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Department of Physiology, University of Bergen, Bergen N-5009, Norway
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Volume exclusion, i.e., the space not available
for a specific probe, may be dependant on the probe charge. Therefore,
interstitial exclusion was measured for positively and negatively
charged immunoglobulin (IgG) in skin and muscle of rats by using a
continuous infusion method (30). Steady-state
concentration of 125I-labeled IgG 1 (pI = 8.7) and
131I- labeled IgG 4 (pI = 6.6) was maintained by
infusion of tracer for 120-168 h with an implanted osmotic pump.
At the end of the infusion period and before tissue sampling, the rat
was anesthetized and nephrectomized, and 51Cr-labeled EDTA
was injected and allowed 4 h for equilibration to measure
interstitial fluid volume (Vi). Interstitial fluid was
isolated from skin and muscle by using nylon wicks implanted post
mortem. The relative IgG available space was measured as the ratio
between labeled IgG and 51Cr-labeled EDTA wick fluid
equivalent spaces, and relative excluded volume fraction
(Ve/Vi) was calculated as 1 
Va/Vi. Ve/Vi
in hindlimb skin averaged 0.37 ± 0.05 (SE) and 0.65 ± 0.06 (P < 0.01) for IgG 1 and 4, respectively, with
corresponding figures of 0.24 ± 0.05 and 0.51 ± 0.04 (P < 0.01) in hindlimb muscle (n = 9 for both tissues). These experiments suggest that fixed negative
charges, most likely glycosaminoglycans, influence distribution of
macromolecules in the interstitium and therefore affect interstitial
fluid balance.
extracellular fluid volume; extracellular matrix; immunoglobulin G space; bound immunoglobulin G; immunoglobulin G subclasses
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE INTERSTITIUM MAY BE DEFINED as the space located between the capillary walls and the cells. As reviewed by Aukland and Reed (3), the basic structure is similar in all tissues: collagen builds the fiber framework that contains a gel phase made up of glycosaminoglycans (GAG), a salt solution, and proteins derived from plasma. The amount of interstitium varies from ~50% of wet weight in skin to 10% in skeletal muscle. In the interstitium, the presence of substances like collagen and GAG limits the space accessible for plasma proteins and other macromolecules simply due to the fact that two materials cannot occupy the same space at the same time. The resulting phenomenon is called volume exclusion (9). As a consequence of exclusion, the concentration of the plasma proteins in the accessible space is higher than that calculated from tissue protein mass and total interstitial fluid volume. As stated by Aukland and Reed (3), the physiological importance of the exclusion phenomenon is twofold: 1) a more rapid approach to a new steady state after a change in transcapillary fluid flow, and 2) less transfer of interstitial protein to plasma for a given capillary hyperfiltration. Interstitial exclusion thus influences plasma volume regulation but does not determine steady-state protein concentration or colloid osmotic pressure of free interstitial fluid or lymph. These are set solely by relative net influx of protein and fluid.
Apart from being of importance for interstitial fluid balance, the study of exclusion phenomena may tell us about the structural organization of the interstitium. The distribution of a specific probe in the interstitial fluid is determined in part by both its size and charge (3, 9), but it will also be influenced by the composition of the tissue, i.e., by the amounts of structural components like collagen and hyaluronan, as shown in a recent study in hypothyroid rats (33).
Because of the wide variations in previous albumin exclusion data, we decided to develop an alternative method on the basis of establishing steady-state levels of 125I-labeled homologous albumin in plasma and interstitial fluid, with subsequent sampling of free interstitial fluid from skin, skeletal muscle, and tendon by subcutaneous, intramuscular, and intratendon wicks (30). By using this method, we compared the value for albumin to that of the larger molecule immunoglobulin (IgG) (32). It was somewhat unexpected to find that that the excluded volume was not different from that of albumin for any of the tissues studied. These data may be explained if steric factors, which might be expected to increase IgG exclusion relative to albumin, were balanced by electrostatic difference, which would increase albumin exclusion relative to IgG.
Studies of the lung (14, 25) have suggested that the charge of the probe determines the distribution volume of macromolecules. Skin and muscle are important for fluid balance, and to see if charge affects the probe distribution volume, we measured distribution volume of positively and negatively charged IgG in these tissues. We found that the negative tracer was excluded from a volume almost twice that of the positive tracer in skin and even more in muscle, suggesting that the interstitium acts as a negatively charged matrix and that interstitial collagen is less important than previously anticipated in determining excluded volume. Part of this work has been presented briefly elsewhere (35).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The experiments were performed on anesthetized female Wistar-Møller rats, 212-249 g, fed a standard laboratory diet. While the rats were anesthetized, body temperature was maintained at 37-38°C with a heat lamp. The rats were not fasted before or during the experiments. All of the experiments were performed in accordance with recommendations given by the Norwegian State Commission for Laboratory Animals and were approved by the local ethical committee.
Measurement of distribution volumes. The present method for measurement of interstitial exclusion is on the basis of reaching a steady-state tracer concentration in the interstitium by a slow, continuous infusion of tracer solution of 1 µl/h for 5-7 days. This method has been described in detail in a previous paper (30), and therefore only a brief description is given here.
We used two isotypes of human IgG known to have different isoelectric points but similar molecular masses of 146 kDa (16) as macromolecular probes. Human IgG 1 and 4 (lambda purified) was obtained from The Binding Site (Birmingham, UK). The pI of these substances, as determined by isoelectric focusing on a vertical minigel system (CBS Scientific), averaged 8.7 and 6.6, respectively. Precasted isoelectric focusing gels (Novex, pH 3-10) were run for 1 h at 100 V, 1 h at 200 V, and 30 min at 500 V, fixed for 60 min (12 g trichloroacetic acid and 3.5 g sulfosalicylic acid in 100 ml distilled water), and stained with Novex colloidal blue stain. The pH gradient profile was determined by using 11 marker proteins with pI ranging from 3.5 to 9.3 (Pharmacia Biotech Broad pI Calibration Kit). In preliminary experiments it was established that the label on the probe did not affect the results, in accordance with the observations of others (25). In the results reported below, IgG 1 was labeled with 125I (125I-IgG 1) and IgG 4 with 131I (131I-IgG 4) by using the Iodogen method. Free radiolabel was removed by dialyzing the probes dissolved in a volume of ~1 ml against 2,000 ml PBS overnight. The 125I-IgG 1 and 131I-IgG 4 stock solutions were mixed, the total radioactivity was adjusted to ~4 MBq/ml, and a mixture of the tracer solutions was filled into an osmotic pump (total volume ~200 µl, pumping rate 1 µl/h, model 2001, Alzet). The filled pump was incubated overnight in a beaker containing sodium azide (0.02%) kept at 37°C. The next day, the rat was anesthetized with a 1:1 mixture of fentanyl/fluanisone (Hypnorm) and midazolam (Dormicum), 2.5 ml/kg, and a polyethylene (PE)-60 catheter filled with isotope mixture solution was inserted into the right jugular vein by using a sterile technique. A 0.035-ml bolus of tracer solution was given, and the catheter was connected to the preincubated osmotic pump. The pump was tunneled to the interscapular region, the wound was closed with clips, and the rat was transferred to its cage to wake up. Blood samples (40-80 µl) were collected in hematocrit tubes every 24 (n = 5) or 48 h (n = 4) and were obtained by scalpel incision of the distal part of one lateral tail vein with the rat in a perspex cylinder. On the final day of the experiment, the rat was anesthetized intraperitoneally with pentobarbital sodium (50 mg/kg). After a tracheotomy was performed, a PE-50 catheterwas inserted in the left carotid artery for measurement of blood pressure. Both kidney pedicles were then ligated via flank incisions, and a PE-50 catheter was inserted into the right jugular vein. A 150-µl blood sample was obtained by orbital puncture, and ~0.15 MBq 51Cr-labeled EDTA (51Cr-EDTA) was injected intravenously for measurement of interstitial fluid volume (Vi). Four hours after the injection of 51Cr-EDTA, a final blood sample of 0.5-0.7 ml was obtained by cardiac puncture, and the rat was euthanized with saturated KCl intravenously. The skin was closely clipped on both hindlimbs and the back, and the rat transferred to a chamber kept at 100% relative humidity at all times for implantation of wicks used to isolate interstitial fluid. Dry wicks were inserted post mortem in back subcutis and hindlimb skin and muscle as described in previous publications (31, 34). After a 20-min implantation period, the wick ends, along with any bloodstained portions, were cut off and the remaining sections transferred to glass vials filled with 1 ml 0.02% sodium azide for elution. After the wick implantation period, the rat was taken out of the humidity chamber, hair was carefully removed from the hind legs and lower back with fine clippers, and the skin was washed and dried. Paired (left and right side) 0.3 to 1.2 g samples taken from hindlimb and back skin, and from both legs of lateral and medial gastrocnemius, tibialis anterior, and semimembranosus muscles. Tissue samples were placed in tared covered vials and weighed. Aliquots of plasma from the blood samples were made up to 1 ml in the same type of vials used for wick samples. Samples were counted in a gamma-counter (LKB, model 1282, Compugamma) using window settings of 290-350 keV for 51Cr, 350-470 keV for 131I, and 15-75 keV for 125I. Standards were counted in every experiment, and spillover as well as background and decay during the period of measurement were automatically corrected for. After counting, tissue samples were dried at 60°C to constant weight (change <1 mg in 24 h). Tissue water content (Vw) was taken to be the difference between wet and dry weights. In a separate series of experiments (n = 6 rats), plasma volume was measured as the distribution volume of 125I-labeled human serum albumin (Institute of Energy Technology; Kjeller, Norway) to be entered as a correction factor in the equation used for calculation of exclusion (see Eq. 4). After anesthesia, ~0.2 MBq of this tracer solution was injected intravenously and allowed 5 min of equilibration. At the end of this period, a ~0.5-ml blood sample was taken by cardiac puncture, and the rat was euthanized by injection of KCl intracardially. Tissue samples were taken for determination of radioactivity as described above.Elution of isotope from tissue. In our previous study (32) on interstitial exclusion of IgG, we found that 17-29% of the tracer remained in the various tissues studied after 48 h of elution. To be able to correct for tracer bound to the tissue (i.e., not free in the interstitial fluid), we measured "free"- and "bound"-labeled IgG 1 and IgG 4 in all individual samples in all rats by elution. The tissues were minced with a scalpel and recounted to determine radioactivity, and 10 ml of 0.02% azide in 0.15 M saline were added. The samples were shaken vigorously and left in an agitator for 24 h at room temperature. After centrifugation and removal of as much supernatant as possible, a new aliquot of sodium azide was added and the procedure was repeated. The counts remaining in each individual tissue sample after elution for 48 h divided by the initial total counts of the same sample, with correction for isotope decay occurring during the extraction procedure, was used to correct for tissue binding of tracer.
Characterization of iodine-labeled isotopes. During labeled IgG infusion over several days, some of the tracer is metabolized and 125I and 131I is either excreted as free iodide or incorporated in tyrosine (20). To see whether 125I or 131I was incorporated in other plasma proteins and whether the isotope was changed during the infusion period, plasma obtained from rats subjected to 125I-IgG 1 and 131I-IgG 4 infusion for 168 h was compared with freshly prepared stock solution by high-performance liquid chromatography with the use of a Superdex 200 HR 10/30 column (Pharmacia-Biotech) with optimal separation range of 10-600 kDa. Elution was with 0.005 M phosphate buffer, pH 7.6 in 0.15 M NaCl. Successive fractions of 1.0 ml were collected and counted in the gamma-counter.
Calculations.
The tissue intravascular plasma volume (Vv) and
extracellular fluid volumes (Vx) were calculated as the 5 min and 4 h, respectively, plasma equivalent distribution
volumes of 125I-labeled human serum albumin (HSA) and
51Cr -EDTA, each equal to the counts per gram of
tissue divided by counts per milliliter of terminal plasma. Thus
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(1) |
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(2) |
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(3) |
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(4) |
Vg,w. Expressed as a fraction of
Vi (fractional excluded volume)
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(5) |
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(6) |
Statistics. Values for paired tissue and fluid samples (e.g., left and right leg skin) were combined and averaged. Data are given as means ± SE and were compared with two-tailed Student's t-tests, using paired comparisons when appropriate. Differences were accepted as statistically significant at the P < 0.05 level.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Establishment of steady-state levels of labeled IgG.
All rats recovered from anesthesia within 1-2 h after osmotic pump
implantation. The average relative concentrations of tracers in plasma
during the infusion period are shown in Fig.
1. In the plot all individual values have
been normalized to the corresponding final value (four at 120 h
and five at 168 h). The first value at 24 h tended to be
higher than 1.0, due to a slightly higher bolus dose than previously
used (0.035 vs. 0.025 ml), which was chosen because of low initial
concentrations in previous experiments using IgG (32).
However, although there was some scatter in the plasma values, none of
the concentrations during the experiments differed significantly from
the final value, i.e., from 1.0. Furthermore, the plasma tracer
concentration after induction of anesthesia and before injection of
51Cr-EDTA did not differ significantly from that in the
final plasma sampled before ending the experiment.
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Tissue elution experiments. Early in the experiments, it became clear that a substantial fraction of tracer was not extractable from the tissue, in accordance with previous observations on native, homologous IgG (32). The unelutable IgG is unspecifically bound to the tissue and not free in the interstitial fluid. If this fraction is not taken into account when calculating the IgG distribution volumes, these volumes will be overestimated and consequently the interstitial exclusion underestimated. We therefore measured this fraction in all individual samples and corrected it for the bound tracer.
Table 2 presents the data for the tissue elution experiments. The 51Cr-EDTA was 98-99% extracted in skin and slightly lower in muscle, 96-97%. However, the extractable fractions of labeled IgG were significantly lower. Thus, for 125I-IgG 1, the extraction ranged from 61 to 65% in the tissues studied, whereas the corresponding range for 131I-IgG 4 was 72-76%.
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Interstitial distribution volumes of IgG 1 and IgG 4.
The relative wick fluid equivalent distribution volumes for the IgG
tracers were calculated from Eq. 6, and by multiplying these
by the interstitial fluid volume, Vi, the absolute
distribution volumes for these tracers were found. As evident from
Table 3, these volumes were all larger
than the corresponding plasma equivalent spaces (see Table 1), as
expected if sieving of protein occurs at the capillary wall.
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Characteristics of tracer IgG in plasma. Samples of stock 125I-IgG 1 and 131I-IgG 4 of the infusion mixture remaining in the osmotic pump after ended experiment and of plasma after 120 and 168 h of tracer infusion were applied to a Superdex 200 column (Pharmacia). The tracers did not change during their stay for up to 168 h in the infusion pump, as shown by nearly identical elution patterns of tracer stock solution and solution remaining in the pump.
Figure 4 shows the effluent 125I and 131I radioactivity from the column after applying plasma sampled after 168 h of infusion. The tracers eluted in the same volume as the stock solutions. Furthermore, the circulation in the animal did not lead to dimers or isotope degradation products. Practically no radioactive degradation products could be detected in final plasma, as shown in Fig. 4.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Although the presence of fixed negative charges in the interstitium may indicate an effect on the distribution volume of macromolecular probes, such effect has to our knowledge not yet been fully documented in skin and muscle. In the present study, we found that the negatively charged IgG 4 was excluded from a volume almost twice that of the positively charged IgG 1 and even more in muscle. This finding shows the significant influence of these fixed charges in distribution of plasma proteins in the interstitium and suggest less influence of collagen in determining excluded volume for charged macromolecules than previously anticipated (8, 9).
Methodological considerations. The procedure we used for evaluating interstitial distribution and exclusion of subclasses of IgG is based on the following: 1) establishing steady-state concentrations of a tracer-labeled protein in plasma and tissues by intravenous infusion for several days with an implanted osmotic pump, 2) subsequently establishing steady-state concentrations of an extracellular volume tracer, 3) sampling interstitial fluid from specific tissues by implantation of nylon wicks post mortem, and 4) sampling the tissues themselves and assaying them as well as plasma and wick fluid for the radioactive tracers. By calculating ratios of IgG distribution volume to interstitial fluid volume, thereby canceling out the volume of wick fluid from Eq. 6, the evaluation of tracer protein distribution and exclusion is based solely on relative counts from gamma-emitting tracers of the entire tissue samples and wicks. Potential errors involved in tissue extraction, chemical or immunoassays, evaporative losses, and measurement of very small weights or volumes are eliminated. This method and potential sources of error has been evaluated for infusion of tracer albumin and IgG in control rats, and the same considerations apply here (30, 32).
The most fundamental requirement of this method is that steady-state distribution of tracer between plasma and tissue is established. The method does not therefore measure uptake rate and exchange of substances across the capillary wall, which is also affected by charge of the macromolecular probe (5, 17, 25). In control rats, stable levels were reached in skin and muscle at 72 h for albumin (30), whereas a somewhat longer time was needed for rat IgG (32). Because of problems in the previous study of reaching a stable IgG level in plasma, we increased the initial bolus dose of tracer and thereby managed to have a stable plasma concentration during the experiment. All of the probes used in this type of study have to be evaluated with respect to attainment of steady state at the end of the experiment, and the similar 125I-IgG 1 and 131I-IgG 4 distribution volumes after 120 and 168 h of isotope infusion suggest that steady state was reached for both probes in our experiments. Another fundamental requirement is that we obtain a sample representative for interstitial fluid. The wick method has been used extensively in skin, and post mortem implantation has been shown to avoid inflammatory effects and to yield fluid closely resembling other estimates of free interstitial fluid composition (2, 18, 31). In previous studies (31, 34) we have shown that an implantation time of 20 min is sufficient to obtain a reliable tissue fluid sample. Wicks implanted directly into muscle become contaminated with intracellular proteins, but this problem as well as blood contamination is avoided by intramuscular wick implantation with a catheter (34). Fluid isolated from wicks in the present study should thus be representative for interstitial fluid. Our method assumes that the extravascular tracer 51Cr-EDTA distributes in the whole extracellular fluid phase and is not itself excluded from this fluid phase or bound to the tissue or components in plasma. We were able to elute 96-99% of this tracer from the tissue, suggesting that binding of 51Cr-EDTA is a minor problem. In a study on fluid distribution spaces in the tail tendon, Aukland (1) found that 51Cr-EDTA was excluded from 8% of total tendon water. In that study, intracellular water could account for a major part of the water inaccessible to 51Cr-EDTA, suggesting that the tracer distributes in the whole extracellular phase. We chose to use two macromolecular probes in the same osmotic pump, whereas only one probe has been used in previous experiments. Because we were technically unable to separate more than three tracers in the same experiment, we had to measure Vp in separate experiments to correct for intravascular tracer (see Eqs. 4 and 6). The error introduced by using average plasma instead of the actual Vv is negligible because Vv is small compared with IgG and interstitial fluid volume. An alternative approach would be to measure Vg,w for IgG 1 or IgG 4 and Vv and thereby Ve/Vi in separate experiments. Actually, the interindividual variation in Vg,w and Vi is considerably larger than the variation in Vv, and therefore the present approach is an improvement because interindividual variation in Vg,w and Vi is avoided. Use of a revised version of Eq. 6 to calculate available, and thereby excluded, volumes led to no changes in these volumes for skin and <4% change in muscle compared with the equation described originally (30). In preliminary experiments we used labeled lactate dehydrogenase (LDH) 1 and 5 as macromolecular probes. These have identical molecular weight, are negatively and positively, respectively, charged at physiological pH, and have been used in distribution volume and exclusion studies in the lung (14, 25). Although these substances behaved as expected in vitro, they were degraded within hours of circulation in the plasma of rats. This appeared as LDH degradation products and a high level of free 125I/131I in plasma. We therefore sought an alternative and chose subclasses of IgG known to have different pI but identical molecular weight (16). One problem with these probes is that they derive from human myelomas and may therefore act as an antigen and elicit an immunological response. We infused trace amounts (fractions of a milligram) of IgG, and even though the antibody response is dependent on the dose of antigen (24), it is not unlikely that antibodies were generated during an antigen infusion period of up to 7 days (4), thereby contributing to cellular uptake and a high unelutable fraction (see below). If this had led to probe degradation or alteration that started some days after initiation of the experiment, we would not expect it would be possible to maintain the stable tracer concentration as actually observed. Furthermore, the probes were unaltered and no probe degradation products (including free 125/131I) were observed in plasma after 1 wk of infusion, suggesting that the use of human IgG did not influence the results. Another problem observed by using the present IgG probes was the substantial unspecific binding to the tissue. We were able to remove 96-99% of the 51Cr-EDTA by elution, whereas only 61-67% of IgG 1 and 72-76% of IgG 4 could be eluted from the various tissues sampled. It therefore seems that up to 39% of the labeled IgG in our tissue samples is not free in the interstitial fluid but is bound in some way within the tissue. This binding and uptake process seems to be influenced by the charge of the molecule because the degree of binding was significantly higher for the positively than for the negatively charged IgG. Unspecific binding may develop over time because only on average 2% of IgG was unelutable in homogenized rabbit skin after 8 h of equilibration (27). However, unspecific binding of IgG may be a problem also in studies of short duration as shown by an adsorption fraction to the peritoneal surface of 5-10% after 5 min of IgG equilibration in peritoneal fluid (12). Whether homogenization of the tissue influences the elutable fraction of IgG or whether there are species differences is not known, but the discrepancy between the data from rabbit skin referred to above and our data might suggest so. Unspecific binding of tracer was also a problem of similar magnitude in a previous study (32) by using homologous IgG, and on the basis of other studies (10, 15) we concluded that the unelutable tracer most likely was bound to lymphocytes or were taken up by macrophages. Charge of the probes seems to influence this potential cellular uptake mechanism less than their respective distribution in the interstitial fluid. If not corrected, tracer binding will lead to an overestimation of the available volume (wick fluid as well as plasma equivalent) and to an underestimation of excluded volume. We therefore eluted all samples to be able to correct each sample individually and by doing so should get a reliable estimate of tracer IgG free in the interstitial fluid.Comparison with previous studies. Several investigators (13, 17, 29) studied the transvascular transport of differently charged molecules, but only a few studies have addressed tissue distribution of charged macromolecules. Parker and co-workers (25) measured the plasma-lymph exchange and tissue distribution of anionic LDH 1 (pI 5.0) and cationic LDH 5 (pI 7.9) in the lung in experiments lasting up to 24 h (14, 25), and, although obtained in another tissue and species, their results have relevance to our data. They found that the anionic LDH had a higher relative concentration in lymph than the cationic, whereas the cationic had a higher tissue distribution volume. Calculating available volume as lymph equivalent space, they found an average Ve/Vi of 0.47 for the anionic LDH 1 and 0.21 for the cationic LDH 5. One problem in these studies was the substantial amount of free label after 20 h of infusion (8-12%). This finding may suggest tracer degradation during the experiment, but no chromatography data of plasma were presented. With these reservations, these data corresponds well to ours for skin and muscle showing a distribution volume for the cationic probe about twice that of the anionic probe. Furthermore, our results are supported by data presented in an abstract by Bell (5). He made albumin more positive (pI 7.6) and found a distribution volume in rabbit hind paw skin of the charge modified after 33-76 h of infusion >60% than that of native albumin (pI 5.0).
The background for the present studies was the finding of not significantly different excluded volumes of albumin (molecular mass 66 kDa, Stokes-Einstein radius, 3.53 nm) (9) and the significantly larger molecule IgG (molecular mass 160 kDa, Stokes-Einstein radius, 5.61 nm) (9), suggesting that charge influenced Ve/Vi significantly. In Fig. 5 we have plotted the present data in the same plot as those data obtained in our previous studies (30, 32). The fractional exclusion for the negatively charged IgG 4 is significantly greater than that for native albumin, native IgG, and IgG 1 in all tissues studied. The excluded volume for the positively IgG 4 tended to be lower than the corresponding values for albumin and IgG in all tissues, but was only significantly lower than Ve/Vi for rat IgG in hindlimb muscle and for albumin as well as IgG in tibialis anterior muscle. Although we have shown here a significant charge effect on Ve/Vi, these data also show a significant effect of probe size. Thus the use of a more anionic probe (IgG 4) than the close-to-neutral native rat IgG (O. Tenstad, unpublished observations) led to great difference in Ve/Vi for the former probe and albumin. An even lower pI of the IgG 4 probe similar to that of rat albumin of pI 5.7 (22) would most likely given even higher Ve/Vi for that probe and show the effect of molecular size fully. If we compare the individual tissues studied, the not significant difference in Ve/Vi for rat IgG and positive IgG 4 in skin, which was different in both of the muscle groups sampled, suggest a different distribution of the excluding agents in these two types of tissue.
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Physiological implications of the data. The major excluding agent of the interstitium is collagen, but hyaluronan also contributes significantly to exclusion (3, 9, 11). In previous studies we used in vitro data obtained by others to predict what excluded volume fraction to expect for albumin and compare that with measured values (30, 33). Whereas measured values for muscle are within the range of what to be expected, Ve/Vi for skin is significantly lower than expected from calculations, suggesting a more dense organization of collagen in vivo than in vitro. For IgG, these type of calculations are more difficult due to more sparse in vitro IgG exclusion data.
Hyaluronan has a strong negatively charge at physiological pH, whereas collagen bears a small positive charge (21). In vitro studies on both hyaluronan and collagen show that exclusion increases with molecular size and that the influence from charge is small (19, 23, 26). In a previous paper we were puzzled by the lack of difference between Ve/Vi for IgG and albumin and suggested that the greater molecular size of collagen was compensated by the more negative charge of albumin. The present experiments clearly demonstrate a substantial charge effect. Furthermore, it also suggests a stronger influence of hyaluronan as excluding agent of the interstitium than previously anticipated from in vivo as well as in vitro experiments. On the basis of the assumption that albumin and hyaluronan are in osmotic equilibrium in the tissue, Reed and co-workers (28) calculated that collagen was responsible for two-thirds and hyaluronan for one-third of the albumin exclusion in skin. Whether this ratio is correct is not known but at least shows that hyaluronan has to be considered when calculating exclusion. In conclusion, because of tissue binding, it is more complex to measure the distribution volume of IgG than of albumin, but as a major plasma protein, the excluded volume of IgG is of interest. On the basis of our studies on the distribution volume of native IgG, the differently charged subclasses IgG 1 and IgG 4, being cationic and anionic at physiological pH, may be used as probes to study the effect of charge on distribution volume of two molecules with identical size. The IgG probes were present as free in interstitial fluid but were also bound to or taken up by cells. We studied the fraction that is free in the interstitial fluid and subject to interstitial exclusion and found that the negatively charged IgG 4 is excluded from a volume almost twice that of the positively charged IgG 1 in skin and even more in muscle. Whereas previous studies have suggested that the weakly positive collagen is the major excluding agent, our finding of a significant charge effect suggest a more important role of the negatively charged hyaluronan as interstitial excluding agent in skin and muscle. Our observations also support the model of the interstitium as a gel filtration column.| |
ACKNOWLEDGEMENTS |
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The authors thank Bengt Rippe for valuable discussions during the initial phase of this study. The authors thank Sigrid Lepsøe, Else Elsayed, and Kristin Mesteig for expert technical assistance.
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FOOTNOTES |
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This study was supported by The Norwegian Council on Cardiovascular Diseases and The Research Council of Norway.
Address for reprint requests and other correspondence: H. Wiig, Dept. of Physiology, Univ. of Bergen, Årstadvn 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.
Received 5 July 2000; accepted in final form 8 November 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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) and 131I- labeled
IgG 4 (131I-IgG 4; 
) relative to that in
final plasma at different durations of tracer infusion.
n = 4 for experiments lasting up to 120 h,
n = 5 for 168 h. Values are means ± SE.
and 
, respectively)
after 120 (n = 4) and 168 h (n = 5) of tracer infusion. Values are means ± SE.
) and
131I-IgG 4 (