- Email: [email protected]
Single-tracer technique to evaluate pulmonary edema and its application to detect the effect of hexamethylene diisocyanate trimer aerosol exposures
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
69,461-470(1983)
Single-Tracer Technique to Evaluate Pulmonary Edema and Its Application to Detect the Effect of Hexamethylene Diisocyanate Trimer Aerosol Exposures’ J. E. VALENTINI,
K.-L. WONG, AND Y. ALARIE’
The Toxicology Laboratory, Department of Industrial Environmental Health Sciences, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Received December 12, 1982; accepted March 3, 1983 Single-Tracer Technique to Evaluate Pulmonary Edema and Its Application to Detect the Effect of Hexamethylene Diisocyanate Trimer Aerosol Exposures. VALENTINI, J. E., WONG, K.-L., AND ALAR& Y. (1983). Toxicol. Appl. Pharmacol. 69,46 I-470. Two hours after a fourhour exposure to hexamethylene diisocyanate trimer (HDIt) aerosol between 2.5 and 39 ms/ m3, mice were injected iv with “Cr-EDTA (chromium ethylenediaminetetraacetate). Ten minutes later the lung was lavaged. A larger amount of %r-EDTA was detected in the lung lavage of HDIt mice than of controls in a concentration-related fashion. The concentration-response curve was shifted to the left compared with that constructed using lung weight increase as response. Kinetic studies of the plasma level of “Cr-EDTA revealed a three-exponential profile in normal mice, and similar plasma levels were obtained with mice exposed to 18-24 mg/m’ HDIt. However, both the amount of “Cr-EDTA in the alveolar space and concentration in the pulmonary extravascular compartment were higher in HDIt-exposed mice than in controls. The data of “Cr-EDTA distribution in the lung were fitted with a three-compartment model. According to the model, HDIt exposures increase the permeability constants of “Cr-EDTA transport into the alveolar space from blood which accounts for the larger amount of “Cr-EDTA in the lung lavage of HDIt-exposed mice. This “Cr-EDTA injection and lung lavage technique is a sensitive method for detecting pulmonary edema.
Pulmonary edema develops when the net rate of fluid movement from the lung capillaries to the interstitium exceeds the rate of lymphatic return to the systemic circulation. This situation may occur as the result of an increase in the net transmural pressure gradient (Levine et al., 1965; Glauser et al., 1974) or as a result of an increase in the permeability of the capillaries or alveolar epithelium, such as following administration of alloxan (Nelson et al., 1978; Peterson et al., 1978). In spite of its complexity, measurements of increased alveolar capillary permeability have ’ Presented at the Annual Meeting of the Society of Toxicology, Las Vegas, March 1983. 2 To whom correspondence should be addressed.
been attempted by several investigators. Crone ( 1963) treated the lungs as a single Krogh cylinder and assumed that a diffusible tracer permeates the capillary wall by a first-order diffusional process and does not return. A multiple-indicator dilution technique was reported by Harris et al., (1976). These investigators developed an effective numerical technique for determining pulmonary capillary permeability from tracer transport measurements. However, the complicated transport phenomena offer serious obstacles to the development of realistic transcapillary models even with the new computational techniques available (Wall et al., 1981), and the interpretation of the results is sometimes difficult (Murphy, 1980). 461
0041-008X/83
$3.00
Copyright 0 1983 by Academic Press, Inc. All rights of reproductnx~ in any form reserved
462
VALENTINI,
WONG, AND ALARIE
Jones et al. (1978, 1979) described another type of approach in which they followed the kinetics of elimination of two tracers from the alveolar space into blood and used such techniques to evaluate the damage created by instillation of hydrochloric acid. With such an approach, they reported an increased rate of transfer of one of the tracers across the alveolar-capillary walls in chronic smokers (Jones et al., 1980). Other investigators have used the increase in protein content or radiolabeled albumin in lung lavage to detect pulmonary edema produced by pulmonary irritants such as nitrogen dioxide or ozone (Selgrade et al., 198 1; Alpert et al., 1971). Recently, Maitani and Suzuki (198 1) have reported that pulmonary edema resulting from ozone exposures can also be detected with an increase in the CajMg ratio in the lung. This study was undertaken to develop a technique for detecting low level of edema in the lung by measuring changes in permeability with “Cr chromium ethylenediaminetetraacetate (“0-EDTA) as a tracer. 51Cr-EDTA was selected as the tracer because this chromium chelate does not dissociate under physiological conditions (Mellor, 1964) and EDTA is not metabolized (Foreman et al., 1953) so that the movement of EDTA can be followed by tracing the “Cr isotope.
METHODS Animals Male Swiss-Webster mice weighing 24-29 g were used in all experiments. They were obtained from Hilltop Laboratories, Scottdale, Pennsylvania, or bred in our facility with adults obtained from this source. Injections of “Cr-EDTA
and Lung Lavage
All injections were made via the tail vein. The tracer solution was prepared by dilution ( 1:100) of “Cr-EDTA solution received from New England Nuclear (Boston) (in 0.005 M EDTA at pH 7 and specific activity of 330
mCi/mg Cr) with isotonic phosphate buffer, pH 7.4, so that the final concentration was 6.8 X IO-l2 mCi/ml and contained 4 X 10m6mol/l of Cr-EDTA (total chromium). A bolus of diluted solution (0. I ml, equivalent to 6.8 &i) was injected in each mouse. This concentration was used for all studies except for the kinetic studies of “Cr-EDTA in normal mice when the specific activity was doubled. The isotope counting procedure of all samples was performed with a Packard-Auto Gamma Counter Model 52 10 at an energy peak of 320 keV. Gross counts were corrected for background to yield net cpm. At appropriate times, as indicated below, the mice were killed by cervical dislocation. The trachea was cannulated immediately, following skin incision, with polyethylene tubing (0.86-mm i.d.) to which a I-ml syringe containing this amount of saline solution was attached. The plunger was then pushed forward 0.15 ml and then drawn back 0.1 ml. After 20 strokes, the plunger was drawn back as far as possible, collecting 0.5 to 0.7 ml of the l-ml lavage fluid. The volume used for counting was adjusted to 0.5 ml.
Measurement
of
Pulmonary Blood Volume
Eight control mice were injected via the tail vein with 0.1 ml suspension of “Cr-labeled red blood cells. After injection, 10 mitt, animals were killed, and 50 ~1 of blood was drawn from the orbital venous plexus by the technique of Sorg and Buckner ( 1964). The thoracic cavity of each animal was carefully opened (to avoid hemorrhage as much as possible) and the lung was excised. Each lung was washed 10 times with 40 ml of saline each time to remove all traces of blood on the surfaces. The amounts of “Cr radioactivity in the lungs as well as in the blood samples were determined. From these values, the blood volume in the lung was calculated. The labeling of RBC was carried out by mixing 2 ml of whole blood drawn from four control mice with 0.3 ml of 5’CrCl, in citrate buffer, pH 7.4 (0.04 &ml, 100 pCi/ml). After 45 min ofincubation at room temperature. the mixture was centrifuged and the supematant fraction was discarded. The RBC in the pellet were washed three times with saline. After the final wash, the RBC pellet was suspended in saline to normal hematocrit prior to injection.
Measurement of Lung Water Content and Hematocrit Eight control mice were killed by cervical dislocation. Their lungs were removed and trimmed. The lungs were rinsed in saline, blotted, weighed before and after drying for 48 hr at 120°C. The water content was calculated from the weight loss. Hematocrit was determined by centrifugation in capillary tubes of blood samples obtained from the orbital venous plexus.
TECHNIQUE Determination
of the Distribution
TO EVALUATE
of “Cr-EDTA
in Blood
A group of four control mice were injected with 0.1 ml “Cr-EDTA and killed after 10 min. Blood samples were taken by cardiac puncture, and the amount of “CrEDTA was measured in whole blood and in plasma following centrifugation of the blood. Kinetic Studies of s’Cr-EDTA in Plasma and Extravascular Space in Normal Mice and Mice Exposed to HDIt
The variation with time of “Cr-EDTA in plasma and extravascular space of the lung following a 0. l-ml bolus tail vein injection, was determined at 10, 15, 20, 30, 40, 60, and 90 min after injection. Four normal mice were used for each time interval. Mice were killed by cervical dislocation, a blood sample was obtained via heart puncture, and the lungs were carefully removed from the thorax and extensively washed 10 times with 40 ml of saline each time. The amounts of “Cr-EDTA were then determined in the blood, plasma, and lung. By knowing the 5’CrEDTA concentration in the blood and the pulmonary blood volume, the amount of “0-EDTA in the extravascular space of the lung at various times alter 5’CrEDTA injections can be calculated. Concentrations of “Cr-EDTA in the plasma of normal mice were also measured at 1,2,3, and 5 min following “Cr-EDTA injection. The effect of HDIt exposure was studied in groups of four mice exposed at 18 to 24 mg/m3 for 4 hr with the exposure procedures described below. Two hours following exposures, “0-EDTA injections were performed. Determinations of “Cr-EDTA in blood, plasma, and lung were made at 10, 20, 40, 60, and 90 min following injection in the same manner as for control mice. Kinetic Studies of “Cr-EDTA in Lung Lavage Normal Mice and Mice Exposed to HDIt
Fluid
PULMONARY
EDEMA
463
prior studies (Weyel et af., 1982). Different aerosol concentrations were generated from 0.08 to 1% solutions (w/v) of HDIt in acetone preparedprior to each experiment. These solutions were fed with a syringe pump at 0.22 ml/min into a Pitt No. 1 aerosol generator as previously described (Weyel et al., 1982). The output of the generator was introduced into a glass exposure chamber where only the head of the animals protruded inside. The chamber was made of a glass cylinder with a volume of 4.6 liters. It was similar to the chamber described by Barrow et al. (1977) but twice the length in order to accommodate eight mice instead of four. The airtlow through the chamber was maintained at 20 liters/min for all experiments. The aerosol concentrations were evaluated gravimetrically by drawing known amounts of chamber air through preweighed 47-mm membrane filters, 0.4-pm pore size, from the Millipore Corp. (Bedford, Mass.). The particle size was evaluated with an Andersen mini impactor and was 0.6-pm aerodynamic equivalent diameter with a geometric standard deviation of 2.4, similar to prior experiments (Weyel et al., 1982). The concentration of acetone in the chamber was also similar to the results from the prior studies (Weyel et al.. 1982) varying between 2800 and 3000 ppm for all experiments. (a) Concentration-response relationships. Five groups of eight mice were exposed for 4 hr to HDIt aerosols at various concentrations (2.5 to 39 me/m’). Two hours following exposure, they were injected with “0-EDTA and killed 10 min afterwards. Lung lavages were performed as described above. Twenty nonexposed animals were used as control and one group of eight animals exposed to acetone alone at 3000 ppm was also used. (b) Time-response relationships. Five groups of eight animals were exposed to HDIt at 35 to 40 mg/m’ for 4 hr. Animals were injected with “Cr-EDTA, at 24, 33, 48, 72, and 240 hr after exposure. After injection, IO min, lung lavages were performed as described above.
in
RESULTS Groups of four normal mice were injected with 5’CrEDTA and killed at 10, 1520, 30,40, and 60 min after injection. Lung lavage was performed as described above, and the amount ofS’Cr-EDTA was determined. The same procedure was followed for groups of mice exposed to HDIt at 18 to 24 mg/m3 for a period of 4 hr. Two hours following exposure, “Cr-EDTA was injected and groups of four mice were killed at 10,20,40, and 60 min following injections.
Exposure
to HDIt
A commercial sample of HDIt (Desmodur-N, Des-N, supplied by Mobay Chemical Corporation, Pittsburgh, Pa.) was used containing 1.13% free hexamethylene diisocyanate. This supply was from the same batch used in
“Cr-EDTA in Lung Lavages following Exposure to HDIt at Various Concentrations The results for this series of experiments are presented in Table 1 and Fig. 1. There was more variation in the control (34% coefficient of variation) than desirable, probably due to the fact that recovery of 5’Cr-EDTA requires that two operations be performed correctly: injection of the tracer and adequate performance of lung lavages. Nevertheless a significant increase in “Cr-EDTA was observed for all HDIt exposure groups when compared to the nonexposed group used as a
464
VALENTINI,
WONG, AND ALARIE TABLE
1
AMOUNT OF “Cr-EDTA IN LUNG LAVAGES OF CONTROL ANIMALS, ANIMALS EXPOSED TO 3000 ppm ACETONE FOR 4 hr, AND ANIMALS EXFTXED TO VARIOUS CONCENTRATIONS OF HDIt AEROSOU FOR 4 hr AND KILLED 2 hr AFTER EXPOSURE
HDIt exposure concentration (mg/m’)
Number of animals
Nonexmsed (control) Acetoned 2.5 + Acetone 5.6 + Acetone 13 + Acetone 23 f Acetone 39 + Acetone
20 8 8 6’ 8 7 8
5’Cr-EDTA” (net cpm + SD) 2920 + 3860 k 41lO+ 6480 + 10950 k 14590 k 17140 f
980 1080 840 1550 2950 3250 3270
Ratio’ of exposed/control 1.3 1.4 2.2 3.8 5.0 5.9
Ratio’ of exposed/acetone 1.1
1.7 2.8 3.8 4.4
GIn 0.5 ml lung lavage fluid. The lavage was performed 10 min after injection. ’ The ratio of 5’Cr-EDTA in HDIt exposed mice vs controls. All groups exposed to HDIt had higher net cpm than the control group (p -=z0.05). ‘The ratio of “Cr-EDTA in HDIt exposed mice vs acetone-exposed mice. All groups exposed to HDIt had higher net cpm than the acetone group (p < 0.05) except the group exposed to 2.5 mg/m’. d Acetone was at 2800 to 3000 ppm for all groups. p When less than eight animals are reported, the results of one or two animals were omitted because of technical failure in 51Cr-EDTA injection or in performing lung lavages.
control group and for all groups above 2.5 mg/m3 when compared to the acetone aloneexposed group which can also be used as a control group. The results from two of eight exposed animals at 5.6 mg/m3 and one of eight animals exposed to 23 mg/m3 were omitted because of obvious technical failure in “CrEDTA injections or in performance of lung lavages. Although we did not expect any difference between the nonexposed control animals and acetone-exposed mice, the concentration-response curve was shifted to the right when the ratios of “Cr-EDTA in lung lavages of HDIt groups versus the acetone group were compared with that versus the nonexposed group (Fig. 1). For all sections below the term “control” will refer to normal animals rather than acetone-exposed animals. 5’Cr-EDTA in Lung Lavage Fluid at Dlflerent Times following Exposures to 35-40 mg/ m3 HDIt These results are presented in Fig. 2. It can be seen that the amount of “Cr-EDTA in
lung lavages, when 5’Cr-EDTA was injected at various times following exposures, declined slowly and returned close to control level after 2 days of exposure and remained at this level at 10 days postexposure. Kinetic Studies of “Cr-EDTA in Plasma of Normal Mice and Mice Exposed to HDIt The results of distribution of “Cr-EDTA following injection revealed that > 90% of “0-EDTA in blood was in the plasma. It was assumed that all 5’Cr-EDTA in blood is in the plasma for the kinetics described below. The results of concentrations of “Cr-EDTA in plasma of normal mice and mice exposed to HDIt, obtained at various times following injection of “0-EDTA, are presented in Fig. 3. To describe the time course of “Cr-EDTA concentrations in plasma of the control animals, a k-exponential equation was obtained with curve stripping. It can be seen that, although the initial time intervals were not studied in the group exposed to HDIt, the data are comparable to the control group. The
TECHNIQUE
TO EVALUATE
PULMONARY
465
EDEMA
6-
5-
4-
:3 $ Z0
Ir I
2
I I ,,,,ll
5
,
IO
20
HDI,(MG/
,
,,,,,,I
40
m
M3 1
FIG. I. The RATIO of the mean amount of “Cr-EDTA in lung lavages performed 10 min after “Cr-EDTA was injected 2 hr following an exposure to various concentrations of HDIt (6 to 8 mice at each concentration) versus the mean of acetone-exposed mice (8 mice) or normal mice (20 mice). For comparison purpose, the data from Weyel et al., (I 982) were included. The ratio of the lung weight of mice 24 hr after HDIt exposure versus that of control mice and the maximum respiratory frequency during HDIt exposure as a .fraction of the preexposure frequency were plotted from this reference. Linear regression lines were fitted with the least squares method.
IO
20
30
40
50 60 MINUTES
70
6.0
90
100
FIG. 3. Concentration of “0-EDTA in plasma at various times after injection of r’Cr-EDTA in control mice and mice exposed to HDIt at 18 to 24 mg/m’. The line was hand-drawn. The control data were fitted with an equation, y = 43.095e?‘.655’ + 9.085e-0.075’ + 6.748e-0.0’9’ by a curve stripping technique.
coefficient of variation for all data points was around 25%.
Kinetic Studiesof “Cr-EDTA in the Interstitial and Alveolar Spacesin Normal Mice and Mice Exposed to HDIt The pulmonary blood volume (PBV) in mice evaluated with labeled red blood cells was found to be 1.88 f 0.43 pi/g of body weight and the average volume of lung water (LW) was found to be 4.16 f 0.38 &g of body weight. Thus an “apparent pulmonary interstitial fluid volume” (PIV) was calculated to be 3.03 PI/g body weight by the expression PIV = LW - PBV( 1 - hematocrit).
TIME
(Days)
FIG. 2. The RATIO ofthe mean amount of “Cr-EDTA in lung lavages performed 10 min after “Cr-EDTA was injected at various times following a 4-hr exposure of HDIt at 40 mg/m3. The mean of normal mice was used as the denominator in the calculation of the RATIO.
This approach assumed that all the water in the lung was distributed between the plasma and the interstital compartment with negligible amount of water in the alveolar space and cells. For each animal from the control group and the group exposed to HDIt, knowing their respective body weights (BW), PBV, and the concentration of 5‘Cr-EDTA in plasma, [’ ‘Cr-
466
VALENTINI,
WONG, AND ALARIE
EDTA],, parameters about the interstitial and alveolar compartment (IAC) can be calculated: Amount
in IAC
= (counts in lung) - PBV - BW X ( 1 - hematocrit) - [’ ‘Cr-EDTA], Concentration
,
in IAC, 0
[“Cr-EDTA],*c
= AmiIy:
Kinetic Studies of “Cr-EDTA in Lung Lavage Fluid in Normal and HDIt-Exposed Animals The net counts of ‘Cr-EDTA in lung lavage fluid (0.5 ml) are presented in Fig. 5 for the controls and the group exposed to HDIt. The data points were fitted by least squares linear regression analysis and yielded a P/z of 15 min for the control group and 16 min for exposures to HDIt. “Cr-EDTA in lung lavage of the
0t
0 CONTROL 0 HOlt
30
40
50 60 MINUTES
70
20
60
I
so
30
40
50
60
70
MINUTES
The calculated values of [5’Cr-EDTA]iAc for controls and for animals exposed to HDIt are presented as a function of time after 5’CrEDTA injection in Fig. 4. The data points were fitted by least square linear regression analysis and yielded a P/z of 77 min for the exposed group and 105 min for the control group. The [“Cr-EDTA],,c was significantly (p < 0.05) higher in the HDIt exposed animals than in controls at all time intervals.
I=
IO
i?
100
FIG. 4. Concentration of “0-EDTA in the extravascular compartment in the lung of controls and mice exposed to 18-24 mg/m3 HDIt as a function of time after injection of SICr-EDTA. Linear mgression analysis resulted in y = 1.484 - O.O03t, r = -0.85 for controls and y 7 I .886 - O.O04f,r = -0.93 for the exposed group, where y = log (concn).
FIG. 5. The amount of “Cr-EDTA in lung lavage of controls and mice exposed to 18-24 mgJm3 HDIt at various times after iv injection of “Cr-EDTA. Linear regression analysis resulted in y = 3.623 - 0.046t and y = 4.088 - 0.044t for controls and HDIt group, respectively, where y = log (amount).
exposed group was significantly higher (p < 0.05) than for control group at all time intervals as found at the IO-min time intervals given in Table 1. Modeling Lung
of “Cr-EDTA
Distribution
in the
From the results given above a compartmental model can be suggested based on the following assumptions and findings: (a) After tail vein injection, 51Cr-EDTA distributes itself among three compartments in the lung: pulmonary blood, pulmonary interstitial space, and a layer of alveolar epithelium and pulmonary surfactant which can be defined as alveolar space. This view is a reasonable assumption considering that Volf et al. ( 197 1) presented data suggesting that EDTA distributes mainly in the extracellular water. (b) “Cr-EDTA behaves as a free compound in all three compartments, since it is known that binding of 5’ Cr-EDTA to plasma protein is negligible (Stacy and Thorburn, 1966; Garnett et al., 1967) and EDTA is not taken up by the red blood cells (Foreman et al., 1953; Gamett et al., 1967). (c) The concentration of “Cr-EDTA in the pulmonary interstitial and alveolar spaces [“Cr-EDTA]i,c can be calculated from the equation given above.
TECHNIQUETO EVALUATEPULMONARYEDEMA
467
systemof differential equationswas blood(plasma)is known at any onetime after solvedby a Runge-Kutta Vemer fifth- and (d) The concentration
of “0-EDTA
in
The
sixth-order method (Hull et al., 1976) which allowed for the calculation of the theoretical CB = [“Cr-EDTA],r,,, = 43.095e-0.65s’ values of [YI] and YII respectively at any time. The constants, ki, where i = l-4 and V,, were + 9.805e-0,075’ + 6.248e0--0’9’ > adjusted minimizing the squares of the difvalid for t > 1 min, from Fig. 3. Thus, the ferences between the experimental and theodistribution of 5’Cr-EDTA can be depicted as retical values. The procedure was performed shown in Fig. 6. The differential equations for the experimental values from the control describing the distribution of “Cr-EDTA as group as well as from the HDIt group. The a function of time would be numerical solutions of the above equations and the minimization procedure were carried d[YI]/dt = k, [CB] - k2 [YI] out on the DEC10 computer of the University + k4.YII/I’a - k3[YI] of Pittsburgh. The results are described in Fig. d[YII]/dt = kXIYI] - kq’YII/C/a; 7, comparing the experimental with the theoso retical value which were calculated with k, dYII/dt = k3 - VJYI] - k4 - YII from Table 2. The experimental and theoretical values agree very well. Both k, and k3 where were higher in the HDIt-exposed groups than [YI] = concentration of “Cr-EDTA in in- for the control group and more so for k3 than terstitial space = (amount in IAC k, . Thus according to the model, the greater - counts in lung lavage)/PIV . body amount of tracer in lung lavage of HDIt mice weight than in controls reflected an increased per[YII] = concentration of “Cr-EDTA in al- meability of the barrier between the interstiveolar space tium and alveolar space in HDIt mice. YII = amount of “Cr-EDTA in alveolar space = counts in lung lavage k, , k2, = first-order rate constants in min-’ DISCUSSION k k4 I’, = apparent volume of distribution of The most common and simple method of “0-EDTA in the alveolar space in measuring pulmonary edema is by the mea4. surement of wet lung weight increases. Unfortunately increases in wet lung weight are sensitive for only moderate or severe pulmonary edema and other methods will have to be used in the detection of minor pulmonary edema. Numerous methods have been developed for that purpose but they all suffer from some kind of drawbacks. For instance, the volume of extravascular water in lungs can be determined by either the indicator dilution method (Goresky et al., 1969) or the soluble inert gas method (Glauser et al., 1974; Peterson et al., 1978). However, the indicator dilution method is technically too complicated FIG. 6. Three-compartment model for the distribution of “Cr-EDTA in lungs. The symbols k14 represent the since it involves the cannulation of pulmonary rate constants for the transfer of “Cr-EDTA between the artery and aorta and serial blood sampling at compartments. l- or 2-set intervals, while the soluble inert injection
as given by the equation:
468
VALENTINI, WONG, AND ALARIE
10,000
et al., 1978) or injection into the right atrium (Harris et al., 1976) and serial blood sampling. o.-.o PREDICTED Comparatively speaking the method presented in this report is much simpler since only one radioactive tracer was used, making tracer analysis a simple matter and avoiding serial blood sampling. Not only is the present single-tracer method simple, but it is also very sensitive. This view becomes apparent when the present method 8. CONTROL MICE is compared with methods based on wet lung 0-o PREDICTED weight which was used by many investigators l + EXPERIMENTAL as a standard of comparison for any new method of detecting pulmonary edema (Goresky et al. 1 1969; Glauser et al. ( 1974; Peterson et al., 1978). Such a comparison is depicted in Fig. 1 in which the data of wet lung weight increases produced 24 hr after HDlt exposure, I I 1 1 I 1 1 taken from a report by Weyel et al. (1982). I 0 IO 20 30 40 50 60 TO MINUTES were included. The increase in “Cr-EDTA in FIG. 7. The amount of 5’Cr-EDTA in the alveolar space lung lavage was much more sensitive than inas predicted by the model versus the experimental data creases in wet lung weight in detecting the in (A) mice exposed to IS-24 mg/m’ HDIt; and(B) control pulmonary irritating effect of HDlt with the mice at various times after iv injection of “Cr-EDTA. concentration-response curve of the former For the HDIt-exposed mice, the model prediction was based on k, = 2.4 and k, = 6.6. while k, = 1.9 and k3 shifted one order of magnitude to the left compared with that based on increases in wet = 3.4 were used in the model for controls. lung weight. The “Cr-EDTA method can detect an increase in permeability at an HDlt gas method requires expensive analytical concentration that is not high enough to proequipment such as a mass spectrometer or duce gross pulmonary edema. else breath-holding maneuvers will need to be Alpert et al. ( 197 1), reported a method very performed on the test animals. In addition, similar to ours for the detection of pulmonary the soluble inert gas method consistently overestimates the volume of extravascular TABLE 2 water in lungs by 10 to 15% (Glauser et al., VALUESOFTHE FIRST-ORDER RATECONSTANTS 1974; Peterson et al., 1978). FOR THE DISTRIBUTION MODEL OF “0-EDTA IN Pulmonary edema can be classified, acCONTROL AND HDltExPosED ANIMALS cording to the cause, into two broad categories: hemodynamic edema and permeability A B edema. The methods mentioned above can k Control HDIt exposed’ WA detect edema due to either of the two causes. 2.40 1.26 1.90 There are no methods that will detect he- kb 0.28 0.31 1.10 kz modynamic pulmonary edema specifically, 6.60 1.94 3.40 kj but several methods specific for permeability 5.10 5.10 1.00 x-, 10.00 IO.00 1.00 pulmonary edema have been reported (Harris V,’ et al., 1976; Jones et al., 1978; Nelson et al., 0 Exposure at 18 to 24 mum’ for 4 hr. 1978). All of these procedures involve the adb .L were first-order rate constants in min-‘. ministration of more than one tracer via trac V. was the apparent volume of distribution of “Crcheal instillation (Jones et al., 1978; Nelson EDTA in the alveolar space in microliters. A.
t
HDlt
MICE
1
I
TECHNIQUE
TO
EVALUATE
edema produced by ozone except that 13’1albumin was used as the tracer instead of 51CrEDTA. Since ‘Cr-EDTA is a much smaller molecule than 13’1-albumin (MW 362 versus 60,000), we believe that our single-tracer method should be able to detect a smaller increase in permeability than the method using ‘311-albumin because a smaller tracer stands a better chance in transversing the diffusion barrier when small leaks start to appear leading to an increase in tracer in lung lavage that may not be detectable if a larger tracer such as albumin is used. We propose that 51Cr-EDTA distributes in the lung according to a three-compartment model which was also shown to be true for the distribution of radioactive albumin by Gee and Staub (1977). As shown in Table 2 and Fig. 7, the pulmonary distribution of “CrEDTA can be closely assimilated with the three-compartmental model when the transfer rate constants, k, and k2, were larger than k3 and k4, respectively. This finding indicates that for the transfer of “Cr-EDTA between pulmonary blood and the alveolar space the transport of “Cr-EDTA across the capillary endothelium is the rate limiting step. The capillary endothelium has also been assumed to be the major barrier for the distribution of urea in the lung by Brigham et al. ( 1979). Another evidence to support that transport across the capillary endothelium is rate limiting for the pulmonary distribution of “CrEDTA is that in the control mice the half-life of the decline of alveolar “Cr-EDTA was only 15 min (Fig. 5) compared with 105 min for the decline of “Cr-EDTA in the interstitial and alveolar spaces combined (Fig. 4). This result means that as 5’Cr-EDTA leaves the alveolar space to return to the blood it accumulates in the interstital space because the capillary endothelium is the major barrier for the re-entry of “Cr-EDTA into the capillaries. The slow rate of decline of “Cr-EDTA in the interstitial and alveolar spaces that we found in mice was comparable to that reported by Jones et al., (1978) in rabbits. The plasma level of “Cr-EDTA decreased with time in a n-i-exponential fashion with a
PULMONARY
EDEMA
469
short terminal half-life (approximately 32 min) in both control and HDIt- exposed mice indicating a rapid elimination. Since the plasma level in the HDIt mice coincided with that in controls, this finding ruled out the possibility that a greater amount of 51Cr-EDTA in the lung lavage of the HDIt mice was due to a higher plasma level. The model suggests that the greater amount in the lung lavage of the HDIt mice was a result of increased permeabilities in both the capillary endothelium and alveolar epithelium due to HDIt exposures. The increased permeabilities could be due to communicating channels not normally present being induced by pulmonary irritation caused by HDIt. This suggestion is a distinct possibility since it has been shown by Simani et al. (1974) that tight junctions in airway epithelium became freely permeable to horseradish peroxidase after inhalation of cigarette smoke in guinea pigs. The prediction by the model shown in Fig. 7 is based on the assumption that the volume of alveolar fluid (V,) in mice was 10 ~1. This assumption is comparable to the estimated volume of alveolar fluid in humans (Ma&in, 1954) on a per gram body weight basis. The assumption that the volumes of alveolar fluid were the same in controls and in mice exposed to HDIt at 18-24 mg/m3 is valid because the HDIt concentration was not high enough to produce alveolar flooding. The three-compartment model indicates that HDIt exposures make the capillary endothelium and alveolar epithelium more permeable which may lead to pulmonary edema. We conclude that the single-tracer technique with “Cr-EDTA is a simple and sensitive technique that can be used to detect even minor permeability pulmonary edema. ACKNOWLEDGEMENTS The authors wish to thank Professor H. S. Borovetz of the University of Pittsburgh, School of Medicine, for his helpful discussion regarding the experimental and computational phases of the present work. This work was supported under research Grant 1 ROl ES02747-01 from the National Institute of Environmental Health Sciences.
470
VALENTINI,
WONG, AND ALARIE
We also thank Dr. D. A. Weyel for his help in generation ofthe aerosol and Mrs. M. F. Stock for technical assistance and preparing the figures.
REFERENCES ALPERT, S., SCHWARTZ, B., LEE, S. D., AND LEWIS, T. (1971). Alveolar protein accumulation: A sensitive indicator of low level oxidant toxicity. Arch. Intern. Med. 28,69-73.
BARROW,C. S., ALARIE, T., WARRICK, J. C., AND STOCK, M. Y. (1977). Comparison of the sensory irritation response in mice to chlorine and hydrogen chloride. Arch. Environ.
Health
32, 68-76.
BRIGHAM, K. L., SNELL, J. D., JR., HARRIS, T. R., MARSHALL, S., HAYNES, J., BOWERS,R. E., ANDPERRY, J. ( 1979). Indicator dilution lung water and vascular permeability in humans. Cir. Res. 44, 523-530. CRONE, C. (1963). Permeability of capillaries in various organs determined by use of the indicator diffusion method. Acta Physiol. Stand. 58, 293-305. FOREMAN, H., VIER, M., AND MAGEE, M. (1953). The metabolism of C14-labelled ethylenediaminetetraacetic acid in the rat. J. Biol. Chem. 203, 1045-1053. GARNET-~, E. S., PARSONS, V., AND VEALL, N. (1967). Measurement of glomerular filtration-rate in man using a “Cr/edetic acid complex. Lancet i, 8 18-8 19. GEE, M. H. AND STAUB, N. C. (1977). Role of bulk tlow in protein permeability of the dog lung alveolar membrane. J. Appl. Physiol.: Respir. Environ. Exercise Physiol. 42, 144-149. GLAUSER, F. L., WILSON, A. F., HOSHIKO, M., WATANABE, AND DAVIS, J. (1974). Pulmonary parenchymal tissue (Vt) changes in pulmonary edema. .I. App/. Physiol. 36, 648-652.
GORESKY, C. A., CRONIN, R. F. P., AND WANGEL, B. E. ( 1969). Indicator dilution measurements of extravascular water in the lungs. J. Clin. Invest. 48, 487-501. HARRIS, T. R., ROWLET~, R. D., ANDBRINGHAM, K. L. (1976). The identification of pulmonary capillary permeability from multiple-indicator data. Microvasc. Res. 12, 177-196.
HULL, T. E., ENRIGHT, W. H., AND JACKSON, K. R. (1976). User’sguidefor DVERK. A subroutine for solving NON-STIFF ODE’5 TR No. 100. Department of Computer Science, University of Toronto. JONES,J. G., BERRY, M., HULANDS, G. H., ANDCRAWLEY, J. C. W. (1978). The time course and degree of change in alveolar-capillary membrane permeability induced by aspiration of hydrochloric acid and hypotonic saline. Amer. Rev. Respir. Dis. 118, 1007-1013. JONES,J. G., GROSSMAN, R. F., BERRY, M., SLAVIN, G., HULANDS, G. H., AND MINTY, B. (1979). Alveolarcapillary membrane permeability. Correlation with
functional, radiographic, and postmortem changes after fluid aspiration. Amer. Rev. Respir. Dis. 120, 399-4 10. JONES,J. G., MINTY, B. D., LAWLER, P., HULANDS, G.. CRAWLEY. J. C. W.. AND VEALL, N. (1980). Increased alveolar epithelial permeability in cigarette smokers. Lancet 8159. 66-68. LEVINE, 0. R., MELLINS, R. B., AND FISHMAN, A. P. (1965). Quantitative assessment of pulmonary edema. Cir. Res. 17, 4 14-426. MACKLIN, C. C. (1954). The pulmonary alveolar mucoid film and the pneumocytes. Lancet 1, 1099-I 104. MAITANI, T. AND SUZUKI. K. T. ( 198 1). Metal content ratio as a sensitive indicator of pulmonary edema. Taxicol. Lett.
9, 295-300.
MELLOR, D. P. ( 1964). In Chelating Agents and Metal Chelates (F. P. Dwyer and D. P. Mellor, eds.). p. 41. Academic Press, New York. MURPHY, J. J. (1980). Characterization ofpulmonary injury induced by oleic acid infusion. M. S. thesis. University of Pittsburgh. NELSON, R. M., MCINTYRE, 8. R., AND EGAN, E. A. (1978). Solute permeability of the alveolar epithelium in alloxan edema in dogs. J. Appl. Physiol.: Respir. Environ.
Exercise
Physiol.
44, 353-357.
PETERSON, B. T., PETRINI, M. F., HYDE, R. W., AND SCHREINER,B. F. (1978). Pulmonary tissue volume in dogs during pulmonary edema. J. Appl. Physiol.: Respir. Environ.
Exercise
Physiol.
44, 782-795.
SELGRADE, M., MOLE, L. M., MILLER, F. J., HATCH, G. E., AND GARDNER, D. E. (1981). Effect of Nor inhalation and vitamin C deficiency on protein and lipid accumulation in the lung. Environ. Res. 26,422437.
SIMANI, A. S., INOUE, S., AND HOGC, J. C. (1974). Penetration of the respiratory epithelium of guinea pigs following exposure to cigarette smoke. Lab. Invest. 31, 75-81.
SORG, D: A. AND BUCKNER, B. (1964). A simple method of obtaining venous blood from small laboratory animals. Proc. Sot. Exp. Biol. Med. 115, 113 l-l 132. STACY, B. D. AND THORBURN, G. D. (1966). Chromium5 1 ethylenediaminetetraacetate for estimation of glomerular filtration rate. Science 152, 1076- 1077. VOLF, V., VLADAR, M., AND SEIDEL, A.. (1971). Distribution of labelled calcium-yttrium- and chromiumchelates of EDTA in rats. Arch. Int. Pharmacodyn. 190, 110-123.
WALL, C., III, BOROVETZ, H., MURPHY, J. J., AND HARDESTY, R. (1981). System parameter identification in transport models using the fast fourier transform (FIT). Comp.
Biomed.
Res. 14, 670-68
1.
WEYEL. D. A., RODNEY, B. S., AND ALARIE, Y. (1982). Sensory irritation, pulmonary irritation, and acute lethality of a polymeric isocyanate and sensory irritation of 2,6-toluene diisocyanate. Toxicol. Appl. Pharmacol. 64, 423-430.
69,461-470(1983)
Single-Tracer Technique to Evaluate Pulmonary Edema and Its Application to Detect the Effect of Hexamethylene Diisocyanate Trimer Aerosol Exposures’ J. E. VALENTINI,
K.-L. WONG, AND Y. ALARIE’
The Toxicology Laboratory, Department of Industrial Environmental Health Sciences, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Received December 12, 1982; accepted March 3, 1983 Single-Tracer Technique to Evaluate Pulmonary Edema and Its Application to Detect the Effect of Hexamethylene Diisocyanate Trimer Aerosol Exposures. VALENTINI, J. E., WONG, K.-L., AND ALAR& Y. (1983). Toxicol. Appl. Pharmacol. 69,46 I-470. Two hours after a fourhour exposure to hexamethylene diisocyanate trimer (HDIt) aerosol between 2.5 and 39 ms/ m3, mice were injected iv with “Cr-EDTA (chromium ethylenediaminetetraacetate). Ten minutes later the lung was lavaged. A larger amount of %r-EDTA was detected in the lung lavage of HDIt mice than of controls in a concentration-related fashion. The concentration-response curve was shifted to the left compared with that constructed using lung weight increase as response. Kinetic studies of the plasma level of “Cr-EDTA revealed a three-exponential profile in normal mice, and similar plasma levels were obtained with mice exposed to 18-24 mg/m’ HDIt. However, both the amount of “Cr-EDTA in the alveolar space and concentration in the pulmonary extravascular compartment were higher in HDIt-exposed mice than in controls. The data of “Cr-EDTA distribution in the lung were fitted with a three-compartment model. According to the model, HDIt exposures increase the permeability constants of “Cr-EDTA transport into the alveolar space from blood which accounts for the larger amount of “Cr-EDTA in the lung lavage of HDIt-exposed mice. This “Cr-EDTA injection and lung lavage technique is a sensitive method for detecting pulmonary edema.
Pulmonary edema develops when the net rate of fluid movement from the lung capillaries to the interstitium exceeds the rate of lymphatic return to the systemic circulation. This situation may occur as the result of an increase in the net transmural pressure gradient (Levine et al., 1965; Glauser et al., 1974) or as a result of an increase in the permeability of the capillaries or alveolar epithelium, such as following administration of alloxan (Nelson et al., 1978; Peterson et al., 1978). In spite of its complexity, measurements of increased alveolar capillary permeability have ’ Presented at the Annual Meeting of the Society of Toxicology, Las Vegas, March 1983. 2 To whom correspondence should be addressed.
been attempted by several investigators. Crone ( 1963) treated the lungs as a single Krogh cylinder and assumed that a diffusible tracer permeates the capillary wall by a first-order diffusional process and does not return. A multiple-indicator dilution technique was reported by Harris et al., (1976). These investigators developed an effective numerical technique for determining pulmonary capillary permeability from tracer transport measurements. However, the complicated transport phenomena offer serious obstacles to the development of realistic transcapillary models even with the new computational techniques available (Wall et al., 1981), and the interpretation of the results is sometimes difficult (Murphy, 1980). 461
0041-008X/83
$3.00
Copyright 0 1983 by Academic Press, Inc. All rights of reproductnx~ in any form reserved
462
VALENTINI,
WONG, AND ALARIE
Jones et al. (1978, 1979) described another type of approach in which they followed the kinetics of elimination of two tracers from the alveolar space into blood and used such techniques to evaluate the damage created by instillation of hydrochloric acid. With such an approach, they reported an increased rate of transfer of one of the tracers across the alveolar-capillary walls in chronic smokers (Jones et al., 1980). Other investigators have used the increase in protein content or radiolabeled albumin in lung lavage to detect pulmonary edema produced by pulmonary irritants such as nitrogen dioxide or ozone (Selgrade et al., 198 1; Alpert et al., 1971). Recently, Maitani and Suzuki (198 1) have reported that pulmonary edema resulting from ozone exposures can also be detected with an increase in the CajMg ratio in the lung. This study was undertaken to develop a technique for detecting low level of edema in the lung by measuring changes in permeability with “Cr chromium ethylenediaminetetraacetate (“0-EDTA) as a tracer. 51Cr-EDTA was selected as the tracer because this chromium chelate does not dissociate under physiological conditions (Mellor, 1964) and EDTA is not metabolized (Foreman et al., 1953) so that the movement of EDTA can be followed by tracing the “Cr isotope.
METHODS Animals Male Swiss-Webster mice weighing 24-29 g were used in all experiments. They were obtained from Hilltop Laboratories, Scottdale, Pennsylvania, or bred in our facility with adults obtained from this source. Injections of “Cr-EDTA
and Lung Lavage
All injections were made via the tail vein. The tracer solution was prepared by dilution ( 1:100) of “Cr-EDTA solution received from New England Nuclear (Boston) (in 0.005 M EDTA at pH 7 and specific activity of 330
mCi/mg Cr) with isotonic phosphate buffer, pH 7.4, so that the final concentration was 6.8 X IO-l2 mCi/ml and contained 4 X 10m6mol/l of Cr-EDTA (total chromium). A bolus of diluted solution (0. I ml, equivalent to 6.8 &i) was injected in each mouse. This concentration was used for all studies except for the kinetic studies of “Cr-EDTA in normal mice when the specific activity was doubled. The isotope counting procedure of all samples was performed with a Packard-Auto Gamma Counter Model 52 10 at an energy peak of 320 keV. Gross counts were corrected for background to yield net cpm. At appropriate times, as indicated below, the mice were killed by cervical dislocation. The trachea was cannulated immediately, following skin incision, with polyethylene tubing (0.86-mm i.d.) to which a I-ml syringe containing this amount of saline solution was attached. The plunger was then pushed forward 0.15 ml and then drawn back 0.1 ml. After 20 strokes, the plunger was drawn back as far as possible, collecting 0.5 to 0.7 ml of the l-ml lavage fluid. The volume used for counting was adjusted to 0.5 ml.
Measurement
of
Pulmonary Blood Volume
Eight control mice were injected via the tail vein with 0.1 ml suspension of “Cr-labeled red blood cells. After injection, 10 mitt, animals were killed, and 50 ~1 of blood was drawn from the orbital venous plexus by the technique of Sorg and Buckner ( 1964). The thoracic cavity of each animal was carefully opened (to avoid hemorrhage as much as possible) and the lung was excised. Each lung was washed 10 times with 40 ml of saline each time to remove all traces of blood on the surfaces. The amounts of “Cr radioactivity in the lungs as well as in the blood samples were determined. From these values, the blood volume in the lung was calculated. The labeling of RBC was carried out by mixing 2 ml of whole blood drawn from four control mice with 0.3 ml of 5’CrCl, in citrate buffer, pH 7.4 (0.04 &ml, 100 pCi/ml). After 45 min ofincubation at room temperature. the mixture was centrifuged and the supematant fraction was discarded. The RBC in the pellet were washed three times with saline. After the final wash, the RBC pellet was suspended in saline to normal hematocrit prior to injection.
Measurement of Lung Water Content and Hematocrit Eight control mice were killed by cervical dislocation. Their lungs were removed and trimmed. The lungs were rinsed in saline, blotted, weighed before and after drying for 48 hr at 120°C. The water content was calculated from the weight loss. Hematocrit was determined by centrifugation in capillary tubes of blood samples obtained from the orbital venous plexus.
TECHNIQUE Determination
of the Distribution
TO EVALUATE
of “Cr-EDTA
in Blood
A group of four control mice were injected with 0.1 ml “Cr-EDTA and killed after 10 min. Blood samples were taken by cardiac puncture, and the amount of “CrEDTA was measured in whole blood and in plasma following centrifugation of the blood. Kinetic Studies of s’Cr-EDTA in Plasma and Extravascular Space in Normal Mice and Mice Exposed to HDIt
The variation with time of “Cr-EDTA in plasma and extravascular space of the lung following a 0. l-ml bolus tail vein injection, was determined at 10, 15, 20, 30, 40, 60, and 90 min after injection. Four normal mice were used for each time interval. Mice were killed by cervical dislocation, a blood sample was obtained via heart puncture, and the lungs were carefully removed from the thorax and extensively washed 10 times with 40 ml of saline each time. The amounts of “Cr-EDTA were then determined in the blood, plasma, and lung. By knowing the 5’CrEDTA concentration in the blood and the pulmonary blood volume, the amount of “0-EDTA in the extravascular space of the lung at various times alter 5’CrEDTA injections can be calculated. Concentrations of “Cr-EDTA in the plasma of normal mice were also measured at 1,2,3, and 5 min following “Cr-EDTA injection. The effect of HDIt exposure was studied in groups of four mice exposed at 18 to 24 mg/m3 for 4 hr with the exposure procedures described below. Two hours following exposures, “0-EDTA injections were performed. Determinations of “Cr-EDTA in blood, plasma, and lung were made at 10, 20, 40, 60, and 90 min following injection in the same manner as for control mice. Kinetic Studies of “Cr-EDTA in Lung Lavage Normal Mice and Mice Exposed to HDIt
Fluid
PULMONARY
EDEMA
463
prior studies (Weyel et af., 1982). Different aerosol concentrations were generated from 0.08 to 1% solutions (w/v) of HDIt in acetone preparedprior to each experiment. These solutions were fed with a syringe pump at 0.22 ml/min into a Pitt No. 1 aerosol generator as previously described (Weyel et al., 1982). The output of the generator was introduced into a glass exposure chamber where only the head of the animals protruded inside. The chamber was made of a glass cylinder with a volume of 4.6 liters. It was similar to the chamber described by Barrow et al. (1977) but twice the length in order to accommodate eight mice instead of four. The airtlow through the chamber was maintained at 20 liters/min for all experiments. The aerosol concentrations were evaluated gravimetrically by drawing known amounts of chamber air through preweighed 47-mm membrane filters, 0.4-pm pore size, from the Millipore Corp. (Bedford, Mass.). The particle size was evaluated with an Andersen mini impactor and was 0.6-pm aerodynamic equivalent diameter with a geometric standard deviation of 2.4, similar to prior experiments (Weyel et al., 1982). The concentration of acetone in the chamber was also similar to the results from the prior studies (Weyel et al.. 1982) varying between 2800 and 3000 ppm for all experiments. (a) Concentration-response relationships. Five groups of eight mice were exposed for 4 hr to HDIt aerosols at various concentrations (2.5 to 39 me/m’). Two hours following exposure, they were injected with “0-EDTA and killed 10 min afterwards. Lung lavages were performed as described above. Twenty nonexposed animals were used as control and one group of eight animals exposed to acetone alone at 3000 ppm was also used. (b) Time-response relationships. Five groups of eight animals were exposed to HDIt at 35 to 40 mg/m’ for 4 hr. Animals were injected with “Cr-EDTA, at 24, 33, 48, 72, and 240 hr after exposure. After injection, IO min, lung lavages were performed as described above.
in
RESULTS Groups of four normal mice were injected with 5’CrEDTA and killed at 10, 1520, 30,40, and 60 min after injection. Lung lavage was performed as described above, and the amount ofS’Cr-EDTA was determined. The same procedure was followed for groups of mice exposed to HDIt at 18 to 24 mg/m3 for a period of 4 hr. Two hours following exposure, “Cr-EDTA was injected and groups of four mice were killed at 10,20,40, and 60 min following injections.
Exposure
to HDIt
A commercial sample of HDIt (Desmodur-N, Des-N, supplied by Mobay Chemical Corporation, Pittsburgh, Pa.) was used containing 1.13% free hexamethylene diisocyanate. This supply was from the same batch used in
“Cr-EDTA in Lung Lavages following Exposure to HDIt at Various Concentrations The results for this series of experiments are presented in Table 1 and Fig. 1. There was more variation in the control (34% coefficient of variation) than desirable, probably due to the fact that recovery of 5’Cr-EDTA requires that two operations be performed correctly: injection of the tracer and adequate performance of lung lavages. Nevertheless a significant increase in “Cr-EDTA was observed for all HDIt exposure groups when compared to the nonexposed group used as a
464
VALENTINI,
WONG, AND ALARIE TABLE
1
AMOUNT OF “Cr-EDTA IN LUNG LAVAGES OF CONTROL ANIMALS, ANIMALS EXPOSED TO 3000 ppm ACETONE FOR 4 hr, AND ANIMALS EXFTXED TO VARIOUS CONCENTRATIONS OF HDIt AEROSOU FOR 4 hr AND KILLED 2 hr AFTER EXPOSURE
HDIt exposure concentration (mg/m’)
Number of animals
Nonexmsed (control) Acetoned 2.5 + Acetone 5.6 + Acetone 13 + Acetone 23 f Acetone 39 + Acetone
20 8 8 6’ 8 7 8
5’Cr-EDTA” (net cpm + SD) 2920 + 3860 k 41lO+ 6480 + 10950 k 14590 k 17140 f
980 1080 840 1550 2950 3250 3270
Ratio’ of exposed/control 1.3 1.4 2.2 3.8 5.0 5.9
Ratio’ of exposed/acetone 1.1
1.7 2.8 3.8 4.4
GIn 0.5 ml lung lavage fluid. The lavage was performed 10 min after injection. ’ The ratio of 5’Cr-EDTA in HDIt exposed mice vs controls. All groups exposed to HDIt had higher net cpm than the control group (p -=z0.05). ‘The ratio of “Cr-EDTA in HDIt exposed mice vs acetone-exposed mice. All groups exposed to HDIt had higher net cpm than the acetone group (p < 0.05) except the group exposed to 2.5 mg/m’. d Acetone was at 2800 to 3000 ppm for all groups. p When less than eight animals are reported, the results of one or two animals were omitted because of technical failure in 51Cr-EDTA injection or in performing lung lavages.
control group and for all groups above 2.5 mg/m3 when compared to the acetone aloneexposed group which can also be used as a control group. The results from two of eight exposed animals at 5.6 mg/m3 and one of eight animals exposed to 23 mg/m3 were omitted because of obvious technical failure in “CrEDTA injections or in performance of lung lavages. Although we did not expect any difference between the nonexposed control animals and acetone-exposed mice, the concentration-response curve was shifted to the right when the ratios of “Cr-EDTA in lung lavages of HDIt groups versus the acetone group were compared with that versus the nonexposed group (Fig. 1). For all sections below the term “control” will refer to normal animals rather than acetone-exposed animals. 5’Cr-EDTA in Lung Lavage Fluid at Dlflerent Times following Exposures to 35-40 mg/ m3 HDIt These results are presented in Fig. 2. It can be seen that the amount of “Cr-EDTA in
lung lavages, when 5’Cr-EDTA was injected at various times following exposures, declined slowly and returned close to control level after 2 days of exposure and remained at this level at 10 days postexposure. Kinetic Studies of “Cr-EDTA in Plasma of Normal Mice and Mice Exposed to HDIt The results of distribution of “Cr-EDTA following injection revealed that > 90% of “0-EDTA in blood was in the plasma. It was assumed that all 5’Cr-EDTA in blood is in the plasma for the kinetics described below. The results of concentrations of “Cr-EDTA in plasma of normal mice and mice exposed to HDIt, obtained at various times following injection of “0-EDTA, are presented in Fig. 3. To describe the time course of “Cr-EDTA concentrations in plasma of the control animals, a k-exponential equation was obtained with curve stripping. It can be seen that, although the initial time intervals were not studied in the group exposed to HDIt, the data are comparable to the control group. The
TECHNIQUE
TO EVALUATE
PULMONARY
465
EDEMA
6-
5-
4-
:3 $ Z0
Ir I
2
I I ,,,,ll
5
,
IO
20
HDI,(MG/
,
,,,,,,I
40
m
M3 1
FIG. I. The RATIO of the mean amount of “Cr-EDTA in lung lavages performed 10 min after “Cr-EDTA was injected 2 hr following an exposure to various concentrations of HDIt (6 to 8 mice at each concentration) versus the mean of acetone-exposed mice (8 mice) or normal mice (20 mice). For comparison purpose, the data from Weyel et al., (I 982) were included. The ratio of the lung weight of mice 24 hr after HDIt exposure versus that of control mice and the maximum respiratory frequency during HDIt exposure as a .fraction of the preexposure frequency were plotted from this reference. Linear regression lines were fitted with the least squares method.
IO
20
30
40
50 60 MINUTES
70
6.0
90
100
FIG. 3. Concentration of “0-EDTA in plasma at various times after injection of r’Cr-EDTA in control mice and mice exposed to HDIt at 18 to 24 mg/m’. The line was hand-drawn. The control data were fitted with an equation, y = 43.095e?‘.655’ + 9.085e-0.075’ + 6.748e-0.0’9’ by a curve stripping technique.
coefficient of variation for all data points was around 25%.
Kinetic Studiesof “Cr-EDTA in the Interstitial and Alveolar Spacesin Normal Mice and Mice Exposed to HDIt The pulmonary blood volume (PBV) in mice evaluated with labeled red blood cells was found to be 1.88 f 0.43 pi/g of body weight and the average volume of lung water (LW) was found to be 4.16 f 0.38 &g of body weight. Thus an “apparent pulmonary interstitial fluid volume” (PIV) was calculated to be 3.03 PI/g body weight by the expression PIV = LW - PBV( 1 - hematocrit).
TIME
(Days)
FIG. 2. The RATIO ofthe mean amount of “Cr-EDTA in lung lavages performed 10 min after “Cr-EDTA was injected at various times following a 4-hr exposure of HDIt at 40 mg/m3. The mean of normal mice was used as the denominator in the calculation of the RATIO.
This approach assumed that all the water in the lung was distributed between the plasma and the interstital compartment with negligible amount of water in the alveolar space and cells. For each animal from the control group and the group exposed to HDIt, knowing their respective body weights (BW), PBV, and the concentration of 5‘Cr-EDTA in plasma, [’ ‘Cr-
466
VALENTINI,
WONG, AND ALARIE
EDTA],, parameters about the interstitial and alveolar compartment (IAC) can be calculated: Amount
in IAC
= (counts in lung) - PBV - BW X ( 1 - hematocrit) - [’ ‘Cr-EDTA], Concentration
,
in IAC, 0
[“Cr-EDTA],*c
= AmiIy:
Kinetic Studies of “Cr-EDTA in Lung Lavage Fluid in Normal and HDIt-Exposed Animals The net counts of ‘Cr-EDTA in lung lavage fluid (0.5 ml) are presented in Fig. 5 for the controls and the group exposed to HDIt. The data points were fitted by least squares linear regression analysis and yielded a P/z of 15 min for the control group and 16 min for exposures to HDIt. “Cr-EDTA in lung lavage of the
0t
0 CONTROL 0 HOlt
30
40
50 60 MINUTES
70
20
60
I
so
30
40
50
60
70
MINUTES
The calculated values of [5’Cr-EDTA]iAc for controls and for animals exposed to HDIt are presented as a function of time after 5’CrEDTA injection in Fig. 4. The data points were fitted by least square linear regression analysis and yielded a P/z of 77 min for the exposed group and 105 min for the control group. The [“Cr-EDTA],,c was significantly (p < 0.05) higher in the HDIt exposed animals than in controls at all time intervals.
I=
IO
i?
100
FIG. 4. Concentration of “0-EDTA in the extravascular compartment in the lung of controls and mice exposed to 18-24 mg/m3 HDIt as a function of time after injection of SICr-EDTA. Linear mgression analysis resulted in y = 1.484 - O.O03t, r = -0.85 for controls and y 7 I .886 - O.O04f,r = -0.93 for the exposed group, where y = log (concn).
FIG. 5. The amount of “Cr-EDTA in lung lavage of controls and mice exposed to 18-24 mgJm3 HDIt at various times after iv injection of “Cr-EDTA. Linear regression analysis resulted in y = 3.623 - 0.046t and y = 4.088 - 0.044t for controls and HDIt group, respectively, where y = log (amount).
exposed group was significantly higher (p < 0.05) than for control group at all time intervals as found at the IO-min time intervals given in Table 1. Modeling Lung
of “Cr-EDTA
Distribution
in the
From the results given above a compartmental model can be suggested based on the following assumptions and findings: (a) After tail vein injection, 51Cr-EDTA distributes itself among three compartments in the lung: pulmonary blood, pulmonary interstitial space, and a layer of alveolar epithelium and pulmonary surfactant which can be defined as alveolar space. This view is a reasonable assumption considering that Volf et al. ( 197 1) presented data suggesting that EDTA distributes mainly in the extracellular water. (b) “Cr-EDTA behaves as a free compound in all three compartments, since it is known that binding of 5’ Cr-EDTA to plasma protein is negligible (Stacy and Thorburn, 1966; Garnett et al., 1967) and EDTA is not taken up by the red blood cells (Foreman et al., 1953; Gamett et al., 1967). (c) The concentration of “Cr-EDTA in the pulmonary interstitial and alveolar spaces [“Cr-EDTA]i,c can be calculated from the equation given above.
TECHNIQUETO EVALUATEPULMONARYEDEMA
467
systemof differential equationswas blood(plasma)is known at any onetime after solvedby a Runge-Kutta Vemer fifth- and (d) The concentration
of “0-EDTA
in
The
sixth-order method (Hull et al., 1976) which allowed for the calculation of the theoretical CB = [“Cr-EDTA],r,,, = 43.095e-0.65s’ values of [YI] and YII respectively at any time. The constants, ki, where i = l-4 and V,, were + 9.805e-0,075’ + 6.248e0--0’9’ > adjusted minimizing the squares of the difvalid for t > 1 min, from Fig. 3. Thus, the ferences between the experimental and theodistribution of 5’Cr-EDTA can be depicted as retical values. The procedure was performed shown in Fig. 6. The differential equations for the experimental values from the control describing the distribution of “Cr-EDTA as group as well as from the HDIt group. The a function of time would be numerical solutions of the above equations and the minimization procedure were carried d[YI]/dt = k, [CB] - k2 [YI] out on the DEC10 computer of the University + k4.YII/I’a - k3[YI] of Pittsburgh. The results are described in Fig. d[YII]/dt = kXIYI] - kq’YII/C/a; 7, comparing the experimental with the theoso retical value which were calculated with k, dYII/dt = k3 - VJYI] - k4 - YII from Table 2. The experimental and theoretical values agree very well. Both k, and k3 where were higher in the HDIt-exposed groups than [YI] = concentration of “Cr-EDTA in in- for the control group and more so for k3 than terstitial space = (amount in IAC k, . Thus according to the model, the greater - counts in lung lavage)/PIV . body amount of tracer in lung lavage of HDIt mice weight than in controls reflected an increased per[YII] = concentration of “Cr-EDTA in al- meability of the barrier between the interstiveolar space tium and alveolar space in HDIt mice. YII = amount of “Cr-EDTA in alveolar space = counts in lung lavage k, , k2, = first-order rate constants in min-’ DISCUSSION k k4 I’, = apparent volume of distribution of The most common and simple method of “0-EDTA in the alveolar space in measuring pulmonary edema is by the mea4. surement of wet lung weight increases. Unfortunately increases in wet lung weight are sensitive for only moderate or severe pulmonary edema and other methods will have to be used in the detection of minor pulmonary edema. Numerous methods have been developed for that purpose but they all suffer from some kind of drawbacks. For instance, the volume of extravascular water in lungs can be determined by either the indicator dilution method (Goresky et al., 1969) or the soluble inert gas method (Glauser et al., 1974; Peterson et al., 1978). However, the indicator dilution method is technically too complicated FIG. 6. Three-compartment model for the distribution of “Cr-EDTA in lungs. The symbols k14 represent the since it involves the cannulation of pulmonary rate constants for the transfer of “Cr-EDTA between the artery and aorta and serial blood sampling at compartments. l- or 2-set intervals, while the soluble inert injection
as given by the equation:
468
VALENTINI, WONG, AND ALARIE
10,000
et al., 1978) or injection into the right atrium (Harris et al., 1976) and serial blood sampling. o.-.o PREDICTED Comparatively speaking the method presented in this report is much simpler since only one radioactive tracer was used, making tracer analysis a simple matter and avoiding serial blood sampling. Not only is the present single-tracer method simple, but it is also very sensitive. This view becomes apparent when the present method 8. CONTROL MICE is compared with methods based on wet lung 0-o PREDICTED weight which was used by many investigators l + EXPERIMENTAL as a standard of comparison for any new method of detecting pulmonary edema (Goresky et al. 1 1969; Glauser et al. ( 1974; Peterson et al., 1978). Such a comparison is depicted in Fig. 1 in which the data of wet lung weight increases produced 24 hr after HDlt exposure, I I 1 1 I 1 1 taken from a report by Weyel et al. (1982). I 0 IO 20 30 40 50 60 TO MINUTES were included. The increase in “Cr-EDTA in FIG. 7. The amount of 5’Cr-EDTA in the alveolar space lung lavage was much more sensitive than inas predicted by the model versus the experimental data creases in wet lung weight in detecting the in (A) mice exposed to IS-24 mg/m’ HDIt; and(B) control pulmonary irritating effect of HDlt with the mice at various times after iv injection of “Cr-EDTA. concentration-response curve of the former For the HDIt-exposed mice, the model prediction was based on k, = 2.4 and k, = 6.6. while k, = 1.9 and k3 shifted one order of magnitude to the left compared with that based on increases in wet = 3.4 were used in the model for controls. lung weight. The “Cr-EDTA method can detect an increase in permeability at an HDlt gas method requires expensive analytical concentration that is not high enough to proequipment such as a mass spectrometer or duce gross pulmonary edema. else breath-holding maneuvers will need to be Alpert et al. ( 197 1), reported a method very performed on the test animals. In addition, similar to ours for the detection of pulmonary the soluble inert gas method consistently overestimates the volume of extravascular TABLE 2 water in lungs by 10 to 15% (Glauser et al., VALUESOFTHE FIRST-ORDER RATECONSTANTS 1974; Peterson et al., 1978). FOR THE DISTRIBUTION MODEL OF “0-EDTA IN Pulmonary edema can be classified, acCONTROL AND HDltExPosED ANIMALS cording to the cause, into two broad categories: hemodynamic edema and permeability A B edema. The methods mentioned above can k Control HDIt exposed’ WA detect edema due to either of the two causes. 2.40 1.26 1.90 There are no methods that will detect he- kb 0.28 0.31 1.10 kz modynamic pulmonary edema specifically, 6.60 1.94 3.40 kj but several methods specific for permeability 5.10 5.10 1.00 x-, 10.00 IO.00 1.00 pulmonary edema have been reported (Harris V,’ et al., 1976; Jones et al., 1978; Nelson et al., 0 Exposure at 18 to 24 mum’ for 4 hr. 1978). All of these procedures involve the adb .L were first-order rate constants in min-‘. ministration of more than one tracer via trac V. was the apparent volume of distribution of “Crcheal instillation (Jones et al., 1978; Nelson EDTA in the alveolar space in microliters. A.
t
HDlt
MICE
1
I
TECHNIQUE
TO
EVALUATE
edema produced by ozone except that 13’1albumin was used as the tracer instead of 51CrEDTA. Since ‘Cr-EDTA is a much smaller molecule than 13’1-albumin (MW 362 versus 60,000), we believe that our single-tracer method should be able to detect a smaller increase in permeability than the method using ‘311-albumin because a smaller tracer stands a better chance in transversing the diffusion barrier when small leaks start to appear leading to an increase in tracer in lung lavage that may not be detectable if a larger tracer such as albumin is used. We propose that 51Cr-EDTA distributes in the lung according to a three-compartment model which was also shown to be true for the distribution of radioactive albumin by Gee and Staub (1977). As shown in Table 2 and Fig. 7, the pulmonary distribution of “CrEDTA can be closely assimilated with the three-compartmental model when the transfer rate constants, k, and k2, were larger than k3 and k4, respectively. This finding indicates that for the transfer of “Cr-EDTA between pulmonary blood and the alveolar space the transport of “Cr-EDTA across the capillary endothelium is the rate limiting step. The capillary endothelium has also been assumed to be the major barrier for the distribution of urea in the lung by Brigham et al. ( 1979). Another evidence to support that transport across the capillary endothelium is rate limiting for the pulmonary distribution of “CrEDTA is that in the control mice the half-life of the decline of alveolar “Cr-EDTA was only 15 min (Fig. 5) compared with 105 min for the decline of “Cr-EDTA in the interstitial and alveolar spaces combined (Fig. 4). This result means that as 5’Cr-EDTA leaves the alveolar space to return to the blood it accumulates in the interstital space because the capillary endothelium is the major barrier for the re-entry of “Cr-EDTA into the capillaries. The slow rate of decline of “Cr-EDTA in the interstitial and alveolar spaces that we found in mice was comparable to that reported by Jones et al., (1978) in rabbits. The plasma level of “Cr-EDTA decreased with time in a n-i-exponential fashion with a
PULMONARY
EDEMA
469
short terminal half-life (approximately 32 min) in both control and HDIt- exposed mice indicating a rapid elimination. Since the plasma level in the HDIt mice coincided with that in controls, this finding ruled out the possibility that a greater amount of 51Cr-EDTA in the lung lavage of the HDIt mice was due to a higher plasma level. The model suggests that the greater amount in the lung lavage of the HDIt mice was a result of increased permeabilities in both the capillary endothelium and alveolar epithelium due to HDIt exposures. The increased permeabilities could be due to communicating channels not normally present being induced by pulmonary irritation caused by HDIt. This suggestion is a distinct possibility since it has been shown by Simani et al. (1974) that tight junctions in airway epithelium became freely permeable to horseradish peroxidase after inhalation of cigarette smoke in guinea pigs. The prediction by the model shown in Fig. 7 is based on the assumption that the volume of alveolar fluid (V,) in mice was 10 ~1. This assumption is comparable to the estimated volume of alveolar fluid in humans (Ma&in, 1954) on a per gram body weight basis. The assumption that the volumes of alveolar fluid were the same in controls and in mice exposed to HDIt at 18-24 mg/m3 is valid because the HDIt concentration was not high enough to produce alveolar flooding. The three-compartment model indicates that HDIt exposures make the capillary endothelium and alveolar epithelium more permeable which may lead to pulmonary edema. We conclude that the single-tracer technique with “Cr-EDTA is a simple and sensitive technique that can be used to detect even minor permeability pulmonary edema. ACKNOWLEDGEMENTS The authors wish to thank Professor H. S. Borovetz of the University of Pittsburgh, School of Medicine, for his helpful discussion regarding the experimental and computational phases of the present work. This work was supported under research Grant 1 ROl ES02747-01 from the National Institute of Environmental Health Sciences.
470
VALENTINI,
WONG, AND ALARIE
We also thank Dr. D. A. Weyel for his help in generation ofthe aerosol and Mrs. M. F. Stock for technical assistance and preparing the figures.
REFERENCES ALPERT, S., SCHWARTZ, B., LEE, S. D., AND LEWIS, T. (1971). Alveolar protein accumulation: A sensitive indicator of low level oxidant toxicity. Arch. Intern. Med. 28,69-73.
BARROW,C. S., ALARIE, T., WARRICK, J. C., AND STOCK, M. Y. (1977). Comparison of the sensory irritation response in mice to chlorine and hydrogen chloride. Arch. Environ.
Health
32, 68-76.
BRIGHAM, K. L., SNELL, J. D., JR., HARRIS, T. R., MARSHALL, S., HAYNES, J., BOWERS,R. E., ANDPERRY, J. ( 1979). Indicator dilution lung water and vascular permeability in humans. Cir. Res. 44, 523-530. CRONE, C. (1963). Permeability of capillaries in various organs determined by use of the indicator diffusion method. Acta Physiol. Stand. 58, 293-305. FOREMAN, H., VIER, M., AND MAGEE, M. (1953). The metabolism of C14-labelled ethylenediaminetetraacetic acid in the rat. J. Biol. Chem. 203, 1045-1053. GARNET-~, E. S., PARSONS, V., AND VEALL, N. (1967). Measurement of glomerular filtration-rate in man using a “Cr/edetic acid complex. Lancet i, 8 18-8 19. GEE, M. H. AND STAUB, N. C. (1977). Role of bulk tlow in protein permeability of the dog lung alveolar membrane. J. Appl. Physiol.: Respir. Environ. Exercise Physiol. 42, 144-149. GLAUSER, F. L., WILSON, A. F., HOSHIKO, M., WATANABE, AND DAVIS, J. (1974). Pulmonary parenchymal tissue (Vt) changes in pulmonary edema. .I. App/. Physiol. 36, 648-652.
GORESKY, C. A., CRONIN, R. F. P., AND WANGEL, B. E. ( 1969). Indicator dilution measurements of extravascular water in the lungs. J. Clin. Invest. 48, 487-501. HARRIS, T. R., ROWLET~, R. D., ANDBRINGHAM, K. L. (1976). The identification of pulmonary capillary permeability from multiple-indicator data. Microvasc. Res. 12, 177-196.
HULL, T. E., ENRIGHT, W. H., AND JACKSON, K. R. (1976). User’sguidefor DVERK. A subroutine for solving NON-STIFF ODE’5 TR No. 100. Department of Computer Science, University of Toronto. JONES,J. G., BERRY, M., HULANDS, G. H., ANDCRAWLEY, J. C. W. (1978). The time course and degree of change in alveolar-capillary membrane permeability induced by aspiration of hydrochloric acid and hypotonic saline. Amer. Rev. Respir. Dis. 118, 1007-1013. JONES,J. G., GROSSMAN, R. F., BERRY, M., SLAVIN, G., HULANDS, G. H., AND MINTY, B. (1979). Alveolarcapillary membrane permeability. Correlation with
functional, radiographic, and postmortem changes after fluid aspiration. Amer. Rev. Respir. Dis. 120, 399-4 10. JONES,J. G., MINTY, B. D., LAWLER, P., HULANDS, G.. CRAWLEY. J. C. W.. AND VEALL, N. (1980). Increased alveolar epithelial permeability in cigarette smokers. Lancet 8159. 66-68. LEVINE, 0. R., MELLINS, R. B., AND FISHMAN, A. P. (1965). Quantitative assessment of pulmonary edema. Cir. Res. 17, 4 14-426. MACKLIN, C. C. (1954). The pulmonary alveolar mucoid film and the pneumocytes. Lancet 1, 1099-I 104. MAITANI, T. AND SUZUKI. K. T. ( 198 1). Metal content ratio as a sensitive indicator of pulmonary edema. Taxicol. Lett.
9, 295-300.
MELLOR, D. P. ( 1964). In Chelating Agents and Metal Chelates (F. P. Dwyer and D. P. Mellor, eds.). p. 41. Academic Press, New York. MURPHY, J. J. (1980). Characterization ofpulmonary injury induced by oleic acid infusion. M. S. thesis. University of Pittsburgh. NELSON, R. M., MCINTYRE, 8. R., AND EGAN, E. A. (1978). Solute permeability of the alveolar epithelium in alloxan edema in dogs. J. Appl. Physiol.: Respir. Environ.
Exercise
Physiol.
44, 353-357.
PETERSON, B. T., PETRINI, M. F., HYDE, R. W., AND SCHREINER,B. F. (1978). Pulmonary tissue volume in dogs during pulmonary edema. J. Appl. Physiol.: Respir. Environ.
Exercise
Physiol.
44, 782-795.
SELGRADE, M., MOLE, L. M., MILLER, F. J., HATCH, G. E., AND GARDNER, D. E. (1981). Effect of Nor inhalation and vitamin C deficiency on protein and lipid accumulation in the lung. Environ. Res. 26,422437.
SIMANI, A. S., INOUE, S., AND HOGC, J. C. (1974). Penetration of the respiratory epithelium of guinea pigs following exposure to cigarette smoke. Lab. Invest. 31, 75-81.
SORG, D: A. AND BUCKNER, B. (1964). A simple method of obtaining venous blood from small laboratory animals. Proc. Sot. Exp. Biol. Med. 115, 113 l-l 132. STACY, B. D. AND THORBURN, G. D. (1966). Chromium5 1 ethylenediaminetetraacetate for estimation of glomerular filtration rate. Science 152, 1076- 1077. VOLF, V., VLADAR, M., AND SEIDEL, A.. (1971). Distribution of labelled calcium-yttrium- and chromiumchelates of EDTA in rats. Arch. Int. Pharmacodyn. 190, 110-123.
WALL, C., III, BOROVETZ, H., MURPHY, J. J., AND HARDESTY, R. (1981). System parameter identification in transport models using the fast fourier transform (FIT). Comp.
Biomed.
Res. 14, 670-68
1.
WEYEL. D. A., RODNEY, B. S., AND ALARIE, Y. (1982). Sensory irritation, pulmonary irritation, and acute lethality of a polymeric isocyanate and sensory irritation of 2,6-toluene diisocyanate. Toxicol. Appl. Pharmacol. 64, 423-430.