Native Fluorescence of Extravasated Proteins A New Method for Quantitation Vascular Leakage
ANNA
MILLER-LARSSON
Macromolecular method tracer
AND RALPH BRAITSAND
vascular
leakage
that does not require or dye to the animals.
the concentration orescence
fluorescence.
concentration
of 0.1-120 to the
or radioactive
rescence;
diluted
is quantified
of vascular proteins
plasma.
leakage
measured
Native
is determined by their
fluorescence
at 340 nm, is almost
Fg/mL.
The results obtained from
two other
by a new
macromolecular native
leakage;
due
of protein
in the PNF assay were found
conventional
assays where
was used as the external macromolecular
Vascular
by flu-
of proteins
exclusively
The PNF assay can be used in the range
albumin
Bradykinin;
lumen
of an exogenous
results
Rat trachea;
Key Words:
plasma
against
to tryptophan
rescent
The magnitude
at 295 nm and measured
excited
to be equivalent
into rat tracheal
the administration
of extravasated
and assayed
(PNF), when
of Macromolecular
Inflammation;
Protein
fluo-
tracer.
native
fluo-
Budesonide
INTRODUCTION
The native fluorescence of proteins (PNF) was discovered by Shore and Pardee in 1956 and shortly thereafter described in detail by Teale and Weber (1957). Since then it has been extensively investigated and used mainly in studies of protein structure. Konev and Kozunin
(1961) were
precise
of protein
determination
the first to use PNF in quantitative content
in milk. Resch, Imm,
Ferber,
assay for the Wallach,
and
Fischer, (1971) found PNF to be eminently suited for quantitative determination of soluble and membrane proteins. However, up to now, PNF-based techniques have not won any wider application in quantitative protein assay. Instead, the methods with
proteins
labeled
by different
markers
(colored,
fluorescent,
radioactive),
de-
veloped before the discovery of PNF, have been preferred. We present here a method to quantify the macromolecular vascular leakage into rat tracheal lumen by means of the native fluorescence of the extravasated endogenous proteins, thus without the need of administration of an exogenous macromolecular tracer or dye to the animals. This method is compared with two other conventional and well-established assays where fluorescent or radioactive albumin is used as external From
the Research
Address
reprint
macromolecular
and Development
requests
tracers.
Department,
to: Anna Miller-Larsson
AB Draco, AB Draco,
Lund,
Sweden.
Pharmacology
1 Dpt. Box 34 S-221 00 Lund,
Sweden. Received
September,
1990;
revised
and accepted October,
1990.
251 Journal of Pharmacological 0 1991
Elsevier
Science
Methods Publishing
25, 251-262 Co.,
Inc.,
0160/91/$3.50
(1991)
655 Avenue
of the Americas.
New
York.
NY 10010
252
A. Miller-Larsson and R. Brat&and Physical
Background
Nearly all proteins lower temperatures.
to PNF Assay fluoresce upon excitation in the ultraviolet region at room or This fluorescence is due to their aromatic amino acids, mainly
tryptophan and tyrosine (native or intrinsic fluorescence, PNF) or various prosthetic groups (extrinsic fluorescence). (For a more indepth description, see Udenfriend, 1962,1969; Barenbojm, Domanskii, 1983; Hudson et al., 1985).
and Turoverov,
1969; Guilbault,
1973; Lakowicz,
The fluorescence of tryptophan and tyrosine is due to the presence of indole or phenol rings, respectively, in their structure. The quantum yield (number of quanta emitted
per quantum
of absorbed
energy)
of free amino
acids in solution
depends
on the state of ionization of their amino, carboxyl, and hydroxyl groups. PNF is not a simple function of the fluorescent properties of free amino acids and its number in the polypeptide chain. The contribution of a component single aromatic residue to PNF is closely connected with the chemical and spatial structure of a protein: amino acid sequence, position of aromatic residue in the polypeptide chain, and three-dimensional structure of protein. PNF is generally
excited
at the absorption
maximum
near 280 nm (or at longer
wavelengths), and in tryptophan-containing proteins is strongly dominated by the tryptophan fluorescence. In proteins the quantum yield of tryptophan is highest upon excitation at 295-305 nm. In this region tryptophan fluorescence is selectively stimulated with practically no interference from tyrosine fluorescence. The emission maximum of tryptophan is dependent on solvent polarity and other environmental factors that affect the specific In water, the emission At neutral pH, protein
interaction
between
the indole
ring and the solvent.
maximum for free tryptophan occurs at around 340-350 nm. native fluorescence decreases with increasing temperature;
for tryptophan it decreases by about 2% per degree near room temperature. The quantum yield for tryptophan is nearly constant for external medium pH values 48. Thus PNF is relatively insensitive to pH changes inside the physiological range (Figure I). Because PNF is dependent on many environmental factors, the quantitative interpretation of PNF requires appropriate and stable conditions as well as comparison to a protein standard in the actual environment. The quantitative protein assay can be applied only in the range of protein concentration where fluorescence
emission
mL) and therefore
is linearly cannot
effect), for example. Fluorometric methods
related
to protein
be used for undiluted for the monitoring
concentration, plasma
of proteins
about
0.1-120
Fg/
(due to a self-quenching are superior
to absorption
spectroscopy techniques due to their sensitivity (lower detection threshold by two to three orders of magnitude) and specificity. They are more specific because both absorption and emission spectrum are the discriminating factors. Thus interference with other principles is less likely; the native fluorescence of protein does not interfere with the fluorescence of nucleic acids (in aqueous solutions, at room temperature, and at the physiological range of pH).
Quantitation of Vascular Leakage into Trachea
Fluorescence
295-340
nm
lOOO-
loo-
10-
‘I (a / ’ O.l-
Plasma
(6
dilutions
Uogf
I
I
t
I
-6
I
-5
-4
-3
-2
FIGURE 1. linear relationship between concentration of plasma proteins (from 10e6 to 1.5 x 10m3 plasma dilutions) and intensity of protein native fluorescence at excitation-emission wavelength 295-340 nm, (bandwidth IO-10 nm); (A) in NaCl (at pH = 6.2-6.6) and (0) at a constant pH of 7.4 in phosphate buffer. Mean values + SEM, n = 3. Regression line drawn for NaCI. Recordings at 10T6 plasma dilution are on the threshold of detection. METHODS Experiments
The detailed description of the method for continuous permeability studies in rat trachea, measured by fluorescein isothiocyanate (FITWabeled rat albumin, has been presented previously (Miller-Larsson and Brat&and, 1990). In the present series, the permeability has been measured simultaneously with three methods: FITCrat albumin, 13’l-human albumin, and with the PNF assay as described below. In male Sprague-Dawley anesthetized and spontaneously breathing rats (300-400 g), an in situ segment of trachea (between larynx and manubrium sterni) was perfused (0.1 mUmin) with normal saline. Perfusion fluid was collected continuously throughout the experiment (each sample collected for 10 min). Blood samples were taken every 60 min. Perfusate and blood samples were centrifuged at -7200 g for
253
254
A. Miller-Lawson
and R. Brattsand
IO min to remove cells and debris. The animals were maintained at a constant perature (37.5” ? 0.5”C), with perfusion fluid maintained at room temperature + 2°C).
tem(22”
In the first series of experiments (eight rats) 1 mg of FITC-rat albumin (1 mL in saline) and about 1 MBq of 13’1-human albumin (-0.3-0.6 mg) was administered i.v. After initial perfusion with saline for IO-15 min, the trachea was perfused with budesonide perfusion
(BUD) with
1 mg/mL
saline.
or BUD vehicle
At appropriate
for 50 min,
moments,
and this was followed
saline was replaced
by
for IO min by
bradykinin (BK) 2 x IO-’ M, 2 x 10d6 M or 2 x 10V5 M to induce vascular leakage. BK was applied either as a single dose or three doses were administered in increasing concentration with I-hr intervals between each dose. (Reactivity to BK is completely recovered 1 hr after previous BK application, data not shown). The first BK dose was administered 90 min after the start of perfusion with BUD or BUD vehicle. The magnitude of macromolecular vascular after the BK challenge, was determined periment. FITC-Albumin The amount
Assay (FAM) of FITC-albumin
leakage into the tracheal lumen before and simultaneously by three assays in each ex-
in 1 mL perfusate
(one sample)
was determined
by
the FITC fluorescence assayed against a standard curve obtained with 10~3-10-2 dilutions of plasma, FITC fluorescence of the samples and diluted plasma was mea= 520 nm (bandwidth IO-IO nm, quartz cuvette, sured at A,, = 490 nm and A,, LS-5 luminescence
spectrometer,
Perkin-Elmer).
1311-Albumin Assay (IAM) The amount of 13’ l-albumin in each perfusate sample was indicated by the isotope 13’1 gamma activity (measured in a Nal detector, Harshaw) and by its relationship to plasma activity. PNF Assay. The concentration was measured
of plasma
by their
native
proteins
in the tenfold-diluted
fluorescence
at A,,
perfusate
= 295 nm and A,,
samples = 340 nm
(bandwidth IO-IO nm, quartz cuvette, LS-5 luminescence spectrometer, PerkinElmer; pH of the samples was in the range of 6-7). The fluorescence of the samples was interpolated on a standard curve that expressed the relationship between the fluorescence and the concentration of proteins in plasma (Figure 1). The magnitude of vascular leakage was expressed as a volume (FL) of extravasated plasma. Due to multiple administration of BK in some rats, 14 pairs of data results (before and after BK), were obtained from eight animals for each assay. The data pairs were divided into the two groups of seven controls and seven BUD-treated animals (BTA). In the second series of experiments, after 20 min perfusion with modified Locke buffer (pH = 7.01, the trachea was perfused with carbacholine (six rats), phenylephrine (three rats), or salbutamol (three rats), with increasing concentrations in the range 3 x 1O-8-3 x IO-* M, 40 min with each concentration (increment factor = IO). FAM and PNF assays were
used simultaneously
for evaluation
of leakage.
Quantitation In the third
series of experiments
(three
of Vascular leakage into Trachea
rats), FITC-dextran
(70 kDa),
in an i.v.
dose of loo-250 @rat (0.2-0.5 mL in saline), was used as a macromolecular tracer instead of FITC-albumin. After 70-130 min perfusion with modified Locke buffer (pH = 7.0), BK 2 x lop6 M was perfused for IO min followed by 40 min perfusion with buffer. In the fourth
series of experiments
the second series FITC-albumin.
but with
(three
FITC-dextran
rats), carbacholine
was perfused
as the macromolecular
tracer
as in
instead
of
In the third and fourth series, FITC-dextran assay (analogous to FAM) was applied simultaneously with the PNF assay. During perfusion with carbacholine, secreted mucus was collected
from the mouth
and nose and its PNF was measured.
Drugs and Solutions Bradykinin (acetate salt), carbacholine (carbamylcholine (L-phenylephrine hydrochloride), and salbutamol (sulfate
chloride), phenylephrine salt) were purchased from
Sigma Chemicals (St. Louis, Missouri, USA); budesonide (batch No. 145) from Astra (Sodertalje, Sweden); human serum 1311-albumin from Kemiintresse (Stockholm, Sweden); FITC-albumin (rat) and FITC-dextran (70 kD) from Bioflor (Uppsala, Sweden). Bradykinin
was prepared
modified Locke buffer diluted directly before
as a stock
2 x lop4
solution
M in normal
saline
(or
in a third series of experiments), stored at 4”C, and further application. Budesonide was suspended in 99.5% ethanol,
further diluted in normal saline (a final ethanol 20-30 min, and used on the day of preparation.
concentration -0.3%), sonified for Modified Locke buffer (concentra-
tions in g/L: NaCl, 9.0; KCI, 0.42; CaCI,*2H,O, 0.32; NaHC03, 0.15) was buffered with Hepes 5.96 g/L (Sigma Chemicals, St. Louis, Missouri, USA) and titrated to pH = 7.0 by 1 M NaOH. Carbacholine, phenylephrine, and salbutamol were dissolved to a final concentration Statistical
in modified
Locke buffer
before
application.
Analysis
Comparison
between
the three
assays used was done
in the following
two ways.
1. Calibration approach. The validity of PNF assay was assessed against IAM and FAM assays, which were treated as precise reference methods. Slope values of regression lines and correlation coefficients were calculated for the each pair of methods. The significance of the regression (Pearson 2. Parametric
analysis.
of correlation coefficients and Hartley, 1976).
This approach
allows
was assessed
both numerical
estimation
by the t test of the bias
between methods (mean of between-method differences) and assessment of its significance (Altman and Bland, 1983, Bland and Altman, 1986). Differences between measurements obtained in each two assays (methodi - methodi) were calculated and regressed on the measurement 2). The independence of these two variables relation
coefficient
averages ((methodi + methodi)/ was tested (null hypothesis: cor-
r = 0). If the between-method
differences
were
dependent
255
256
A. Miller-Larsson and R. Brattsand on the size of measurements, was applied
in attempt
then a logarithmic
to remove
transformation
this association.
of the raw data
For independent
variables
(original or log-transformed), the mean of the between-method differences, its standard deviation, and 95% confidence interval (95% conf. int.) for the bias were calculated. The hypothesis that the calculated bias is not significantly different from zero (null hypothesis: bias = 0) was examined by the paired t test. The
procedures
described
above
mental data as well as separately sidered significant at p < 0.05.
were
applied
for controls
for the whole
range
and BTA. The differences
of experiwere
con-
RESULTS Two experimental parameters were analyzed: Basal leakage was defined as the plasma volume lumen
during
10 min of perfusion
without
basal leakage and extra leakage. that extravasated into the tracheal
the addition
of any external
inflammatory
stimulus. The basal leakage was measured IO-20 min before BK challenge. Extra leakage was defined as the total surplus leakage (basal leakage subtracted) obtained after BK challenge. The extra leakage lasted for about 30 min and was calculated over this period. Extra leakage The absolute concentration. 2 x 10-6,
values of extra leakage obtained, are dependent They range between -0.5-2 FL for BK 2 x IO-’
M, and -9-27
on the applied BK M, -4-7 ~J_Lfor BK
t.r,Lfor BK 2 x 10e5 M. The dose-response
relationship
for BK 2 x IO-’ M-2 x lop5 M, obtained by the three applied assays in one of the experiments is presented in Figure 2. The wide range of BK concentration was applied in order to investigate if the level of leakage influences the between-method differences. As shown in Figure 3, the absolute values of extra leakage are tightly gathered along the line of identity (methodi = methodi). Correlation coefficients calculated
for each pair of methods
are in the range 0.994-0.999;
the slope of the
regression lines equals between 1.01 and 1.34. The highest correlation coefficient and the slope value closest to 1 are obtained for the pair PNF/IAM. All calculated correlation coefficients are significant at p < 0.001. No obvious between-method differences are seen between controls and BTA. When the analysis of between-method differences against the average of each two methods is performed, the best agreement between methods is obtained for the pair PNF/IAM, where the between-method measurement size (absolute value of leakage).
differences are independent of the The bias between PNF and IAM (PNF-
IAM = -0.10 FL) is not significant (p > 0.2) and the 95% conf. int. is between -0.29 and 0.09 PL. The same close agreement is also valid inside the groups of controls and BTA. For the pairs PNF/FAM and IAM/FAM, the between method differences increase with the absolute value of leakage in such a way that PNF > FAM and IAM > FAM. For the pair IAM/FAM, the log-transformation removes this association of variables. The calculated bias between IAM and FAM (IAM = 1 .I1 FAM,
Quantitation
of Vascular leakage into Trachea
~1 plasma leakage 16
*
IAM
0
FAM
n
PNF
8
6
4
2
FIGURE 2. Dose-response relationship obtained in one of the experiments for BK 2 X ‘IO-’ M, 2 x 10S6 M and 2 x lo-’ M by the three assays: IAM, FAM, and PNF. The arrow indicates the moment of ‘3’l-albumin and FITC-albumin i.v. administration.
95% conf. int. 0.98-1.26) is found to be nonsignificant (p > 0.05) for the whole range of measurements, as well as separately for controls and BTA. The relation of between-method differences against their average is very similar for IAM/FAM and PNFIFAM;
the slope
of regression
equals
0.2 for both
pairs.
Nevertheless,
associ-
ation between variables remains for PNF/FAM after the log-transformation, thus the significance of the between-method differences cannot be assessed for this pair of methods. Basal leakage The absolute values of basal leakage, obtained in the three assays tested vary between -0.5-2 FL (with one value around 2.5-3 p,L) and are tightly gathered along the line of identity in Figure 3. Correlation coefficients calculated for each pair of methods range between 0.940 and 0.985; the slope of the regression lines equals between culated correlation coefficients are significant at p < 0.001. between-method For all the three
differences
between
controls
0.94 and 0.98. All calThere are no obvious
and BTA.
pairs of assays, the between-method
differences
are independent
257
258 leakage
(@I
Extra leakage
30
0 0
PNF/IAM
AA
PNF/FAM
0 n IAM/FAM
Basal leakage leakage
(pl) Line of identity
0 0
PNF/IAM
AA
PNFIFAM
0 n IAWFAM
FIGURE 3. The absolute values of extra (upper) and basal (lower) leakage, obtained in PNF, IAM, and FAM assays are tightly gathered along the line of identity (methodi = method+. Comparison for the pairs PNWIAM, PNFfFAM, and IAMIFAM as ordinate-abscissa, respectively. Open symbols, controls; filled symbols, BTA.
Quantitation
of Vascular leakage into Trachea
of the absolute value of leakage. When the whole range of data is considered, there is no significant bias between the methods PNF and IAM (PNF-IAM = 0.09 FL, p > 0.1,95% conf. int. from -0.05 to 0.23 pL), nor between PNF and FAM (PNF-FAM -0.08 pL, p > 0.1, 95% conf. int. from -0.22 to 0.05 FL). On the other hand, t=he bias between IAM and FAM is significant at p < 0.001 (IAM-FAM = -0.18 pL, 95% conf. int. from -0.27 to -0.09 p.L). No pair of assays is completely free of significant between-method differences calculated inside the groups of controls and BTA. They are all of approximately the same size, about +0.2 FL, that is, 13%-15% of the mean Influence
leakage
value
(1.3-1.5
PL).
of Secretion
Carbacholine did not influence the level of basal leakage in either assay (PNF and FAM tested) up to the concentration of 3 x 1O-5 M and 40 min perfusion. At 3 x 10e4 M, a tendency
to higher
leakage
was noticed
in both assays (Figure 4). During
1
1pL plasma
L
FAM .._.._)_._._
PNF
3x10s8
3x10-7
3x10-6
3x10-5
3x10-4
3x10-3
3x10-2M
l-l 10 min
CARBACHOLINE
FIGURE 4. The effect of carbacholine 3 x lo-‘-3 x lo-* M on the basal leakage values calculated by PNF and FAM, in three separate experiments. The first two measurements, in each experiment, are obtained in modified Locke buffer (pH = 7.0) as a control to carbacholine. The start concentration of carbacholine is different in each experiment. The figure is drawn with different positions of baseline (zero leakage value) for each experimental curve, and individual baselines are not plotted (see Figure 2 for typical position of baseline).
259
260
A. Miller-Larsson and R. Brattsand perfusion with carbacholine 3 x lop3 M and 3 x IO-’ M, strong mucus secretion from the mouth and nose was seen. During this period, the leakage became transiently higher in both assays and afterwards decreased rapidly below the previous baseline (Figure 4). Neither phenylephrine nor salbutamol up to the concentration 3 x 1O-2 M and 40 min perfusion, secretion.
increased
the leakage
or induced
any external
There was no difference between FITC-dextran and PNF assays for the leakage induced by BK at 2 x 1O-6 M. Under basal conditions (perfusion with buffer) and during perfusion with carbacholine, FITC-dextran leakage was on average -0.2 FL lower (independent of carbacholine concentration), but it generally followed oscillations
of plasma
protein
leakage.
DISCUSSION The consistency of the three methods used for quantitation of macromolecular
applied proves that the PNF assay can be vascular leakage. Application of the PNF
assay makes it possible to avoid several disadvantages connected with the methods where external macromolecular tracer is used. These are as follows: 1) The question of instability of the complex macromolecule-tracer; 2) The tapering of macromolecular tracer in plasma with time and the necessity of its monitoring; 3) The destructiveness of the tracer in vitro with time, light exposure, and monitoring; and 4) The potential hazard combined when working with the radioactive tracers. All three assays appear to be equivalent for evaluation of BK-induced leakage (extra leakage). Also for the basal leakage, there is a close agreement between PNF and IAM as well as between PNF and FAM. At the same time, FAM gives significantly higher
results (p < 0.001) than IAM for the whole
for groups
of controls
also when
compared
range of data as well as separately
and BTA. In BTA rats, FAM gives higher with
basal leakage
values
PNF.
The higher values of basal leakage obtained by FAM cannot be explained by the instability of the FITC-albumin conjugate, because in such a case higher values of extra leakage would be expected as well. Nor can the difference be explained by the native fluorescence of rat’s own plasma proteins, as there is already correction for this effect in the assay (perfusate samples are assayed against diluted plasma). It is plausible that the higher basal leakage obtained in FAM assay is caused by the presence in the tracheal lumen of an additional protein component (or components) of nonplasma origin, which potentiates FITC fluorescence. We have observed that the fluorescence of FITC-albumin (and FITC-dextran) somewhat increases with the total protein content in the external medium (also at the stable physiological pH). This problem has been previously described for FITC-labeled human gamma-globulin by Tenegerdy (1965) and Tenegerdy and Chin-an Chang (1966). This potentiation effect cannot be explained by simple algebraic addition of FITC and plasma fluorescence, and it seems to be characteristic for FITC-conjugate because fluorescence of unreacted FITC molecule is generally decreased by the environmental plasma proteins. The presence of a protein component of nonplasma origin in trachel lumen under
Quantitation
of Vascular Leakage into Trachea
basal conditions is supported also by the significantly higher value of basal leakage in PNF assay than in IAM in the group of controls. It is possible that this component is inhibited by BUD as no difference between PNF and IAM was obtained in BTA. To investigate the possibility that this component could be of secretory origin (from mucus glands or epithelial mucus cells) and to assess its magnitude, we perfused the powerful secretagogues, carbacholine and phenylephrine, in high concentrations in a separate series of experiments. Furthermore, salbutamol was perfused because this substance has been reported to enhance strongly the secretion/transport of albumin into the lumen of ferret trachea (Webber and Widdicombe, 1989). Nevertheless, even at these conditions, no significant differences were observed between FAM and PNF assays (IAM not tested). The possibility that labeled albumin incorporates into secretion and in this way appears in tracheal perfusate (hence the consistency between the assays) must be excluded. This is because, even in the presence of a high concentration of carbacholine, FITC-dextran leakage closely followed plasma protein leakage; plasma leakage exceeded FITC-dextran leakage by a negligible -0.2 PL which is the level of between-method differences (when PNF, IAM, and FAM are compared). The conclusion is drawn that the luminal amount of exogenous albumin and exogenous dextran, is proportional to the amount of extravasated plasma, thus there is no support for an albumin-specific exudation/secretion into the tracheal lumen of the rat. The PNF of the collected mucus, secreted during the perfusion with carbacholine, was found to be about 20 times lower than the PNF of plasma. Hence, more than 4 PL of pure mucus needs to be present in a I-mL perfusate sample to exceed the level of be~een-method differences for basal leakage (4 PL of mucus would be indicated as 0.2 FL higher “leakage”). Thus, although PNF assay is a total protein assay, it seems to be quite insensitive to the presence of possible secretory proteins in the tracheal lumen. This makes the PNF assay a relatively accurate and simple analytical method for quantitation of macromolecular vascular leakage in the present rat model. However, the limitation remains that the PNF assay may give erroneous results in the presence of high concentration of endogenous proteins of nonplasma origin. Also the studies of mediators and drugs that exert their own PNF, may have to be restricted to lower concentrations of such agents. The best way to neutralize any possible background and interfering fluorescence is to relate the measured fluorescence to a standard curve obtained in the actual studied environment. The close congruity between the methods applied demonstrates that the proteins present in tracheal lumen after inflammatory challenge are, by an overwhelming majority, of vascular origin. The agreement between the albumin assays (IAM and FAM) and the protein assay (PNF) shows that the relationship between albumin and total plasma protein content in the tracheal lumen after inflammatory challenge is about the same as that in the plasma. This finding supports the view of the bulk character of plasma exudation (Persson and Erjefalt, 1988) and is, in the rat, not consistent with the hypothesis of a specific active transport of albumin across the tracheal wall (Webber and Widdicombe, 1989). In conclusion, the PNF assay gives an accurate quantitation of macromolecular
261
262
A. Miller-Larsson and R. Brattsand vascular leakage; its results are equivalent to the results of labeled albumin assays (IAM and FAM). In the present rat model, the possible interference from other nonplasma proteins is, after inflammatory challenge, apparently negligible in the PNF assay. It makes the PNF assay suitable for studies of induced vascular permeability follow
to macromolecules and its susceptibility to drug action. When the aim is to thoroughly a defined vascular leakage under basal conditions, the IAM assay
may be the method
of first
choice.
assay seems to be preferable Draco
is a subsidiary
of leakage thank
of A6 Astra,
measurement
Professor
Sweden.
by radioactive
Stella O’Donnell
However,
even under
this
condition
the PNF
to the FAM assay. We thank
albumin
who offered
lngrid
Erjefalt for the introduction
and for her helpful
valuable
discussion.
to the method
We would
like also to
suggestions.
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