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Tumor Biology |
Department of Radiation Oncology [S. N. E., A. A. G., J. M. P., M. W. D.], Kenan Plastic Surgery Research Laboratories [C. C. P., B. K.], Department of Biomedical Engineering [N. A. W., M. W. D., B. K.], and Cancer Center Biostatistics [J. S. S.], Duke University Medical Center, Durham, North Carolina, 27710
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As the perfusion fluid is pumped slowly through the microdialysis probe, analytes in the perfusate and the interstitium have the opportunity to approach equilibrium. The degree of equilibration between microdialysate and interstitium is dependent on physical and physiological factors (1) . The predominant factors that affect recovery are perfusion flow rate, diffusivity of analytes through the tissue and capillary wall, metabolism, length of the dialysis membrane (2 , 3) , and properties of the dialysis membrane, such as composition and pore size (3, 4, 5, 6) .
Microdialysis is often performed under nonequilibrium conditions. As a result, microdialysate concentrations of a solute or analyte of interest may be only a fraction of the unbound concentrations in the extracellular space in the region of the implanted probe. The concentration of the analyte of interest in the dialysate would then be less than that in the extracellular fluid. The term recovery is used to reflect the ratio of the concentration obtained in microdialysate to the concentration in the extracellular fluid surrounding the probe (7) . Changes in recovery during an experiment (because of blood flow or capillary permeability) could yield changes in microdialysate composition that may not reflect altered extracellular concentrations.
The extent to which the microdialysate equilibrates with the interstitium must be quantified to accurately interpret the measured microdialysate concentrations of an analyte. When equilibration is incomplete, the recovery of a given analyte must be determined to allow estimation of true extracellular concentrations. The ratio of the concentration of analyte in the microdialysate to its concentration in the surrounding medium is commonly expressed as a percentage and is typically termed RR5 (1) . Knowing the RR allows conversion of microdialysate concentrations into absolute interstitial concentrations (3) . When using microdialysis for quantitative measurements of tissue concentrations, RR must be determined for each probe in vivo (1 , 2) .
One method to measure extracellular concentrations in vivo is the no net flux method. If the concentration of analyte in the perfusate equals the extracellular concentration, then the net flux of analyte into or out of the probe is zero. The no net flux method uses known concentrations of a test analyte or marker in the perfusate (8) at a range of concentrations above and below the projected tissue concentrations and assumes steady-state interstitial analyte concentrations. The true extracellular concentration of the marker can be estimated as the point where inflow concentration equals outflow concentration (that is, where no net flux occurs), assuming analytes have achieved steady-state concentrations.
A recovery marker is used to determine the efficiency of mass transport across a given microdialysis probe. If we assume a linear relationship between marker recovery and the recovery of other analytes, then the recovery for the marker can then be used to convert the measured concentration of analyte in the microdialysate to an absolute tissue concentration. For example, if a marker recovery of 50% is measured and if the analyte of interested has similar diffusion characteristics as the marker, then all of the analyte concentrations measured in the microdialysate would be divided by 50% to yield the estimated absolute analyte concentration in the tissue. Recovery markers that have been used by other investigators in normal tissues are glucose and urea (9, 10, 11) . Because tissues other than the liver are not expected to produce or consume urea and because urea diffuses readily throughout tissues, urea plasma concentration may be a relatively accurate measure of interstitial urea concentrations in normal tissues. Tumor and ischemic tissues pose a particularly difficult problem because of high consumption and poor delivery of nutrients. In tumors, interstitial glucose levels are variable (12 , 13) , making glucose inappropriate as a tumor recovery marker.
In contrast, urea is produced in the liver by the urea cycle and is a highly diffusible molecule that freely distributes throughout the body water compartment. Similar to normal tissues, urea does not appear to be consumed or produced in neoplastic tissues. Guillino et al. (13) suggest minimal differences between the tumor interstitial urea levels and blood urea levels flowing in and out of tissue-isolated tumors showing that it is not consumed or produced there.
The aim of our study was to use the no net flux method to determine the extracellular concentration of urea in tumors. Our hypothesis was that a linear relationship exists between tumor and PUs over a physiological range. If so, urea can be used as a recovery marker for tumor microdialysis. An application of this study was to measure interstitial glucose concentrations during i.v. glucose infusion to determine the efficiency of glucose transport in tumors during hyperglycemia.
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MATERIALS AND METHODS |
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Anesthesia Methods and Monitoring.
For both the no net flux and hyperglycemia studies, rats were anesthetized with i.p. injections of sodium pentobarbital (Nembutal; 50 mg/kg). The right femoral artery was cannulated to monitor the status of the animals during anesthesia. Blood pressures (systolic, diastolic, and mean pressures) and heart rate were determined from the arterial pulsatile waveform from a standard pressure transducer and monitored continuously (BPA 190b Blood Pressure Analyzer; Micro-Med., Inc., Louisville, KY).
A femoral vein catheter allowed repeated dosing of anesthetic during the procedures and for euthanasia. In addition, the glucose was administered i.v. Anesthesia was maintained with repeated dosing of Nembutal as needed (0.5–1.0 mg of Nembutal). At the end of the experiment, the animal was euthanized with an i.v. injection of an overdose of Nembutal (15 mg).
Animals breathed room air spontaneously, and respiratory rate was monitored routinely. Animals were kept normothermic by placing them on heated water blankets during the experiment (Baxter K MOD 100 Heat Therapy Pump; Baxter Healthcare Corp, Deerfield, IL). Body temperature was monitored by rectal thermometer. Tumor blood flow was monitored with a laser Doppler flowmeter (LDF; Oxford Optronix, Oxford, United Kingdom).
Microdialysis Probes.
Commercially available microdialysis probes with a membrane length of 10 mm, a molecular weight cutoff of Mr 100,000, and an outer diameter of 0.5 mm (model CMA/20; CMA Microdialysis, Solna, Sweden) were used for the no net flux study. CMA/20 Microdialysis probes with a membrane length of 10 mm, a molecular weight cutoff of Mr 20,000, and an outer diameter of 0.5 mm (model number 830-9571; CMA Microdialysis) were used for the hyperglycemia study.
Microdialysis preparation and set-up were similar for both studies. The probes were soaked in lactated Ringer’s solution before placement in tissue. The inlet and outlet lines of the probe were connected to FEP Teflon tubing (BAS Bioanalytical Systems Inc., West Lafayette, IN) with flanged tubing connectors. Tubing connectors were soaked in ethanol before use. The tubing is designed to minimize adsorption of proteins and other substances to its walls.
To place the microdialysis probe into the neoplastic tissue, a small stab incision was made in the skin of the rodent with a #11 scalpel blade. A plastic split tubing (CMA Microdialysis) was placed over a sharp needle and inserted together into the tumor. The needle was removed from the split tubing, and the microdialysis probe was inserted into the plastic tubing. The split tubing could then be separated, leaving the probe in the tumor. Finally, the probe was sutured to skin. Probe placement was confirmed by gross inspection after each experiment.
No Net Flux Study Perfusate.
Urea (Sigma Chemical Co., St, Louis, MO) was prepared in lactated Ringers’ solution (Abbott Laboratories, North Chicago, IL) to concentrations of 0.83, 2.5, 5.0, and 13.33 mM. A 1-ml gastight syringe (Bee Stinger; BAS Bioanalytical Systems Inc.) was filled with perfusate at ambient temperature. The loaded syringe was placed in a microinfusion syringe pump (model number MD-1001; BAS Bioanalytical Systems Inc.). The syringe pump was connected to a microinfusion controller to produce a constant flow rate (Controller BAS Beehive; BAS Bioanalytical Systems Inc.).
No Net Flux Study Protocol.
Before the start of each experiment, the microdialysis probe was flushed at 2 µl/min to purge the tubing and probes of air bubbles and to ensure that fluid was flowing through the complete system. The perfusate flow rate was maintained at 2 µl/min for the duration of the experiment. Dialysate samples were collected by a microfraction collector (CMA model 142). At least four dialysate samples were collected during 15-min intervals from the tumor probes for each perfusate solution.
After the probe was inserted, perfusate was allowed to flow for 30 min to allow for steady state to be established. Thus, the first two 15-min samples (t = -45 and -30 min), which reflect alterations caused by insertion trauma, were discarded. The next two dialysate samples (t = -15 and 0 min) were used in no net flux analysis. There was a delay of 
2 min before concentration changes in the perfusate reached the tissues and the collection vial. This delay is attributable to diffusion, the low flow rate, and the tubing volume between the syringe, the probe, and collection vial (1
, 11)
. After changing the perfusate to the next concentration of urea, another 30 min was allowed to reach steady state conditions, and the next two dialysate samples were taken for inclusion in no net flux analyses.
Syringes were rinsed with distilled water between each perfusate change. After each collection interval, samples were immediately capped and stored on ice until the end of the experiment of the day, then stored at -20°C until glucose and urea analysis by the CMA/600 Microdialysis Analyzer (CMA Microdialysis). A check of samples analyzed both immediately and after storage at -20°C indicated no systematic changes in composition.
Blood Sampling and Plasma Metabolite Collection.
During the no net flux study, blood samples (0.1 ml) were collected from the arterial line at 60 min intervals for a minimum of four samples/experiment. The blood samples were collected in heparinized microhematocrit tubes and promptly centrifuged for 5 min to obtain plasma. Hematocrit was recorded, and the plasma was transferred to dialysate collection vials. The plasma samples were immediately capped and stored with the dialysate samples.
For the hyperglycemia study, blood samples were drawn into heparinized capillary tubes from the tail vein by nicking the tail. Blood glucose values were determined 15 min before the onset of the glucose infusion and at 15 min intervals after the induction of hyperglycemia for 75 min. Glucose was infused from 8 to 10 min, and the first blood sample was collected 10 min after induction. A drop of each blood sample was analyzed by an Accu-checkIII glucometer (Boehringer Mannheim, Indianapolis, IN) to obtain blood glucose values. The remaining blood was filled into heparinized microhematocrit tubes and handled as described above.
No Net Flux Calculations.
Measurement of absolute extracellular urea concentration in tumor was done according to the equilibrium method (8)
. For each dialysate sample, the urea concentration in the perfusate at that time was subtracted from the measured urea concentration in the dialysate to yield the net change in the perfusate urea concentration as it traversed the probe. For each rat, there was a linear relationship between the net change in urea concentration and the urea concentration in the perfusate. Using the regression line, the perfusate urea concentration not resulting in any increase or decrease in concentration (i.e., no net flux) was determined. The estimated urea concentration at which there is no gradient across the dialysis membrane represents the extracellular concentration of urea.
Hyperglycemia Study Protocol.
Microdialysis probes CMA/20 (PC, membrane length, 10 mm; molecular weight cutoff, Mr 20,000) were used to collect dialysate from tumor. Normosol (Abbott Laboratories, North Chicago, IL) was perfused at 2 µl/min. Before placement in tissue, the microdialysis probe was flushed at 2–8 µl/min to purge the tubing and probes of air bubbles and to ensure that fluid was flowing through the complete system. After probe insertion into the tumor, perfusate flow rate was maintained at 2 µl/min for the duration of the experiment, and 45 min was allowed for steady-state conditions to be reached (t = -60 to -15).
Glucose solutions were prepared as 20% weight per volume with glucose (
-D-(+) Glucose; Sigma Chemical Co.) in Normosol. Glucose was given through the femoral vein at a dose of 1 g/kg. The infusion time was 8–10 min, depending on body weight. Our group has shown that this moderate level of hyperglycemia has no effect on tumor blood flow (14)
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Dialysate samples were collected before, during, and after administration of glucose with a CMA/142 microfraction collector (CMA Microdialysis) over 15-min intervals. The microdialysate sample collected from t = -15 to 0 min established baseline analyte concentrations. Samples were collected for 75 min after the start of glucose infusion (t = 0 to 75 min). Blood samples were drawn from the tail vein. Samples were handled, stored, and analyzed in a manner similar to the no net flux study samples.
Urea and Glucose Measurements.
The CMA/600 Microdialysis Analyzer (CMA Microdialysis) was used for urea and glucose measurements of microdialysate and plasma samples. The Analyzer is a clinical chemistry analyzer that uses enzymatic reagents and colorimetric measurement (CMA Microdialysis).
Recovery.
RR is defined as the percentage of urea collected from the ISF and takes into account the concentration of urea in the perfusate (inflow). RR was calculated with the following formula:
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For the hyperglycemia study, RR for each probe was determined using urea as a recovery marker for glucose. To do this, the relationship between plasma and TUs determined by the no net flux study was used. TUCalc allowed RR to be determined. Because there was no urea in the perfusate, [urea] inflow is zero, and the RR formula simplifies to the following equation.
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Data Analysis.
The primary objective of the no net flux study was to determine whether there is a linear relationship between the levels of urea in tumor and plasma. Ordinary least squares regression was used to address this objective. Because it is possible that RR is influenced by urea concentration or by MBP, secondary analyses included exploring the differences in RR across urea concentrations and the correlation between the RR and MBP. For these analyses, a Kruskal-Wallis test and Spearman rank correlation were used. The PUs were stable over the course of the experiment. Nonparametric tests, the Kruskal-Wallis test and Spearman rank correlation, were used for these analyses.
For the hyperglycemia study, all of the data are expressed as medians and quartile ranges. Significant differences in glucose and urea before and after hyperglycemia, as well as between tumor and plasma, were tested with Wilcoxon matched pairs signed-ranks test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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illustrates the raw data from a single rat during a representative no net flux experiment with respect to time. At the start of perfusion, a steady state for microdialysate was reached after 30 min. The next two microdialysate samples were collected for inclusion in no net flux analysis. Similarly, after a change in perfusate urea concentration, steady state was reached after another 30 min. The mean of the third and fourth microdialysate samples collected after each perfusion fluid change was calculated to determine the urea concentration in the dialysate ([urea] outflow). On average, there was no evidence of a change in PU over the course of these experiments (Fig. 2
; P = 0.61).
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No Net Flux Determinations of Tumor Urea.
For each rat, the inflow urea concentrations were plotted against the difference between the inflow and outflow dialysis concentrations. The resulting relationship was linear for all of the rats. Fig. 3
depicts a typical no net flux graph. The concentration at which the difference between inflow and outflow was zero was defined as being equivalent to the urea concentration in the extracellular space.
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. The median recovery rate was 30% (inter-quartile range 27:37%) overall urea concentrations. The ranges and quartiles varied with urea concentration (Fig. 5)
. The lowest median recovery was at [urea] = 5.0 mM and the highest at [urea] = 0.83 mM.
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. When we examined the correlations conditional on urea concentration, we found positive correlations at urea concentrations of 0.83 and 2.5 mM (P < 0.05).
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Hyperglycemia Effects on Glucose Levels.
Median baseline and peak glucose values and 25% and 75% quartiles are presented in Table 2
. Hyperglycemia caused 10- and 3.6-fold increases in tumor and plasma glucose, respectively (Fig. 7)
. Plasma glucose reached its peak within 10 min and remained elevated for <20 min. In contrast, peak glucose in tumor dialysate was observed 20 min later at t = 30 min [1.5 mM (0.7:2.7)]. Lowest plasma glucose values were found at 45 min [6.4 mM (5.83:6.98)]. There was no effect of hyperglycemia on urea concentrations (data not shown.)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A fundamental challenge of microdialysis is to determine the RR and the resulting relationship between the analyte concentration in the microdialysate and in the extracellular fluid. Complete or near-complete equilibrium between microdialysate and interstitium eliminates the need for recalculation or earlier calibration for quantitative results. Such complete equilibrium (100% RR) requires a long dialysis membrane or a very low perfusion flow rate (2) . A low flow rate may minimize the removal of analytes and the interference with normal tissue concentrations and physiology (1) .
In one study, Rosdahl et al. (2) determined the interstitial concentrations of urea and glucose in human adipose tissue and skeletal muscle with microdialysis using a low perfusion flow that resulted in 100% RR. Using a low perfusion flow rate of 0.66 µl/min and a probe with a length of 3 cm, near 100% RR of urea was achieved in skeletal muscle. In adipose tissue, 100% RR of urea was slower than in muscle. Still, at lowest perfusion flow, the concentrations in both tissues were equal. Moreover, microdialysate urea equilibrated with the interstitium more rapidly than glucose and more rapidly in skeletal muscle than in adipose tissue.
We performed near equilibrium studies in two rodent tumors with the intent of determining the extracellular concentration of urea in tumors using a very low perfusion flow rate (data not shown). Our objective was to confirm the relationship between plasma urea and tumor urea as determined with the no net flux study. A low perfusion flow rate of 0.1 µl/min through our 1 cm-long probe was used in two experiments because Rosdahl et al. (2) used 0.16 µl/min in a longer probe. With this low flow rate, we obtained variable results and could not conclude that urea concentrations were at equilibrium. Although longer probes would facilitate equilibration between microdialysate and interstitium, we were limited by the size of the rat tumors to 1 cm-long probes. In future studies of canine, feline, or human patients with larger diameter tumors, 3-cm probes may be used, which could make a near equilibrium study possible.
Although using low flow rate can directly yield interstitial concentrations without any recalculation from an internal standard or a previous calibration, a low flow rate and long probe are not always practical. The demand of low flow rate must be balanced against the difficulty encountered in handling small amounts of sample and the need to obtain enough sample for the analytical technique (1) . Also, as flow rate is decreased, the duration of sampling for the same collection volume will often increase to unrealistically long times, especially if a goal is to measure temporal changes in analytes.
When equilibration between microdialysate and interstitium is incomplete, recovery must be determined in vivo to estimate accurately the analyte concentration in the interstitium. Because of differences in factors such as blood flow, microvascular density, and diffusion coefficient in water and in tissue, in vitro recovery is not considered an accurate predictor of in vivo recovery (1) . Furthermore, recoveries must be determined for each microdialysis probe, because probe-to-probe variability can be high. The no net flux technique is an in vivo calibration method that requires steady-state conditions and a constant flow rate.
The no net flux technique has been used previously as a calibration technique in patients. For example, glycerol production after ingestion of oral glucose was studied with microdialysis in lean and obese men after a no net flux calibration procedure (16) . The 5-h no net flux technique was used to calculate interstitial glycerol concentrations before the study. Estimating interstitial glycerol concentrations for a patient allowed RR for each probe to be determined. As glycerol values were followed during the experiment, microdialysate values could be corrected using the RR value, and quantitative results could be determined.
Whereas this approach provides an accurate estimation of extracellular concentrations and RR, the no net flux calibration period requires steady-state conditions and is time-consuming. In addition to human patients, we plan to perform microdialysis in murine models and veterinary patients. Because animals must typically be anesthetized for their studies, prolonged anesthesia time on the day of the study may not be practical. Our goal was to validate an approach to recovery determination that could be useful in future animal studies.
The choice of recovery marker is important. In normal tissues, both endogenous and exogenous markers can be used. For endogenous markers such as urea and glucose, plasma concentrations have been used to estimate directly interstitial concentrations in normal tissues (10) . The extremely low baseline levels of glucose in tumor observed in the hyperglycemia study are consistent with measurements of others indicating that these tumors consume large amounts of glucose, leading to deficiencies in supply (12 ; 17, 18, 19) . Clearly, glucose is inadequate as a tumor recovery marker, particularly when hyperglycemia is being induced.
In contrast, urea appears to be a suitable recovery marker. First, we found PUs to be stable over time (Fig. 2)
. Second, our study shows that tumor urea appears to be 26% greater than plasma urea. Rosdahl et al. (2)
also found that muscle and adipose urea concentrations appeared to be greater than plasma urea but only by 10%. It has been suggested that trauma-induced changes in local microvascular permeability and/or blood flow could result in a no-net-flux intercept concentration that differs from the distant tissue extracellular concentration.6
During microdialysis studies, blood sampling provides a means to measure PU, which can be used for TUcalc. As demonstrated with the hyperglycemia study, one can quantitatively determine the concentration of an analyte of interest in tumor interstitium.
Of the microdialysis experiments reported in the literature, many studies occur under nonequilibrium conditions and report incomplete recovery. At a perfusion flow rate of 2 µl/min, in vivo recovery with commercially available probes with relatively small membrane surface has been reported as 5–25% (10)
. Higher recovery was reported in human adipose and skeletal muscle (16
, 20
, 21)
. At the flow rate of 2 µl/min, we had a median RR of 30%, with the middle 50% of the values falling between 27% and 37% (see Table 1
) overall urea concentrations. There was one negative RR value (-24%). This result was unexpected. This value occurred at a urea concentration in the perfusate of 5.0 mM ([urea]inflow). In the no net flux analysis of this animal, we estimated the tumor extracellular urea concentration to be 5.7 mM ([urea]ISF). When the urea concentration in the perfusate is almost equal to the extracellular urea concentration in the tumor, a small error in the assay can yield a large change in RR.
We compared RR to MBP to determine whether blood pressure could be associated with the range of observed relative recoveries. In normal tissues, we would expect blood flow to vary somewhat with blood pressure, although autoregulation should minimize this effect. Outside the range of physiological blood pressure, one would expect that changes in blood flow may influence RR. In contrast, absent to poor autoregulation by tumors can result in changes in blood flow with observed changes in blood pressure, thereby altering recovery. We found a positive association of RR and MBP overall urea concentrations (Fig. 6)
, which would be consistent with a reduced perfusion at lower blood pressures. Although the association was statistically significant, it is physiologically a minor effect. It is also unclear why there were positive correlations at only two lower urea concentrations of 0.83 mM (P = 0.04) and 2.5 mM (P = 0.05; Fig. 6
).
Urea is a highly diffusible molecule, which contributes to the high relative recoveries. Urea has been found to equilibrate more quickly than glucose, which was attributed to its small molecular size (2) . A study of i.v. microdialysis found urea to be more diffusible than lactate, creatinine, or glucose (10) . In a related study, we found that the RR of glucose was 68% of urea (15) .
Retrodialysis is commonly used to assess in vivo RR in normal tissues (22) and in neoplastic tissues (23, 24, 25, 26) . The target analyte is perfused before or after a study, and its RR is determined. This method is based on the fact that mass transport efficiency over the membrane is the same in both directions; that is, the percentage delivery of the substance from the perfusate during retrodialysis will be the same as the percentage recovery from the tissue during conventional microdialysis (1 , 27) . Determination of delivery in vivo requires only the perfusate and dialysate concentration and can readily be determined. It has been shown that experimentally determined delivery (retrodialysis) could be used to provide the necessary recovery value for calibration of a probe for in vivo or in vitro studies (23 , 28) . In contrast, the delivery method cannot be used exclusively when treatment or study conditions, such as hyperglycemia or hyperthermia, may significantly change recovery. Ideally, urea could be used as a continuous recovery standard to follow temporal changes in RR in combination with an initial retrodialysis recovery determination of the analyte of interest. This potential was recently validated (29) .
In summary, defining a relationship between plasma and TUs will be extremely valuable in the determination of extracellular concentrations of tumor metabolites and unbound drug levels. However, it must be cautioned that some anesthetics, such as Telazol, can significantly alter tissue urea levels (30) and cause urea to be an inaccurate recovery marker. Microdialysis is a useful sampling technique that allows minimally invasive and continuous sampling of extracellular fluid in living tissues. If microdialysis is to be used for quantitative sampling, the degree of equilibration between microdialysate and interstitium must be determined. If complete or near-complete equilibrium cannot be achieved, the measured recovery can be used to calculate true extracellular concentrations of analytes. Because urea is a freely diffusible molecule, it is appropriate to use it as a recovery marker for quantifying small unbound solutes free in the extracellular space. In addition, the defined relationship between plasma and TUs allows continual determinations of recovery throughout a study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Cancer Institute Grants CA42745 and CA40355, and the Robert Jones Fund. 
2 These two authors contributed equally to this work.
3 This research was submitted by C. C. P. to Friedrich-Alexander Universitaet, Erlangen, Germany, in partial fulfillment of the requirement for doctoral thesis.
4 To whom requests for reprints should be addressed, at Kenan Plastic Surgery Research Labs, Box 3906, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-3929; Fax: (509) 277-6943; E-mail: klitz{at}duke.edu
5 The abbreviations used are: RR, relative recovery; ISF, interstitial fluid; PU, plasma urea concentration; TU, tumor urea concentration; TUcalc, calculated tumor urea concentration; MBP, mean blood pressure.
6 P. Bungay, personal communication.
Received 7/28/00. Accepted 8/27/01.
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represent the urea concentration in the microdialysate samples and are plotted at the end of each 15-min interval. Steady state between the perfusate and tumor interstitium was achieved after 30 min for each perfusate concentration. * indicate the steady-state samples that were used for the no net flux analyses. Blood samples were taken at 1-h intervals. The 
represent the urea concentration in the plasma sample and are plotted at the hourly times the samples were drawn.
