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Department of Physiology, University of Bergen, N-5009 Bergen, Norway
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Access to interstitial fluid is of fundamental importance to understand tumor transcapillary fluid balance, including the distribution of probes and therapeutic agents. Tumors were induced by gavage of 9,10-dimethyl-1,2-benzanthracene to rats, and fluid was isolated after anesthesia by exposing tissue to consecutive centrifugations from 27 to 6,800 g. The observed 51Cr-EDTA (extracellular tracer) tissue fluid-to-plasma ratio obtained from whole tumor or from superficial tumor tissue by centrifugation at 27-424 g was not significantly different from 1.0 (0.92-0.99), suggesting an extracellular origin only. However, fluid collected from excised central tumor parts had a significantly lower ratio (0.66-0.77) for all imposed G forces, suggesting dilution by fluid deriving from a space unavailable for 51Cr-EDTA. The colloid osmotic pressure in tumor fluid was generally higher than in fluid isolated from the subcutis, attributable to less selective capillaries and impaired lymphatic drainage in tumors. HPLC analysis of tumor fluid showed that low-molecular-weight macromolecules not present in arterial plasma were present in tumor fluid obtained by centrifugation and in venous blood draining the tumor, most likely representing proteins derived from tumor cells. We conclude that low-speed centrifugation may be a simple and reliable method to isolate interstitial fluid from tumors.
extracellular fluid; colloid osmotic pressure; collagen; glycosaminoglycans
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ANALYSIS OF INTERSTITIAL FLUID, i.e., the fluid bathing the cells in a tissue, may give significant information on fluid exchange. Proteins dissolved in interstitial fluid will exert a colloid osmotic pressure (COP) opposing the corresponding pressure in plasma and will most likely influence the interstitial fluid pressure. Interstitial fluid is, however, not readily accessible in the normally hydrated state, and therefore various techniques have been developed for tissue fluid sampling (for review see Ref. 2).
Access to interstitial fluid in solid tumors is of special interest because this may have potential therapeutic consequences. As shown by us and several others (26) (for review see Ref. 9), tumors have a high interstitial fluid pressure, which may influence drug delivery (13). One of the determinants of interstitial fluid pressure is the COP of interstitial fluid, again depending on the protein concentration of interstitial fluid. Therefore, access to interstitial fluid may be of fundamental importance to understand transcapillary passage of substances from vessels to interstitium. Furthermore, the emergence of immunoglobulins in solid tumor therapy makes it of interest to measure the concentration and distribution volume of macromolecules as well as other substances in the fluid bathing tumor cells.
Despite the interest, no interstitial fluid sampling method is generally accepted. This may partly be due to the nature of solid tumors. In other tissues, lymph has been accepted as a measure of interstitial fluid (2). Although intratumoral lymph vessels have been observed (11, 18), these have been shown to be nonfunctional (11), making lymph sampling inapplicable in this tissue. In a classical study, Gullino and co-workers (8) sampled interstitial fluid from porous chambers implanted in tumors. Another approach was used by Sylven and Bois (21) who collected fluid from a sectioned surface by using micropipettes, risking the contamination from cellular fluid. In tissues other than solid tumors, wicks have been used for interstitial fluid sampling (2), and in a recent study Stohrer and co-workers (19) sampled fluid from solid tumors by using nylon wicks. Wick implantation in richly vascularized tumor tissue may result in cell and vessel damage leading to contamination of the isolated fluid by intracellular fluid and blood, and therefore alternative methods are recommended. Communication with interstitial fluid may also be obtained by using the microdialysis technique (7), but this is a technique suitable for the study of tumor distribution of small molecules, and again the probe insertion may result in vascular damage.
In a series of studies, Aukland and co-workers (1, 3, 22) have isolated tissue fluid from the cornea and rat tail tendon by centrifugation. These tissues have a low cell content and are rich in collagen, which may restrict the removal of macromolecules when high G force is applied. Tumor tissue has at least an order of magnitude higher hydraulic conductivity than cornea (9, 20), suggesting that tissue fluid could be isolated from tumor tissue by centrifugation. We evaluated centrifugation as a method for tumor fluid isolation in a chemically induced mammary carcinoma and found that fluid isolated this way may be representative for tumor interstitial fluid. This new method may therefore be useful when studying interstitial transcapillary fluid balance and interstitial distribution of probes, including therapeutic agents.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental animals. The experiments were performed in anesthetized female Sprague-Dawley rats, 207-260 g at the time of the experiment, fed a standard laboratory diet. At 7 wk of age, the rats were given 16 µg 9,10-dimethyl-1,2-benzanthracene in olive oil by gavage in the animal facility at M&B, Ry, Denmark. After gavage the rats were housed in isolation for 1 wk and subsequently transferred to our animal facility. The rats were housed under standard animal housing conditions until mammary tumors developed along the mammary crest 6 to 8 wk later. All experiments were performed in accordance with recommendations given by the Norwegian State Commission for Laboratory Animals and were approved by the local ethical committee.
The rats were not fasted before or during the experiments. At the day of the experiment, the rat was anesthetized with pentobarbital (50 mg/kg ip). While the rat was anesthetized, body temperature was maintained at 36.5-37.5°C with a heating lamp. Unless otherwise specified, blood was sampled by cardiac puncture, and the rat was killed by intracardiac injection of saturated potassium chloride.Centrifugation technique. The anesthetized rat was immediately transferred to an incubator kept at room temperature (20-24°C) and 100% relative humidity. Tumors were excised, flushed with saline to remove blood from the surface, blotted gently with tissue paper to remove excess saline, and transferred to 2-ml centrifuge tubes used for isolation of tumor fluid. Tumors weighing 0.25-1.0 g were transferred intact to the tube, and results from these are later referred to as intact tumor. Larger tumors (>1 g) were sectioned, and the tumor sections (~0.5 g) were placed with the intact (i.e., unsectioned) surface facing downwards toward the nylon mesh, later referred to as tumor pole (see RESULTS). In some tumors, central parts (also ~0.5 g) were excised for centrifugation avoiding obviously necrotic areas.
The preweighed centrifuge tubes were provided with a basket of nylon mesh with pore size ~15 × 20 µm designed to keep the sample up from the bottom of the tube (1). The tube was immediately capped, reweighed, and spun in an Eppendorff 5417 R centrifuge placed in a cold room at 4°C, and then immediately brought back into the incubator. The basket containing the sample was transferred to another preweighed centrifuge tube, and the tumor fluid accumulated at the bottom was collected in glass capillaries that were either closed immediately for later analysis or diluted in buffer for HPLC. After being reweighed, the second tube was centrifuged, and the procedure was repeated by centrifugation of the basket in a third, fourth, and sometimes fifth tube. As described previously (1), the protocol provided data on the initial and subsequent weights of the tumor sample, each centrifugation volume, as well as any fluid loss by evaporation. Typical tumor fluid samples were 1-15 µl. To reduce the risk of cell compression, the centrifugation speed was lower than in the experiments with the tail tendon. In initial experiments, the samples were spun at 500 rpm (27 g) for 10 min, and the speed was increased successively in 100-rpm steps until a sample appeared at the bottom of the tube. This usually did not occur until a speed of 800 rpm (68 g) was used, so in later experiments centrifugation speeds of 800, 1,000 (106 g), 2,000 (424 g), 4,000 (1,700 g), and 8,000 rpm (6,800 g) were used. Fluid isolated from tumors by centrifugation was compared with fluid obtained from the subcutis isolated by centrifugation or isolated from dry wicks implanted postmortem. About 0.5 g of skin was excised and placed with the subcutis facing the net, allowing fluid to be collected at the bottom of the tube. Dry wicks were inserted postmortem in the back subcutis and hindlimb skin as described in a previous publication (24). After a 20-min implantation period, the wick ends along with any blood-stained portions were cut off, and the remaining sections were transferred to glass vials filled with 1 ml 0.02% azide saline for elution or were transferred to oil-filled centrifugation tubes provided with a funnel for isolation of native wick fluid (24).Analyses of tumor fluid.
Our aim was to isolate fluid from the tumor interstitium. To estimate
any contribution of cellular fluid, we therefore studied the
concentration ratio of an extracellular tracer in tumor fluid and
plasma. After anesthesia and placement of a polyethylene-50 catheter in
a jugular vein, both kidney pedicles were ligated via flank incisions,
and 60-70 µCi 51Cr-EDTA were injected intravenously
for distribution in the interstitial fluid and measurement of
extracellular fluid volume. After 115 min of tracer equilibration,
3-4 µCi of 125I-labeled human serum albumin were
injected intravenously and allowed 4 min of equilibration. Blood was
sampled by cardiac puncture for isolation of plasma, and the rat was
killed and transferred to the incubator. Tumors were removed and
transferred to centrifuge tubes and were handled as described above.
Tumor fluid isolated in these experiments was pipetted into 1 ml of
saline contained in counting vials. Tissue samples taken from the tumor
and back skin for calculation of plasma equivalent distribution volumes (in
counts · min
1 · g
tissue1/counts · min1 · ml
plasma1) were placed in tared, covered vials and weighed.
Colloid osmotic pressure. The COP in isolated fluid from tumors and in serum was measured in a colloid osmometer designed for submicroliter samples (23), using membranes with cut-off size of 10 kDa.
Characterization of fluid isolated from tumors and subcutis. The distribution of macromolecules in the tumor fluid isolated by centrifugation, elution fluid of intact or homogenized tumors (see RESULTS), wick fluid, and serum was determined by HPLC using Superdex 75 HR 10/30 or Superose 12 HR 10/30 size exclusion columns (Pharmacia-Biotech) with optimal separation range of 3-70 kDa and 10-300 kDa, respectively. About 1 µl of isolated tumor fluid in a total volume of 100 µl eluent (NaP buffer, 0.15 M, pH 7.4) was injected onto the HPLC system using a Gilson 234 autoinjector (200 µl loop). Constant flow of 1 ml/min was obtained by a Spectreseries P2000 pump (Thermo separation products), and the protein concentration in the elution fluid was measured by UV detection at 280 nm (Spectraseries UV100). The UV signal was digitalized, sampled at 2 Hz, and analyzed on a computer using ChromoQuest (version 2.51, ThermoQuest).
Samples of fluid from tumor, subcutis, and plasma and standard proteins ranging from 2.5-200 kDa in molecular mass (Mark12, Invitrogen) were also analyzed by SDS gel electrophoresis using a vertical mini-gel system (CBS Scientific) and 10% Tris-glycine precast gels (Novex). The gels were run at 125 V for about 90 min and stained with colloidal blue (Novex). HPLC of fluid isolated from tumors revealed a UV peak around 40 kDa, which was not present in serum. This fraction was subjected to electrophoresis to increase the resolution. During HPLC, a 2-ml sample of eluate containing this peak was collected from three consecutive tumor centrifugates (68, 106, and 424 g) of two tumors. The content was pooled and freeze-dried to increase the concentration and thereafter dialyzed overnight against distilled water. This substance and pI markers (Pharmacia Biotec Broad pI calibration kit, pH 3.5-9.3) were subjected to an isoelectric focusing gel (Novex pH 3-10) that ran at 100 V for 60 min, 200 V for 60 min, and 500 V for 30 min under native conditions. The gel was fixed in TCA (12 g), and sulfosalicylic acid (3.5 g) was added to 100 ml deionized water and stained with Colloidal blue (Novex).Sampling of tumor venous blood. To examine whether the UV peak around 40 kDa (see Characterization of fluid isolated from tumors and subcutis) was present in venous blood draining the tumor, blood was sampled with micropipettes from surface veins of five tumors in three rats. After anesthesia, the tumor surface was exposed by a skin incision. Under guidance of a Zeiss stereomicroscope (magnification ×40), a micropipette with tip diameter ~50 µm was inserted into a venule draining the tumor. After aspiration of 5-20 µl of blood, the pipette was withdrawn, and the blood was dissolved in 0.5 ml of HPLC buffer. This sample was centrifuged, and the red blood cell-free supernatant was processed for elution in the HPLC column.
Composition of the tumor extracellular matrix. As shown in previous publications, the composition of the extracellular matrix may influence the diffusion properties of the tumor interstitium (14) and the composition of the fluid isolated by centrifugation (1, 3). Therefore, the tumor content of collagen, hyaluronan, and total glycosaminoglycans (assayed as uronic acid) was measured. Analysis of hyaluronan was performed using a radioassay (HA-test 50, Pharmacia Diagnostics; Uppsala, Sweden) after papain digestion of freeze-dried specimens (16).
Uronic acid was measured by the method of Bitter and Muir (5) as described in a previous paper (15), whereas collagen was determined according to the method of Woessner (27), based on the determination of hydroxyproline content as described in a previous publication (15) assuming a hydroxyproline content of 0.91 µmol/1 mg collagen (4).Statistics. Data are given as means ± SE. Data from consecutive centrifugations were compared using one-way ANOVA and with Tukey tests for multiple comparisons. Differences were accepted as statistically significant at the P < 0.05% level.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The centrifugal force needed to isolate any fluid from the tumors varied. In some, 500 rpm (27 g) was sufficient, whereas 800 rpm (68 g) was sufficient for fluid isolation in all tumors. The fluid isolated was straw colored, and sometimes some erythrocytes collected at the bottom of the centrifugation tube, most often in the initial centrifugation (27-68 g) of the consecutive centrifugations. Fluid isolated from the central tumor was more grayish than that of the other tissues and became more viscous at increasing G force.
The weight fractions of fluid isolated from the whole tumor
(n = 19), tumor pole (n = 8), central
tumor (n = 5), and skin (n = 9) are
shown in Fig. 1. In the initial of the
consecutive centrifugations (27-68 g), the isolated
fractions ranged from 0.013-0.017, with a corresponding range of
0.030-0.043 at 106 g. At 424 g, the
accumulated fraction isolated from the central tumor of 0.10 differed
significantly from those derived from the other types of tissue (range
0.050-0.069). At even higher centrifugation speeds, the isolated
fractions from tumor tissue differed significantly from that of the
skin, e.g., with a fraction of 0.191 for central tumor and 0.089 for
skin at 6,800 g.
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Analyses of tumor fluid.
In these experiments we measured recovered tracer that had equilibrated
in extracellular fluid (51Cr-EDTA) or distributed in plasma
(125I-labeled human serum albumin), and the results are
shown in Fig. 2. For the intact, whole
tumor, the concentration of 51Cr-EDTA in tumor fluid
relative to plasma averaged 0.922 (± 0.035), 0.952 (± 0.028), and
0.987 (± 0.028) in consecutive centrifugation at 27-68, 106, and
424 g, respectively (Fig. 2A). None of these ratios differed significantly from 1.0. The corresponding numbers for
the tumor pole were 0.830 (± 0.019), 0.966 (± 0.034), and 0.954 (± 0.063). Of these ratios, only that obtained at the lowest G force
differed significantly from 1.0 (P < 0.001). The
51Cr-EDTA central tumor-to-plasma ratio ranged
0.67-0.77 for the three lowest centrifugation speeds, all
significantly <1.0 (P < 0.001 for all comparisons).
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424; none of the ratios were significantly different from
1.0 (Fig. 2A). Increasing the G force in the consecutive centrifugations to 1,700 and 6,800 g resulted in a
corresponding ratio of 0.85 and 0.72, respectively, both significantly
different from 1.0 (P < 0.001).
The results from the 125I-labeled human serum albumin
recovery in centrifuged fluid are shown in Fig. 2B. For the
intact tumor, the tracer concentration relative to plasma was
0.05-0.06 for the first four consecutive centrifugations (1,700
g), falling to 0.032 at the highest G force (6,800 g). The corresponding average ratios for the pole and
central tumor differed slightly but not significantly from that of the
whole tumor for all centrifugation speeds. In fluid isolated from the
skin, the 125I-labeled human serum albumin ratio ranged
from 0.0130-0.0142, all significantly different from the ratio of
fluid isolated from tumor at all centrifugation speeds.
COP.
The COPs in fluid isolated in subsequent centrifugations from the tumor
and skin are shown in Fig. 3. In the
intact tumor, the average pressure ranged from 14.4-15.9 mmHg for
27-68 to 1,700 g and was 17.7 mmHg at the highest G
force induced by 6,800 g (Fig. 3A). The
corresponding pressure in plasma was 20.5 mmHg (± 0.8, n = 14). All pressures except for that obtained at
6,800 g was significantly different from the COP in plasma.
No data were obtained for the tumor pole at the lowest and highest
centrifugation speed, but for 106-1,700 g, the COP in
the fluid was similar to that of the whole tumor.
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Analyses of isolated fluid.
The elution pattern of fluid isolated from an intact tumor by
centrifugation at 106 g is shown in Fig.
4B, also showing the elution
pattern of plasma for comparison (Fig. 4A). We observe that
for albumin and molecules larger than albumin, the elution pattern is
similar for the globulins and albumin. For molecules smaller than
albumin, the elution pattern of tumor fluid and plasma differed
significantly. In tumors, a macromolecule with molecular weight less
than albumin consistently eluted at 13.4 ml, and such a macromolecule
was never seen in the arterial plasma samples. The area of this peak
ranged from 8.9% to 32.1% (average 16.3%) of the area under the
elution curve when measured in 13 tumors centrifuged at 27-6,800
g and did not change with centrifugation speed. Furthermore,
the relative areas for intact tumor and tumor pole were similar. In
addition, there were some even smaller macromolecules in tumor fluid
that were or were not found in plasma, constituting a variable but
small fraction (<8.3%) of the total area under the elution curve.
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424, the average ratio between albumin in
centrifugate and plasma ranged from 0.70-0.76, with a
corresponding ratio for globulins of 0.70-1.06.
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Fluid volumes and composition. Tumors (n = 7) had a water content of 0.83 ml/g (± 0.002). Hyaluronan and uronic acid in tumors averaged 1.92 (± 0.26) and 1.47 (±0.43) mg/g wet wt, respectively, whereas the mean collagen content was 4.6 mg/g wet wt (± 0.7).
The extracellular and intravascular fluid distribution volumes for intact tumors (n = 11) were 0.40 ± 0.02 and 0.014 ± 0.001 ml/g, respectively, with corresponding volumes in the back skin (n = 7) of 0.46 ± 0.03 and 0.005 ± 0.001 ml/g. The tracer injected could be totally eluted from the tissue. Thus after elution, <1% of the initial counts of 51Cr-EDTA (range 0.40-0.96%) remained in the tumor (n = 5) and skin (n = 4). The corresponding range for 125I-labeled human serum albumin was 0.1-2.3%.| |
DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We have shown that it is possible to isolate tumor fluid even at
low-speed centrifugation. We have evaluated the isolated fluid, and the
major conclusion of our work is that when centrifuging intact tumor or
tumor pole at 424 g (here 2,000 rpm), it is possible to
isolate fluid that is representative for tumor interstitial fluid. At
higher G force, dilution of the centrifugate occurs most likely by
intracellular fluid. Centrifuging excised central tumor tissue results
in a centrifugate where the extracellular tracer is diluted and
contaminated by intracellular proteins and/or tumor degradation
products. In the following, we discuss the basis for our conclusion,
limitations of the method, and implications for further work. In
agreement with previous studies, this fluid had a high protein
concentration and COP (8, 19). Furthermore, our
experiments suggest that typical tumor macromolecules, most likely
proteins, are liberated to the interstitial fluid and general circulation, in agreement with previous work (21).
Evaluation of the centrifugation method.
In previous studies, we used centrifugation to isolate fluid from the
cornea (22) and tail tendon (3), both being
tissues with a low cell content. Tumors are rich in cells, and cell
compression during centrifugation may lead to extrusion of cellular
fluid, resulting in the isolation of a mixture of interstitial and cell fluid. We decided to use 51Cr-EDTA as a probe to show
possible "contamination" of cellular fluid. 51Cr-EDTA
(molecular weight 341) is a substance that is not metabolized and is
not taken up by cells (12). Addition of tumor cell fluid to the centrifuged volume should thus show up as a reduced
51Cr-EDTA concentration in the centrifugate relative to
plasma. For tissue centrifugation at a G force 424, we found that the ratio of 51Cr-EDTA in centrifugate and plasma was not
significantly different from 1.0, suggesting no dilution of
extracellular fluid. Furthermore, the unaltered COP with increasing G
force up to 424 suggests no sieving of macromolecules in this G force
range. The observed gradual reduction of this ratio when exposing the
tissue to higher G forces indicates that a gradual dilution is
occurring, which may be a result of extrusion of cellular fluid and/or
cell damage. The somewhat lower 51Cr-EDTA
centrifugate-to-plasma ratio of in the initial sample isolated at
27-68 g may be a result of flushing of the tumor with saline to remove surface blood.
424 g (2,000 rpm), because the protein concentration was
unrelated to the G force in this range, a finding that most likely is a
result of the composition of the extracellular matrix of the tumor.
Centrifugation at 1,700 and 6,800 g resulted in dilution of
the centrifugate by fluid from a space not accessible to
51Cr-EDTA, most likely the intracellular phase.
Comparison to other available methods. Few other methods have been described for isolation of interstitial fluid. Sylven and Bois (21) created pouches in central and peripheral tumor tissue by blunt dissection and sampled fluid that collected in these by micropipettes. As argued by the authors, the fluid from the periphery was not from tumor only but also from surrounding normal tissues. The fluid from the tumor center was not mixed but included necrotic tissue and may therefore not be representative for interstitial fluid. Another approach was used by Gullino and co-workers (8). They created a chamber within a tumor separated from the tumor tissue by a porous membrane. Fluid draining from the tumor into the chamber could be sampled with a catheter. The advantage of that procedure will be the possibility to sample for longer periods and that fluid is easily obtainable, but capsule implantation may result in scarring around the sampling device, i.e., a connective tissue not representative for tumor tissue. In a recent study published from Jain's group (19) wicks were used to obtain interstitial fluid. Wicks were either inserted simultaneously with the tumors (chronic wicks) or acutely at the termination of the experiment. As pointed out by the authors, one problem with the use of this method in the highly vascular tumor tissue is blood contamination of the isolated wick fluid. Even though the authors claimed that this problem was avoided by cutting off blood-stained wick portions, our experience is that blood contamination is difficult to avoid in tumors. Another difficulty that may be faced when using the wick technique in cell-rich tumor tissue is contamination of wick fluid with intracellular proteins, i.e., mainly proteins with lower molecular weight than albumin, as observed in skeletal muscle (25). These reservations have to be considered when discussing data on tumor interstitial fluid isolated using wicks.
Functional importance of the data. The fluid isolated from tumors by centrifugation had a COP of ~75% of that in plasma, which was higher than the corresponding ratio of 50% found in skin, showing a high protein concentration in the tumor interstitium. Using the implanted chamber technique discussed above in rats, Gullino et al. (8) found an average tumor interstitial fluid protein concentration, 67% of that in plasma, whereas the corresponding ratio for subcutis was ~50%. In mammary carcinomas in mice, Sylven and Bois (21) observed a tumor fluid protein concentration not different from that in plasma, in agreement with the work of Stohrer et al. (19), who found a COP in wick fluid of three of the four tumor types not significantly different from that in plasma. Although there are differences in the absolute numbers, that partly may be related to tumor types, species, and the methods used for sampling, previous as well as the present data suggest that the protein concentration and COP in interstitial fluid of tumors are higher than in normal tissue. This is most likely a consequence of a low size selectivity of tumor capillaries (10, 28) and the lack of functional lymphatics in tumor tissue (11). The functional importance of the low COP gradient across the tumor capillary will be less than in normal tissue for determining net filtration pressure and thereby transcapillary fluid exchange, which will then mainly be governed by the difference in hydrostatic pressure in capillary and interstitial fluid.
In conclusion, we developed a centrifugation method to isolate interstitial fluid from tumors. Centrifugation of tumor tissue at424
g resulted in the isolation of fluid with a similar
concentration of the extracellular tracer 51Cr-EDTA as in
plasma. Our data suggest that typical tumor macromolecules not found in
plasma are dissolved in the interstitial fluid and that the tumor
interstitial fluid COP is higher than in skin. Even though we have
evaluated the procedure in one tumor type only, our data also suggest
that low-speed tumor centrifugation may be a simple and reliable method
when studying interstitial transcapillary fluid balance and
interstitial distribution of probes, including therapeutic agents.
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ACKNOWLEDGEMENTS |
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Financial support from The Research Council of Norway, and expert technical assistance from Wibeke Skytterholm, Birgitte Hageseter, Odd Kolmannskog, and Sigrid Lepsøe are gratefully acknowledged.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Wiig, Dept. of Physiology, Univ. of Bergen, Årstadveien 19, N-5009 Bergen, Norway (E-mail: helge.wiig{at}fys.uib.no).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published September 12, 2002;10.1152/ajpheart.00327.2002
Received 10 April 2002; accepted in final form 10 September 2002.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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