; cuprophan, a regenerated cellulose membrane; and polyacrylonitrile. When microdialysis probes were tested in a stirred in vitro solution, large and statistically significant differences among the three membranes in extraction of acid metabolites (3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, and homovanillic acid) and acetaminophen were found. Polyacrylonitrile had the highest extractions in vitro. In contrast, when microdialysis probes were implanted in vivo (in rat striatum), extraction of acid metabolites and acetaminophen did not differ significantly among the different membranes. These results are consistent with predictions made by a mathematical model of microdialysis and can be explained by the fact that in vitro the main factor limiting extraction is membrane resistance to diffusion, whereas tissue resistance to diffusion plays a more dominant role in vivo. These findings suggest that (aside from differences in surface area), the choice of semipermeable membrane will generally have little effect on in vivo microdialysis results. Furthermore, in vitro measurements of microdialysis probe extractions are not a reliable way of calibrating in vivo performance.

Steady-state theory for quantitative microdialysis of solutes and water in vivo and in vitro.

A mathematical framework was developed to provide a quantitative basis for either in vivo tissue or in vitro microdialysis. Established physiological and mass transport principles were employed to obtain explicit expressions relating dialysate concentration to tissue extracellular concentration for in vivo applications or external medium concentrations for in vitro probe characterization. Some of the important generalizations derived from the modeling framework are: (i) the microdialysis probe can perturb the spatial concentration profile of the substance of interest for a considerable distance from the probe, (ii) for low molecular weight species the tissue is generally more important than the probe membrane in determining the dialysate-to-tissue concentration relationship, (iii) metabolism, intracellular-extracellular and extracellular-microvascular exchange, together with diffusion, determine the role of the tissue in in vivo probe behavior, and, consequently, (iv) in vitro "calibration" procedures could be useful for characterizing the probe, if properly controlled, but have limited applicability to in vivo performance. The validity of the proposed quantitative approach is illustrated by the good agreement obtained between the predictions of a model developed for tritiated water ([3]H2O) in the brain and experimental data taken from the literature for measurements in the caudoputamen of rats. The importance of metabolism and efflux to the microvasculature is illustrated by the wide variation in predicted tissue concentration profiles among [3]H2O, sucrose and dihydroxyphenylacetic acid (DOPAC).

Posterior vitreous fluorophotometry. II. Comparison of diabetic patients and controls with the use of a new analysis procedure.

We used a new computerized procedure to analyze posterior vitreous fluorophotometry scans, obtained with a commercial fluorophotometer (Fluorotron Master Coherent, Palo Alto, Calif), in insulin-dependent diabetic patients and controls. Diabetic patients with minimal retinopathy had significantly higher effective fluorescein vitreous diffusivity and 30-minute 3-mm vitreous fluorescence values than either controls or diabetic patients with no retinopathy. However, there was no significant difference between the groups for the apparent permeability of the blood-retinal barrier to fluorescein. These results suggest the possibility that the higher 3-mm fluorescence levels found in the diabetic patients with minimal retinopathy may result in part from enhanced movement of fluorescein through the vitreous.