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The lung as a metabolic organ
The Lung as a M e t a b o l i c Organ Juan J. Touya, Javad Rahimian, Howard F. Corbus, David E. Grubbs, Katherine M. Savala, Edwin C. Glass, and Leslie R. Bennett Recently, the lung has received increasing attention as a metabolic organ. In this role, the lung modulates the composition of the arterial blood by several mechanisms: removing active substances from the plasma, releasing substances into the plasma, temp o r a r i l y holding substances from circulation, and activating or inactivating substances that pass through the lungs. In this report, the procedures proposed by different investigators for in vivo noninvasive assessment of the lung metabolic functions are reviewed. Most procedures are based on an estimation of the clearance of plasma amines by the lung endothelial cells. This clearance is assessed by measuring the lung uptake or the extraction fraction of an intravenously (IV) injected radiolabeled amine.
Our own procedure, which assesses the number of free pulmonary endothelial amine receptors, is discussed in detail. In our procedure, the number of receptors was computed using the number of injected molecules of amine and determining the lung extraction fraction of the amine during its first pass through the lungs. In goats, using N-isopropylp-iodoamphetamine labeled w i t h ~=Sl as the radiopharmaceutical, the total number of endothelial lung amine receptors was found to be 1.589 • 10=~ The methods for studying the lung metabolic functions, which are discussed in this report can be applied in humans to evaluate either physiological or pathological conditions.
HE LUNG, traditionally viewed as the
vasoactive substances and drugs between compartments were studied by measuring arterialvenous concentration differences in perfused lung preparations (isolated or in situ), in intact animals and in humans? 1 As early as 1975, Chinard 22 recognized that in normal and pathological conditions, lung metabolic functions could be assessed with higher accuracy and precision if invasiveness was avoided. For this purpose, he proposed the use of gamma-emitter radiotracers and external scintillation counters instead of beta-emitter tracers, direct sampling of the perfusate, and scintillation well counters. Although not proven, it has been postulated that alterations of the lung metabolic functions are responsible for a variety of unsuspected dise a s e s . 4'23'24 With the intention of exploring and demonstrating this hypothesis, investigators have been working on the design of noninvasive procedures for in vivo assessment of metabolic activities accomplished by each lung region and by both lungs as a whole organ. 23'25-29 The purpose of this report is to review the procedures proposed by different investigators for noninvasive assay of the lung metabolic functions, and to look for those that may have potential for identifying diseases caused by malfunctions of the lung as a metabolic organ. The specific lung metabolic function assayed by the different investigators, the parameters measured, and the radiolabeled amines used as tracers will be examined. Finally, the method used in
T body s respiratory organ, is receiving increasing attention as a metabolic organ. Aside from being responsible for the exchange of gases (OJCO2), the lung has been shown to regulate a variety of vasoactive circulating substances, such as amines, polypeptides, hormones, and drugs) -6 These lung activities, exercised with the purpose of modulating the plasma composition, are known as the lung metabolic functions. Table 1 lists ~variety of the plasma substances known to be released, removed, activated, inactivated, or modulated by the lungs. Until recently, the lung metabolic functions were mainly studied using invasive techniques. Enzymes and the metabolic pathways involved in the activities of the lung as a metabolic organ were explored in subcellular fractions, tissue slices, homogenates, minces, isolated cells, and cultured cells; the rates of exchange of natural
From the University of California, San Francisco-Fresno Central San Joaquin Valley Medical Education Program; Department of Nuclear Medicine, Saint Agnes Medical Center, Fresno; Department of Biology, California State University, Fresno; and Division of Nuclear Medicine, Department of Radiology, University of California, Los Angeles. Address reprint requests to Juan J. Touya MD, PhD, Department of Nuclear Medicine, Saint Agnes Medical Center, 1303 E Herndon, Fresno, CA 93710. 9 1986 by Grune & Stratton, Inc. 0001-2998/86/1604~004505.00/0
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9 1986 b y G r une & S t r a t t o n . Inc.
Seminars in Nuclear Medicine, Vol XVl, No 4 (October), 1986: pp 296-305
THE LUNG AS A METABOLIC ORGAN
our laboratory, the first-pass dual-indicator dilution technique in which the boluses are injected successively, will be discussed in detail. CLEARANCE OF AMINES
The lung is a complex organ with approximately 40 different constituent cell types, is The most thoroughly studied of all these cells are the epithelial cells, which line the air alveolar spaces, and the pulmonary endothelial cells, which line the functional blood vessels. The pulmonary endothelial cells, because of their direct and continuous access to the entire cardiac output, are considered to be involved in practically all the lung metabolic functions. This access to the whole cardiac output permits the cells to exert an instantaneous control over the chemical composition of the total arterial blood. Currently, most published noninvasive procedures for testing the metabolic functions of the lung limit the assay to the study of functions accomplished by the pulmonary endothelial cell membranes, specifically the clearance of active amines from the plasma. 23'25-27 The pulmonary clearance of active amines has been estimated by measuring the lung uptake 23"25 or the lung extraction fraction 26"3~of an intravenously (IV) injected radiolabeled amine.
Lung Uptake The lung uptake of amines has been defined as the percentage of the injected activity detected in the lungs at a predetermined time3 3 It has been proposed that lung uptake be computed as the ratio, at imaging time, of the counts in the lung fields over the counts in the whole body multiplied by 100.23
Lung Extraction Fraction The lung extraction fraction has been defined as the percentage of the injected radiotracer that is removed by the lung during a predefined time interval. The lung extraction fraction can be computed by several slightly different methods. 31'32 (t) Syrota et al computed the lung extraction fraction of chlorpromazine labeled with 1~C (HC-CPZ) by comparing the lung residue function of 1~C-CPZ with that of a reference vascular tracer. 26 We computed lung extraction fraction of N-isopropyl-p-iodoamphetamine la-
297
Table 1. Handling of Biologically Active Compounds by the Lungs Removed by the lungs Bradykin 7 Adenine nucleotides s Serotonin s Norepinephrine 1~ Prostaglandins E and FT M
Activated by the lungs Angiotensin I TM
Unaffected by passing through the lungs Epinephrine s Oopamine la Angiotensin I114
Vasopressin'S Prostaglandin A ~1
Released by the lungs under stimuli Prostaglandins, eg, prostacyclin16-~a Histamine 14.19 Slow reacting substance of anaphylaxis (SRS-A) z~
beled with ~23I (~23I-IMP) by comparing the impulse response function (IRF) of u3I-IMP with the IRF of a reference vascular tracer. 3~In both approaches, extraction fraction was computed using the following conventional formula: E(t) = [Hr(t) -- HR(t)]/[1 -- Hg(t)]
(1)
where the Hs were either the lung residue functions by Syrota's approach or the superior vena cava-lung IRFs in our approach. In both approaches, T refers to the test tracers (the radioactive-labeled amines), R refers to the reference tracers (the vascular indicators), and t are the time intervals. The information obtained by measuring the uptake or by determining the extraction fraction is similar; nevertheless, the extraction fraction determination is more precise and accurate. Uptake assesses the amount of amine that is retained in the lungs at the time of its measurement; therefore, if the amine has been metabolized in the lung during the interval between injection and imaging, its clearance is underestimated. The extraction fraction assesses the total amount of amine removed by the endothelial cells during the predefined interval of time; therefore, extraction fraction can be independent of the metabolic fate of the amines at the endothelial cell. Initially, most investigators did not determine
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the specific activity of the test tracer, a8'33 They computed uptake and extraction fraction as the percentage of the injected activity detected within the lungs without considering the amount of amine in the bolus. As previously demonstrated, 34 the amines used by most investigators are transported through the pulmonary endothelial cell membranes by saturable mechanisms. Therefore, lung uptake and extraction fraction of these amines are a function of the amount and concentration of the amine in the bolus. THE PULMONARY ENDOTHELIAL RECEPTORS
Many of the metabolic lung functions have a common anatomical and functional sites in the pulmonary endothelial cell membranes: the receptors. Receptors or binding sites are a concept conceived independently by Ehrlich 35 and Langley36 in the earliest part of this century to describe some of the physiological properties of the cell membranes. But, since its original definition, the term receptor has been used with different connotations. In pneumology and cardiology, receptor is generally used in a broader sense than in neurology and psychiatric pharmacology. Receptor, in its broadest sense, is defined as any macrobiomolecule that specifically binds a chemical agent, the ligand, from either inside or outside the cell. 37 Under this definition, a specific cellular recognition site that binds an amine, hormone, or drug is a receptor. In general, neurologists and psychiatric pharmacologists define receptors as macromolecular components that specifically bind a chemical agent, the ligand, and as a consequence of the binding, a known characteristic and predictable biological effect is produced. 3s'39 In a previous report, we proposed that the lung metabolic functions be assessed by in vivo radioassay of the pulmonary endothelial amine binding sites (receptors); 4~in 1985, we described the first procedure for their assay by combining algorithms from two of the most effective high technologies: in vitro radioimmunoassay and in vivo digital imaging) 4 The total number of pulmonary endothelial amine receptors can be assessed in vivo without disturbing the system physiology by a dual radiotracer dilution technique using 123I-IMP as test tracer and determining the number of molecules of IMP extracted by the lungs. The extraction
Fig 1. Goat chest scintigrams obtained at 2 0 seconds after administration of (A) a 0.6 mg bolus and (B) a 150 mg bolus of 12aI-IMP, 3 mCi per bolus. Lung over heart ratios w e r e computed using areas of interest, The ratios w e r e 3.02 in (A) and 1.64 in (B).
fraction of IMP, as shown in Figs 1 and 2, changes with the amount of IMP in the test tracer. When the number of molecules in the IMP bolus is increased, the number of molecules extracted by the lungs also increases. But the percentage of extracted molecules, the activity in the lungs, and the percentage of the injected activity that is detected in the lung, all diminish with the increase in the number of molecules in the bolus. The nonextracted molecules of IMP leave the pulmonary circulation at the same rate as the molecules of the reference tracer, which
THE LUNG AS A METABOLIC ORGAN
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Fig 2. Extracted and nonextracted I~I-IMP as function of the amount injected. This graph plots the results of 62 experiments carried out in goats. The triangles represent the percent of extracted IMP and the circles, the percent of nonextracted IMP. The line through the triangles represents the curve of bound IMP (the bound ligand [Z]) divided by the injected amount of IMP (the total ligand [L]), as a function of the injected amount of IMP (the total ligand [L]). The line through the circles, represents the curve of nonextracted IMP as a function of the amount injected, The amount of IMP passing through the lungs w i t h o u t being retained is considered to be the free ligand (X).
can be demonstrated by a detector placed over the left heart. Results of experiments carried out in goats unequivocally demonstrated that the lung extraction of IMP is done by a saturable carriermediated transport mechanism34 as defined by Oldendorf.4~As shown in Fig 3, when the amount of extracted IMP (Z) is plotted v the amount of injected IMP (L), the curve reaches a plateau at values of approximately 180 mg of IMP per bolus. This plateau indicates saturation of the pulmonary endothelial amine transport mechanisms, and saturation of the transport mechanisms mathematically validates the presence of IMP-amine receptors in the pulmonary endothelial membranes.
Algorithm for Assaying the Number of Pulmonary Endothelial Amine Receptors After determining the IMP extraction fraction and the amount of molecules of IMP in the bolus, the number of molecules of IMP extracted by the endothelial cells can be calculated as the product of the extraction fraction times the amount of IMP (in milligrams) in the bolus, times the
0
0 125 250 Total (L) or free (X) ligand mg
Fig 3. Dependence of the extracted amount of IMP (bound ligand [Z l) on the amount injected (total ligend [L]), and the relationship of the amount extracted with the amount that passes through (the free ligand Ix]). The amounts of 1=I-IMP were calculated in milligrams of IMP. The injected activity and the specific activity of the bolus were used to compute the amounts of bound, free and total IMP, in milligrams.
molecular weight (mol wt) of IMP. In a receptorligand binding system, the number of extracted IMP molecules can be considered to be the bound ligand (Z), the injected IMP, the total ligand (L), and the difference between the amounts injected and extracted--the free ligand (X). In this system, the dissociation constant of the IMPpulmonary endothelial amine-receptor binding reaction can be determined by plotting, in a Scatchard plot, the bound-to-free ligand ratios v the amounts of bound ligand (Fig 4). Knowing the dissociation constant of the amine-receptor binding reaction, the number of endothelial lung amine receptors can be computed by a variety of techniques. We computed the number of lung endothelial amine receptors as follows: NR = R x N/tool wt
(2)
where NR is the total number of receptors, R is the amount of bound amine (in grams) at the plateau of the curve of bound ligand v total injected ligand, and N is Avogadro's number. The lung endothelial amine receptors are the first step in the metabolic chain that controls some of the plasma amines (angiotensin II and bradykinin) with the potential for increasing or
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20
ance of the injected amine, as well as the action of the monoamine oxidase (MAO) on the cleared aliphatic amine, The ability of this technique to assess MAO activity, in addition to its efficiency for assaying the lung endothelial amine receptors, makes it a tool with potential for studying patients with schizophrenia, depression, and migraines.42In these diseases, although not proven, abnormalities in MAO activity have been implicated in the cause of the disturbances.
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Fig 4. Scatchard plot. In 62 experiments performed in goats, the amounts of retained IMP in the lungs were considered to be the bound ligand of a ligand-receptor binding reaction. Under the same conditions, the injected amounts of IMP were considered to be the total ligand. Results of these experiments were plotted for computing the dissociation constant of the IMP-lung endothelial amine receptors binding reaction. The dissociation constant was calculated as the inverse of the slope of the curve and found to be --16.077 rag.
decreasing BP. Therefore, the in vivo noninvasive assay of endothelial amine receptors is the most promising test for further investigation of biological functions that have never before been associated with the lung, THE FATE OF THE ACTIVE AMINES IN THE LUNG Following pulmonary endothelial uptake, amines can be metabolized or released back into the circulation. Little investigation has been done to determine in vivo the fate of the amines following endothelial uptake. Only two procedures have been published on this subject: the determination of the metabolic rate of amines in the lung and the lung release of amines,
Metabolic Rate of Amines in the Lungs Fowler et a123and Gallagher et a125 proposed that the metabolic rate of aliphatic amines could be assessed in the lungs by measuring the amounts of expired "C-CO2 following the IV administration of an aliphatic amine labeled with HC. In this condition, the amount of expired HC-CO: is a function of the lung plasma clear-
Lung Release of Amines Lung release of amines has been defined by Pistolesi et a129as the half-life of the lung washout-time activity curve following the IV administration of a radiolabeled amine, This parameter is simultaneously influenced by the lung uptake, metabolism and release of the amines. Preliminary clinical applications have shown that it is a sensitive index for identifying certain pulmonary diseases. It has been observed that the lung release of amines is prolonged in primary pulmonary hypertension and adult respiratory distress syndrome, whereas it is normal in patients with cardiogenic pulmonary edema, Although lacking specificity, because of the simplicity with which lung release of amines can be measured in humans, the procedure proposed by Pistolesi et al has potential for detecting, in clinical situations, disorders of the lung metabolic functions. RADIOLABELED AMINES USED TO ASSAY LUNG METABOLIC FUNCTIONS
The radioactive amines used by different investigators to explore the lung metabolic functions are listed in Table 2. Three of these amines are labeled with llC, and the other three with iodine: two with 123I and one with 131I. "Coctylamine (I~C-OA), HC-5-hydroxytryptamine (11C-5HT) and HC-CPZ are labeled with 11C, a Table 2. Radioamines Investigated as Lung Metabolic Imaging Agents 1 1 C - o c t y l a m i n e (11C~OA)23
11C_5_hydroxytryptamin e (1!C.5HT)2S 1C_chlorpromazine (11C_CPZ)ZS 12~lqsopropyl-iodoamphetamine (~2~!-IMP)=7 !231-trimethyl-hydroxy-lodobenzyl-propanediamine !231_HIPDM2S 13~l~meta-lodobenzyl.guanidine (13'I.M!BG) 47
THE LUNG AS A METABOLIC ORGAN
cyclotron-produced positron-emitting radionuelide. IIC-OA was selected as a possible lung metabolic imaging agent by Fowler et al, ~3 because of its potential for exploring the amine metabolic pathway far away from the uptake site at the pulmonary endothelial membrane. Unlike most biogenic amines, which are metabolized to water soluble products, aliphatic amines, such as octylamine, are ultimately metabolized to CO2. Therefore, when labeled with IIC, the rate at which they are metabolized can be measured by determining the amount of expired CO2 labeled with llC. HC-5HT was selected by Gatlagher et al es because it was the first natural vasoactive amine known to be removed from the plasma by the lungs. 43 Additionally, its removal process, metabolic pathway, and pharmacological interactions were probably the best known of all the natural amines. 14,44 I~C-CPZ was proposed by Syrota et al as a drug amine prototype, because of its known high tissue to blood ratios when injected into isolated perfused lungs. 26 Theoretically, the use of radiopharmaceuticals labeled with ~C, ~50, or 13N is advantageous, because natural amines, peptides, polypeptides, and drugs can be labeled without producing changes in their molecular structures. Small changes in their molecular structure are known to alter their biological behavior. However, the clinical use of radiopharmaceuticals labeled with ~1C is limited, because of the short physical half-life of "C (20.4 minutes) and the need for special imaging instrumentation not normally available at the community hospitals. Given the disadvantages of positron-labeled radiopharmaceuticals and the present limitations of 99roTe radiopharmaceutical design, many investigators considered it prudent in the beginning of the development of lung metabolic function tests, to use radioiodines as an alternative. The advantages of iodine 123 over iodine 131, when patient dose absortion and imaging resolution were considered, made ~23I-IMP, and 1231HIPDM the most extensively investigated radioamines. In 1981, we introduced Iz3I-IMp27 as an agent for tracing lung metabolic functions because of its initial high lung uptake when administered as a brain perfusion-metabolism imaging agent,
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and because of its availability as an approved drug for research in humans. Iz3I-HIPDM was proposed as another possible radioiodine-labeled metabolic lung imaging agent by Pistolesi et at 29 because it can be prepared by a simple exchange reaction and its biodistribution is similar to that of ~23I-IMP.45'~ Recently, meta-iodobenzyl-guanidine sulfate labeled with ~3~I(13q-MIBG) was tested in vitro as a lung metabolic agent by Slosman et al. Their results seem to demonstrate that the assessment of the MIBG lung extraction could be applied for in vivo detection of lung metabolic disorders;47 however, for in vivo studies MIBG labeled with 123Iappears to be more suitable than ~3q-MIBG. THE FIRST-PASS DUAL-INDICATOR DILUTION TECHNIQUE USING SUCCESSIVE INJECTIONS OF RADIOTRACERS
In 1982,4s we demonstrated the feasibility of measuring lung metabolic functions, in vivo and noninvasively, by the first-pass dual-indicator dilution technique using successive injections of a test tracer and a reference vascular tracer. This procedure is a modification of the Chinard and Enns 49 and Crone ~~ dual-indicator dilution method for analysis of the transport of substances through capillary membranes. The original procedure was modified with the purpose of creating a noninvasive procedure. Noninvasiveness was achieved using gamma-emitter radionuelide-labeled molecules as indicators and sampling their concentration in the different compartments by analysis of rapidly acquired digital images. Because the distribution of photons of different energies cannot be simultaneously imaged separately with standard instrumentation, a second modification was introduced into the original Chinard and Crone method. This second modification consisted of injecting the two radiotracers successively, instead of simultaneously. The adequacy of the successive injections of radiotracers had been previously demonstrated by Fazio et al. 5~
Radiopharmaceuticals When this procedure was applied in goats, we used a bolus of 20 mg of the synthetic amine 123I-IMP, with a specific activity of approximately 150 #Ci/1 mg and a concentration of approximately 1 mCi/1 mL, as test tracer; and a
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bolus of 10 mCi of either red cells labeled with 99mTC or dextran labeled with 99mTC (approximately 87,000 mol wt) as reference (vascular) tracer. The test tracer was injected first and the reference tracer 10 to 15 minutes later. Both tracers were injected in the same IV line. Data Acquisition Images were acquired during the injection of both tracers, in matrices of 64 • 64 • 8, as 0.2-second consecutive frames, for 60 seconds. The detector was positioned in order to visualize the superior vena cava or the right heart without interposition of lung parenchyma between one of these organs and the camera. Data Process The processing steps used to compute the extraction fraction and the number of free amine endothelial receptors were: (1) generation of the time activity curves of the two tracers; (2) removal of the background and noise; (3) computation of the impulse response functions; (4) calculation of extraction fraction; and (5) calculation of the number of receptors. Time activity curves of the test and reference tracers of the lung input (superior vena cava or right ventricle) and of the lung were generated by drawing areas of interest on the acquired images. In the reference and test tracers images, the same sites were sampled by using the same areas of interest in both sets of images. As the first curve processing step, the background produced by the spillover of the first tracer in the pulse height analyzer (PHA) window of the second tracer was subtracted. Following that, the noise was removed. The noise in the input time activity curves and in the lung curve of the reference tracer was removed by curve fitting with gamma variates or Stewart-Hamilton functions. The noise in the lung curve of the test tracer was removed by fitting with a polynomial function of order 7. sz The IRF for the two tracers were calculated by deconvolution of the input and the lung time activity curves using general Fourier technique. The deconvolution was performed constraining the IRF to a predefined monoexponential model. This constraint was initially used because of its simplicity for software implementation. But,
TOUYA ET AL
because the monoexponential modeling of the IRF is less accurate than the non-predefined modeling of the IRF obtained by general Fourier technique, especially for systems with high differences between the input and the output time activity curves, it was abandoned. The pulmonary extraction function of the test tracer was calculated using equation 1, where HT(t) and HR(t) are the normalized IRFs for the test and the reference tracers, respectively. The extraction fraction of the test tracer was determined at the time that 90% of the reference tracer had left the lung compartment. Accuracy and reproducibility of this technique were previously demonstrated in a group of experiments in which a mixture consisting of 99mTc-MAA (macroaggregates of human serum albumin) and 99mTc-dextran in different known proportions were used as test tracers, and boluses of pure 99mTc-dextran as the reference tracers. 53 The IRF of the test tracers showed two components, a fast and a slow slope. The fast slope was due to the washout of 99mTc-dextran from the lung fields and the slow component was due to the trapping (extraction) of 99mTc-MAA within the lung capillary bed. The total number of amine endothelial receptors can be calculated knowing the binding isotherms of the test tracer amine for the specie in consideration. The binding isotherms can be computer generated, using as variables the amounts of injected amine and the percent of free binding sites, and as constants, the dissociation constant of the amine-receptor binding reaction and the total number of binding sites in the lung. 34 FUTURE STUDIES AND APPLICATIONS
The first-pass dual-indicator dilution technique using successive injection of two boluses has permitted in vivo assay of receptors in animals. Currently, this assay is done assuming that equilibrium in the ligand-receptor binding reaction is achieved during the short time interval the bolus is in the lung capillary vessels. Research is in progress on algorithms for computing the number of lung amine endothelial receptors during the dynamic stages of the ligand-receptor binding reaction. Additionally, further investigation is in progress for determining the sensitivity
THE LUNG AS A METABOLIC ORGAN
and specificity of the lung endothelial receptors for the different amines. For clinical applications, the use of two simultaneous radiotracers could be a major inconvenience. Studies designed to analyze the sensitivity and reproducibility of a modified technique in which the IRF of the reference tracer is assumed to be a constant are needed. If these studies demonstrate that the lung extraction of the test tracer can be estimated with reasonable precision by measuring only the IRF of the radioamine test tracer, clinical research on lung metabolic functions could be accelerated. On the contrary, if results of this research are negative, it would be necessary to modify the cameras in order to separately register photons with different energies. Separate photon registration would permit simultaneous injection of more than one radiotracer. Disorders of the lung metabolic functions are considered to be the basic physiopathological abnormality in a variety of unsuspected diseases. The noninvasive assessment of these functions would become important in the diagnosis, followup, and monitoring of the treatments of patients with these diseases. Among those diseases that may be considered to benefit most from the noninvasive evaluation of the metabolic lung functions are: systemic blood hypertension, neonatal and adult respiratory distress syndromes, cystic fibrosis, asthma, and several psychiatric disorders such as depression, schizophrenia, and drug addiction)'4z54'-56 Interest in the lung metabolic functions has increased with the realization
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that the injured lung might influence distant organ function. Thoracic surgeons have learned that systemic manifestations of altered lung metabolic functions are characteristically produced in acute injury and need to be properly treated. 57 In addition, preliminary results of research done in our laboratory on smoke inhalation injuries seem to demonstrate that I2~I-IMP can estimate the degree of endothelial lung injury and can quantify the effects of drugs such as cimetidine on the smoke-injured animal. Endothelial receptors, other than those in the lung, could be assessed in vivo, using a technique similar to the one discussed above. Yet, the lung is the organ where assessment of receptors can be accomplished with the greatest ease, because the radioligand can be injected IV and arrives as an intact bolus to the lungs. To measure receptors in other organs, it will be necessary to inject the radioligands by a catheter placed in the input artery of the organ or to develop a technique to compute the organ input following the IV injection of the tracers) 4 Finally, we predict that further investigation to refine the techniques and to determine specific clinical applications will unveil new knowledge about the lung as a metabolic organ. ACKNOWLEDGMENT
The authors thank Albert Santibanez, Robert Hooten, Ermelinda Holgrein, and Juan P. Medina for their expert technical assistance during the experiments, and Tami Jo McKnight for her assistance in preparing the manuscript. Special thanks to Dr R. Baldwin from Medi-Physic for the supply of radiopharmaceuticals and numerous consultations.
REFERENCES
1. Porter R, Whelen J (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New Series. Amsterdam, Excerpta Medica, 1980 2. Tierney DF: Lung metabolism and biochemistry. Ann Rev Physiol 36:209-23t, 1974 3. Mellins RB: Fleischner lecture: Metabolic functions of the lung and their clinical relevance. A JR 138:999-1009, 1982 4. Block ER, Stalcup SA: Today's practice of cardiopulmonary medicine, metabolic functions of the lung; of what clinical relevance? Chest 81:215-223, 1982 5. Said SI: Metabolic functions of the pulmonary circulation. Circ Res 50:325-333, 1982 (suppl) 6. Ben-Harari RR, Youdim MBH: Commentary, the lung as an endocrine organ. Biochem Pharmacol 32:189-197, 1983
7. Ferreira SH, Vane JR: The disappearance of bradykinin and eledoisin in the circulation and vascular beds of the cat. Br J Pharmacol Chemother 30:417-424, 1967 8. Ryan US, Ryan JW: Correlation between the fine structure of the alveolar-capillary unit and its metabolic activities, in Vane Jr, Bakhle YS (eds): Metabolic functions of the lung. Vol 4 in Series on Lung Biology in Health and Disease, Lenfant C (ed). New York, Marcel Derker, 1977, p 197-232 9. Gaddum JH, Hebb CO, Silver A, Swan AAB: 5Hydroxytryptamine. Pharmacological action and destruction in perfused lungs. Q J Exp Physiol 38:225-262, 1953 10. Nicholas TE, Strum JM, Angelo LS, Junod AF: Site and mechanism of uptake of 3H-l-Norepinephrine by isolated perfused rat lungs. Circ Res 35:670~80, 1974 11. McGiff JC, Terragno NA, Strand JC, Lee JB, Loni-
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gro A J, Ng KKF: Selective passage of prostaglandins across the lung. Nature 216:762-766, 1969 12. Ng KKF, Vane JR: Fate of angiotension I in the circulation. Nature 218:144-150, 1968 13. Youdim MBH, Bakhle YS, Ben-Harari RR: Inactivation of monoamines by the lung, in Porter R, Whelen J (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New Series. Amsterdam, Excerpta Medica, 1980, pp 105-128 14. Vane JR: Introduction, in Porter R, Wheler j (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New Series. Amsterdam, Excerpta Medica, 1980, pp 1-10 15. Simionescu M: Ultrastructural organization of the alveolar-capillary unit, in Porter R, Whelon J (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New Series. Amsterdam, Excerpta Medica, 1980, pp 11-36 16. Armstrong JM, Lattimer N, Moncada S, Vane JR: Comparison of the vasodepressor effects of prostacyclin and 6-OXO-PGF~a with those PGE~ in rats and rabbits. Br J Pharmacol 62:125-130, 1978 17. Waldman HM, Alter I, Kot PA, Rose JC, Ramwell PW: Effect of lung transit on systemic depressor responses to arachidonic acid and prostaeyclin in dogs, J Phamacol Exp Ther 204:289-293, 1978 18. Dusting G J, Moncada S, Van R JR: Recirculation of prostacyclin (PGI~) in the dogs. Br J Phamacol 64:3 i 5-320, 1978 19. Krell RD, McCoy J, Christian P: Accumulation and metabolism of ['4C] histamine by rat lung in vivo. Biochem Pharmacol 27:820-821, 1978 20. Piper P J, Tippins JR, Samhoun MN, Morris HR, Taylor GW, Jones CM: SRS-A and its formation by the lung. Bull Eur Physiopathol Respir 17:571-583, 1981 21. Woods HF, Meredith A, Tucker GT, Shortland JR: Methods for the study of lung metabolism, in Porter R, Whelen J (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New series. Amsterdam, Excerpta Medica, 1980, pp 61-83 22. Chinard FP: Estimation of extravascular lung water by indicator-dilution techniques. Circ Res 37:137-145, 1975 23. Fowler JS, Gallagher BM, Mac Gregor RR, Wolf AP, Ansari AN, Atkins HL, Slatkin DN: Radiopharmaceuticals XIX. HC-labeled octylamine, A potential diagnostic agent for lung structure and function. J Nucl Med 17:752-754, 1976 24. Motutsky H J, Insel PA: Adrenergic receptors in man. Direct identification, physiologic regulation, and clinical alterations. N Engl J Med 307:18-29, 1982 25. Gallagher BM, Christman D, Fowler JS, MacGregor RR, Wolf AP, Sam P, Ansari AN, Atkins H: Radioisotope scintigraphy for the study of dynamics of amine regulation by the human lung. Chest 71:282-284, 1977 (suppl 2) 26. Syrota A, Pascal O, Crouzel M, Kellershohn C: Pulmonary extraction of C-11 chlorpromazine, measured by residue detection in man. J NucI Med 22:145-148, 1981 27. Rahimian J, Bennett LR, Touya J J, Akber SF: Metabolic lung scanning: Demonstration of amine receptors with N-isopropyl-I-123-iodoamphetamine. Clin Nucl Med 6:453, 1981 (abstr)
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28. Mena I, Thompson C, Mason G, Glass EC, Bennett LR: Lung first-pass extraction of N-isopropyl-I123-pamphetamine in patients with chronic obstructive pulmonary disease. Clin Nucl Med 9:35, 1984 29. Pistolesi M, Miniati M, Ghelarducci L, Mazzuca N, Giuntini C, Taccini E, Renzoni G, Pellegrini N, Gilardi MC, Fazio F: Lung release of HIPDM: A new index of lung dysfunction for clinical and experimental studies. J Nucl Med 26:14, 1985 30. Rahimian J, Glass EC, Touya J J, Akber SF, Graham LS, Bennett LR: Measurement of metabolic extraction of tracers in the lung using a multiple indicator dilution technique. J Nucl Med 25:31-37, 1984 31. Guller B, Yipimtsoi T, Orvis AL, Bassingthwaighte JB: Myocardial sodium extraction at varied coronary flows in the dog. Estimation of capillary permeability by residue and outflows detection. Circ Res 37:359-378, 1975 32. Lassen NA, Perl W: Tracer Kinetic Methods in Medical Physiology. New York, Raven, 1979 33. Akber SF, Glass EC, Bennett LR: Scintigraphic demonstration and quantitation of pharmacologic inhibition of pulmonary uptake of N-isopropyl-I-123-p-iodoamphetamine by propranolol. J Nucl Med 26:14, 1985 34. Touya J J, Rahimian J, Grubbs DE, Corbus HF, Bennett LR: A noninvasive procedure for in vivo assay of a lung amine endothelial receptor. J Nucl Med 26:1302-1307, 1985 35. Ehrlich P: Chemotherapeutics: Scientific principles, methods, and results. Lancet 2:445, 1913 36. Langely JN: On the contraction of muscle, chiefly in relation to the presence of "receptive" substances. Part I. J Physiol (Lond) 36:347-384, 1907 37. Lefkowitz R J, Caron MG, Stiles GL: Mechanism of membrane receptor regulation, biochemical, physiological, and clinical insights derived from studies of the adrenergie receptors. N Engl J Med 310:1570-1579, 1984 38. Schafer DE: Measurement of receptor-ligand binding theory and practice, in Lambrecht RM, Rescingo A (eds): Tracer Kinetics and Physiological Modeling. New York, Springer-Verlag, 1983, pp 445-507 39. Lin MT, Chan HK, Chen CF, Teh GW: Involvement of both opiate and catecholaminergic receptors in the behavioral excitation provoked by thyrotropin-releasing hormone: Comparisons with amphetamine. Neuropharmacol 22:463469, 1983 40. Rahimian J, Bennett LR, Grubbs DE, Glass EC, Corbus HF, Touya J J: A noninvasive procedure to measure in vivo lung endothelial receptors. Clin Nucl Med 9:36, 1984 41. Oldendorf WH: The blood-brain barrier. Exp Eye Res 25:177-190, 1977 (suppl) 42. Wyatt R J, Murphy DL, et al: Reduced monoamine oxidase activity in platelets: A possible genetic marker for vulnerability to schizophrenia. Science 179:916-918, 1973 43. Starling EH, Verney EB: The secretion of urine as studied on the isolated kidney. Proc R Soc Lond B Biol Sci 97:321-363, 1925 44. Gillis CN, Cronau LH, Mandel S, Hammond GL: Indicator dilution measurement of 5-Hydroxytryptamine clearance by human lung. J Appl Physiol 46:1178-1183, I979
THE LUNG AS A METABOLIC ORGAN
45. Kung HF, Tramposch KM, Blau M: A new brain perfusion agent: [I-123] HIPDM: N, N, N'-trimethyl-N'[2-hydroxy-3-methyl-5-iodobenzyl]- 1,3-propanediamine. J Nucl Med 24:66-72, 1983 46. Holman BL, Lee RGL, Hill TC, Lovett RD, ListerJames J: A comparison of two cerebral perfusion tracers, N-isopropyl-I-123-p-iodoamphetamine and 1-123, HIPDM, in the human. J Nucl Med 25:25-30, 1984 47. Slosman D, Davidson D, Brill ABK, Alderson PO: Pulmonary accumulation of 1-131-MIBG in the isolated perfused rat lung. J Nucl Med 27:1076, 1986 48. Rahimian J, Glass EC, Touya J J, Akber SF, Graham LS, Bennett LR: Extraction fraction: Noninvasive determination by dual tracer deconvolution analysis. Clin Nucl Med 7:31, 1982 49. Chinard EP, Enns T: Transcapillary pulmonary exchange of water in the dog. Am J Physiol 178:197-202, 1954 50. Crone C: The permeability of capillaries in various organs as determined by use of the "indicator diffusion" method. Acta Physiol Scand 58:292-305, 1963
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51. Fazio F, Jones T, Jones H, Rhodes CG, Hughes JMB: External counting for measuring pulmonary oodema. J Nucl Biol MOd 20:81-83, 1976 52. Rahimian J: Quantitative Assessment of Pulmonary Metabolic Extraction of Tracers Using Multiple Indicator Dilution Technique and Deconvolution Analysis. PhD Dissertation in Medical Physics, University of California, Los Angeles, 1983 53. Rahimian J, Grubbs DE, Gregory R, Santibanez A, Bennett LR, Glass EC, Corbus HF, Touya J J: Accuracy of noninvasive dual tracer technique for measuring pulmonary extraction of plasma substances. MOd Physics 12:673, 1985 54. Bockar J, Roth R, Heninger G: Increased human platelet monoamine-oxidase activity during lithium carbonate therapy. Life Sci 15:2109-2118, 1975 55. Sandier M, Youdim MB, et al: A phenylethylamine oxidizing defect in migraine. Nature 250:335-337, 1974 56. Axelrod J, Weinshilboum R: Catecholamines. N Engl J Med 287:237-242, 1972 57. Addonizio VP, Harken AH: The surgical implications of nonrespiratory lung function. J Surg Res 28:86-95, 1980
Our own procedure, which assesses the number of free pulmonary endothelial amine receptors, is discussed in detail. In our procedure, the number of receptors was computed using the number of injected molecules of amine and determining the lung extraction fraction of the amine during its first pass through the lungs. In goats, using N-isopropylp-iodoamphetamine labeled w i t h ~=Sl as the radiopharmaceutical, the total number of endothelial lung amine receptors was found to be 1.589 • 10=~ The methods for studying the lung metabolic functions, which are discussed in this report can be applied in humans to evaluate either physiological or pathological conditions.
HE LUNG, traditionally viewed as the
vasoactive substances and drugs between compartments were studied by measuring arterialvenous concentration differences in perfused lung preparations (isolated or in situ), in intact animals and in humans? 1 As early as 1975, Chinard 22 recognized that in normal and pathological conditions, lung metabolic functions could be assessed with higher accuracy and precision if invasiveness was avoided. For this purpose, he proposed the use of gamma-emitter radiotracers and external scintillation counters instead of beta-emitter tracers, direct sampling of the perfusate, and scintillation well counters. Although not proven, it has been postulated that alterations of the lung metabolic functions are responsible for a variety of unsuspected dise a s e s . 4'23'24 With the intention of exploring and demonstrating this hypothesis, investigators have been working on the design of noninvasive procedures for in vivo assessment of metabolic activities accomplished by each lung region and by both lungs as a whole organ. 23'25-29 The purpose of this report is to review the procedures proposed by different investigators for noninvasive assay of the lung metabolic functions, and to look for those that may have potential for identifying diseases caused by malfunctions of the lung as a metabolic organ. The specific lung metabolic function assayed by the different investigators, the parameters measured, and the radiolabeled amines used as tracers will be examined. Finally, the method used in
T body s respiratory organ, is receiving increasing attention as a metabolic organ. Aside from being responsible for the exchange of gases (OJCO2), the lung has been shown to regulate a variety of vasoactive circulating substances, such as amines, polypeptides, hormones, and drugs) -6 These lung activities, exercised with the purpose of modulating the plasma composition, are known as the lung metabolic functions. Table 1 lists ~variety of the plasma substances known to be released, removed, activated, inactivated, or modulated by the lungs. Until recently, the lung metabolic functions were mainly studied using invasive techniques. Enzymes and the metabolic pathways involved in the activities of the lung as a metabolic organ were explored in subcellular fractions, tissue slices, homogenates, minces, isolated cells, and cultured cells; the rates of exchange of natural
From the University of California, San Francisco-Fresno Central San Joaquin Valley Medical Education Program; Department of Nuclear Medicine, Saint Agnes Medical Center, Fresno; Department of Biology, California State University, Fresno; and Division of Nuclear Medicine, Department of Radiology, University of California, Los Angeles. Address reprint requests to Juan J. Touya MD, PhD, Department of Nuclear Medicine, Saint Agnes Medical Center, 1303 E Herndon, Fresno, CA 93710. 9 1986 by Grune & Stratton, Inc. 0001-2998/86/1604~004505.00/0
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9 1986 b y G r une & S t r a t t o n . Inc.
Seminars in Nuclear Medicine, Vol XVl, No 4 (October), 1986: pp 296-305
THE LUNG AS A METABOLIC ORGAN
our laboratory, the first-pass dual-indicator dilution technique in which the boluses are injected successively, will be discussed in detail. CLEARANCE OF AMINES
The lung is a complex organ with approximately 40 different constituent cell types, is The most thoroughly studied of all these cells are the epithelial cells, which line the air alveolar spaces, and the pulmonary endothelial cells, which line the functional blood vessels. The pulmonary endothelial cells, because of their direct and continuous access to the entire cardiac output, are considered to be involved in practically all the lung metabolic functions. This access to the whole cardiac output permits the cells to exert an instantaneous control over the chemical composition of the total arterial blood. Currently, most published noninvasive procedures for testing the metabolic functions of the lung limit the assay to the study of functions accomplished by the pulmonary endothelial cell membranes, specifically the clearance of active amines from the plasma. 23'25-27 The pulmonary clearance of active amines has been estimated by measuring the lung uptake 23"25 or the lung extraction fraction 26"3~of an intravenously (IV) injected radiolabeled amine.
Lung Uptake The lung uptake of amines has been defined as the percentage of the injected activity detected in the lungs at a predetermined time3 3 It has been proposed that lung uptake be computed as the ratio, at imaging time, of the counts in the lung fields over the counts in the whole body multiplied by 100.23
Lung Extraction Fraction The lung extraction fraction has been defined as the percentage of the injected radiotracer that is removed by the lung during a predefined time interval. The lung extraction fraction can be computed by several slightly different methods. 31'32 (t) Syrota et al computed the lung extraction fraction of chlorpromazine labeled with 1~C (HC-CPZ) by comparing the lung residue function of 1~C-CPZ with that of a reference vascular tracer. 26 We computed lung extraction fraction of N-isopropyl-p-iodoamphetamine la-
297
Table 1. Handling of Biologically Active Compounds by the Lungs Removed by the lungs Bradykin 7 Adenine nucleotides s Serotonin s Norepinephrine 1~ Prostaglandins E and FT M
Activated by the lungs Angiotensin I TM
Unaffected by passing through the lungs Epinephrine s Oopamine la Angiotensin I114
Vasopressin'S Prostaglandin A ~1
Released by the lungs under stimuli Prostaglandins, eg, prostacyclin16-~a Histamine 14.19 Slow reacting substance of anaphylaxis (SRS-A) z~
beled with ~23I (~23I-IMP) by comparing the impulse response function (IRF) of u3I-IMP with the IRF of a reference vascular tracer. 3~In both approaches, extraction fraction was computed using the following conventional formula: E(t) = [Hr(t) -- HR(t)]/[1 -- Hg(t)]
(1)
where the Hs were either the lung residue functions by Syrota's approach or the superior vena cava-lung IRFs in our approach. In both approaches, T refers to the test tracers (the radioactive-labeled amines), R refers to the reference tracers (the vascular indicators), and t are the time intervals. The information obtained by measuring the uptake or by determining the extraction fraction is similar; nevertheless, the extraction fraction determination is more precise and accurate. Uptake assesses the amount of amine that is retained in the lungs at the time of its measurement; therefore, if the amine has been metabolized in the lung during the interval between injection and imaging, its clearance is underestimated. The extraction fraction assesses the total amount of amine removed by the endothelial cells during the predefined interval of time; therefore, extraction fraction can be independent of the metabolic fate of the amines at the endothelial cell. Initially, most investigators did not determine
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the specific activity of the test tracer, a8'33 They computed uptake and extraction fraction as the percentage of the injected activity detected within the lungs without considering the amount of amine in the bolus. As previously demonstrated, 34 the amines used by most investigators are transported through the pulmonary endothelial cell membranes by saturable mechanisms. Therefore, lung uptake and extraction fraction of these amines are a function of the amount and concentration of the amine in the bolus. THE PULMONARY ENDOTHELIAL RECEPTORS
Many of the metabolic lung functions have a common anatomical and functional sites in the pulmonary endothelial cell membranes: the receptors. Receptors or binding sites are a concept conceived independently by Ehrlich 35 and Langley36 in the earliest part of this century to describe some of the physiological properties of the cell membranes. But, since its original definition, the term receptor has been used with different connotations. In pneumology and cardiology, receptor is generally used in a broader sense than in neurology and psychiatric pharmacology. Receptor, in its broadest sense, is defined as any macrobiomolecule that specifically binds a chemical agent, the ligand, from either inside or outside the cell. 37 Under this definition, a specific cellular recognition site that binds an amine, hormone, or drug is a receptor. In general, neurologists and psychiatric pharmacologists define receptors as macromolecular components that specifically bind a chemical agent, the ligand, and as a consequence of the binding, a known characteristic and predictable biological effect is produced. 3s'39 In a previous report, we proposed that the lung metabolic functions be assessed by in vivo radioassay of the pulmonary endothelial amine binding sites (receptors); 4~in 1985, we described the first procedure for their assay by combining algorithms from two of the most effective high technologies: in vitro radioimmunoassay and in vivo digital imaging) 4 The total number of pulmonary endothelial amine receptors can be assessed in vivo without disturbing the system physiology by a dual radiotracer dilution technique using 123I-IMP as test tracer and determining the number of molecules of IMP extracted by the lungs. The extraction
Fig 1. Goat chest scintigrams obtained at 2 0 seconds after administration of (A) a 0.6 mg bolus and (B) a 150 mg bolus of 12aI-IMP, 3 mCi per bolus. Lung over heart ratios w e r e computed using areas of interest, The ratios w e r e 3.02 in (A) and 1.64 in (B).
fraction of IMP, as shown in Figs 1 and 2, changes with the amount of IMP in the test tracer. When the number of molecules in the IMP bolus is increased, the number of molecules extracted by the lungs also increases. But the percentage of extracted molecules, the activity in the lungs, and the percentage of the injected activity that is detected in the lung, all diminish with the increase in the number of molecules in the bolus. The nonextracted molecules of IMP leave the pulmonary circulation at the same rate as the molecules of the reference tracer, which
THE LUNG AS A METABOLIC ORGAN
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Fig 2. Extracted and nonextracted I~I-IMP as function of the amount injected. This graph plots the results of 62 experiments carried out in goats. The triangles represent the percent of extracted IMP and the circles, the percent of nonextracted IMP. The line through the triangles represents the curve of bound IMP (the bound ligand [Z]) divided by the injected amount of IMP (the total ligand [L]), as a function of the injected amount of IMP (the total ligand [L]). The line through the circles, represents the curve of nonextracted IMP as a function of the amount injected, The amount of IMP passing through the lungs w i t h o u t being retained is considered to be the free ligand (X).
can be demonstrated by a detector placed over the left heart. Results of experiments carried out in goats unequivocally demonstrated that the lung extraction of IMP is done by a saturable carriermediated transport mechanism34 as defined by Oldendorf.4~As shown in Fig 3, when the amount of extracted IMP (Z) is plotted v the amount of injected IMP (L), the curve reaches a plateau at values of approximately 180 mg of IMP per bolus. This plateau indicates saturation of the pulmonary endothelial amine transport mechanisms, and saturation of the transport mechanisms mathematically validates the presence of IMP-amine receptors in the pulmonary endothelial membranes.
Algorithm for Assaying the Number of Pulmonary Endothelial Amine Receptors After determining the IMP extraction fraction and the amount of molecules of IMP in the bolus, the number of molecules of IMP extracted by the endothelial cells can be calculated as the product of the extraction fraction times the amount of IMP (in milligrams) in the bolus, times the
0
0 125 250 Total (L) or free (X) ligand mg
Fig 3. Dependence of the extracted amount of IMP (bound ligand [Z l) on the amount injected (total ligend [L]), and the relationship of the amount extracted with the amount that passes through (the free ligand Ix]). The amounts of 1=I-IMP were calculated in milligrams of IMP. The injected activity and the specific activity of the bolus were used to compute the amounts of bound, free and total IMP, in milligrams.
molecular weight (mol wt) of IMP. In a receptorligand binding system, the number of extracted IMP molecules can be considered to be the bound ligand (Z), the injected IMP, the total ligand (L), and the difference between the amounts injected and extracted--the free ligand (X). In this system, the dissociation constant of the IMPpulmonary endothelial amine-receptor binding reaction can be determined by plotting, in a Scatchard plot, the bound-to-free ligand ratios v the amounts of bound ligand (Fig 4). Knowing the dissociation constant of the amine-receptor binding reaction, the number of endothelial lung amine receptors can be computed by a variety of techniques. We computed the number of lung endothelial amine receptors as follows: NR = R x N/tool wt
(2)
where NR is the total number of receptors, R is the amount of bound amine (in grams) at the plateau of the curve of bound ligand v total injected ligand, and N is Avogadro's number. The lung endothelial amine receptors are the first step in the metabolic chain that controls some of the plasma amines (angiotensin II and bradykinin) with the potential for increasing or
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TOUYA ET AL
20
ance of the injected amine, as well as the action of the monoamine oxidase (MAO) on the cleared aliphatic amine, The ability of this technique to assess MAO activity, in addition to its efficiency for assaying the lung endothelial amine receptors, makes it a tool with potential for studying patients with schizophrenia, depression, and migraines.42In these diseases, although not proven, abnormalities in MAO activity have been implicated in the cause of the disturbances.
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Fig 4. Scatchard plot. In 62 experiments performed in goats, the amounts of retained IMP in the lungs were considered to be the bound ligand of a ligand-receptor binding reaction. Under the same conditions, the injected amounts of IMP were considered to be the total ligand. Results of these experiments were plotted for computing the dissociation constant of the IMP-lung endothelial amine receptors binding reaction. The dissociation constant was calculated as the inverse of the slope of the curve and found to be --16.077 rag.
decreasing BP. Therefore, the in vivo noninvasive assay of endothelial amine receptors is the most promising test for further investigation of biological functions that have never before been associated with the lung, THE FATE OF THE ACTIVE AMINES IN THE LUNG Following pulmonary endothelial uptake, amines can be metabolized or released back into the circulation. Little investigation has been done to determine in vivo the fate of the amines following endothelial uptake. Only two procedures have been published on this subject: the determination of the metabolic rate of amines in the lung and the lung release of amines,
Metabolic Rate of Amines in the Lungs Fowler et a123and Gallagher et a125 proposed that the metabolic rate of aliphatic amines could be assessed in the lungs by measuring the amounts of expired "C-CO2 following the IV administration of an aliphatic amine labeled with HC. In this condition, the amount of expired HC-CO: is a function of the lung plasma clear-
Lung Release of Amines Lung release of amines has been defined by Pistolesi et a129as the half-life of the lung washout-time activity curve following the IV administration of a radiolabeled amine, This parameter is simultaneously influenced by the lung uptake, metabolism and release of the amines. Preliminary clinical applications have shown that it is a sensitive index for identifying certain pulmonary diseases. It has been observed that the lung release of amines is prolonged in primary pulmonary hypertension and adult respiratory distress syndrome, whereas it is normal in patients with cardiogenic pulmonary edema, Although lacking specificity, because of the simplicity with which lung release of amines can be measured in humans, the procedure proposed by Pistolesi et al has potential for detecting, in clinical situations, disorders of the lung metabolic functions. RADIOLABELED AMINES USED TO ASSAY LUNG METABOLIC FUNCTIONS
The radioactive amines used by different investigators to explore the lung metabolic functions are listed in Table 2. Three of these amines are labeled with llC, and the other three with iodine: two with 123I and one with 131I. "Coctylamine (I~C-OA), HC-5-hydroxytryptamine (11C-5HT) and HC-CPZ are labeled with 11C, a Table 2. Radioamines Investigated as Lung Metabolic Imaging Agents 1 1 C - o c t y l a m i n e (11C~OA)23
11C_5_hydroxytryptamin e (1!C.5HT)2S 1C_chlorpromazine (11C_CPZ)ZS 12~lqsopropyl-iodoamphetamine (~2~!-IMP)=7 !231-trimethyl-hydroxy-lodobenzyl-propanediamine !231_HIPDM2S 13~l~meta-lodobenzyl.guanidine (13'I.M!BG) 47
THE LUNG AS A METABOLIC ORGAN
cyclotron-produced positron-emitting radionuelide. IIC-OA was selected as a possible lung metabolic imaging agent by Fowler et al, ~3 because of its potential for exploring the amine metabolic pathway far away from the uptake site at the pulmonary endothelial membrane. Unlike most biogenic amines, which are metabolized to water soluble products, aliphatic amines, such as octylamine, are ultimately metabolized to CO2. Therefore, when labeled with IIC, the rate at which they are metabolized can be measured by determining the amount of expired CO2 labeled with llC. HC-5HT was selected by Gatlagher et al es because it was the first natural vasoactive amine known to be removed from the plasma by the lungs. 43 Additionally, its removal process, metabolic pathway, and pharmacological interactions were probably the best known of all the natural amines. 14,44 I~C-CPZ was proposed by Syrota et al as a drug amine prototype, because of its known high tissue to blood ratios when injected into isolated perfused lungs. 26 Theoretically, the use of radiopharmaceuticals labeled with ~C, ~50, or 13N is advantageous, because natural amines, peptides, polypeptides, and drugs can be labeled without producing changes in their molecular structures. Small changes in their molecular structure are known to alter their biological behavior. However, the clinical use of radiopharmaceuticals labeled with ~1C is limited, because of the short physical half-life of "C (20.4 minutes) and the need for special imaging instrumentation not normally available at the community hospitals. Given the disadvantages of positron-labeled radiopharmaceuticals and the present limitations of 99roTe radiopharmaceutical design, many investigators considered it prudent in the beginning of the development of lung metabolic function tests, to use radioiodines as an alternative. The advantages of iodine 123 over iodine 131, when patient dose absortion and imaging resolution were considered, made ~23I-IMP, and 1231HIPDM the most extensively investigated radioamines. In 1981, we introduced Iz3I-IMp27 as an agent for tracing lung metabolic functions because of its initial high lung uptake when administered as a brain perfusion-metabolism imaging agent,
301
and because of its availability as an approved drug for research in humans. Iz3I-HIPDM was proposed as another possible radioiodine-labeled metabolic lung imaging agent by Pistolesi et at 29 because it can be prepared by a simple exchange reaction and its biodistribution is similar to that of ~23I-IMP.45'~ Recently, meta-iodobenzyl-guanidine sulfate labeled with ~3~I(13q-MIBG) was tested in vitro as a lung metabolic agent by Slosman et al. Their results seem to demonstrate that the assessment of the MIBG lung extraction could be applied for in vivo detection of lung metabolic disorders;47 however, for in vivo studies MIBG labeled with 123Iappears to be more suitable than ~3q-MIBG. THE FIRST-PASS DUAL-INDICATOR DILUTION TECHNIQUE USING SUCCESSIVE INJECTIONS OF RADIOTRACERS
In 1982,4s we demonstrated the feasibility of measuring lung metabolic functions, in vivo and noninvasively, by the first-pass dual-indicator dilution technique using successive injections of a test tracer and a reference vascular tracer. This procedure is a modification of the Chinard and Enns 49 and Crone ~~ dual-indicator dilution method for analysis of the transport of substances through capillary membranes. The original procedure was modified with the purpose of creating a noninvasive procedure. Noninvasiveness was achieved using gamma-emitter radionuelide-labeled molecules as indicators and sampling their concentration in the different compartments by analysis of rapidly acquired digital images. Because the distribution of photons of different energies cannot be simultaneously imaged separately with standard instrumentation, a second modification was introduced into the original Chinard and Crone method. This second modification consisted of injecting the two radiotracers successively, instead of simultaneously. The adequacy of the successive injections of radiotracers had been previously demonstrated by Fazio et al. 5~
Radiopharmaceuticals When this procedure was applied in goats, we used a bolus of 20 mg of the synthetic amine 123I-IMP, with a specific activity of approximately 150 #Ci/1 mg and a concentration of approximately 1 mCi/1 mL, as test tracer; and a
302
bolus of 10 mCi of either red cells labeled with 99mTC or dextran labeled with 99mTC (approximately 87,000 mol wt) as reference (vascular) tracer. The test tracer was injected first and the reference tracer 10 to 15 minutes later. Both tracers were injected in the same IV line. Data Acquisition Images were acquired during the injection of both tracers, in matrices of 64 • 64 • 8, as 0.2-second consecutive frames, for 60 seconds. The detector was positioned in order to visualize the superior vena cava or the right heart without interposition of lung parenchyma between one of these organs and the camera. Data Process The processing steps used to compute the extraction fraction and the number of free amine endothelial receptors were: (1) generation of the time activity curves of the two tracers; (2) removal of the background and noise; (3) computation of the impulse response functions; (4) calculation of extraction fraction; and (5) calculation of the number of receptors. Time activity curves of the test and reference tracers of the lung input (superior vena cava or right ventricle) and of the lung were generated by drawing areas of interest on the acquired images. In the reference and test tracers images, the same sites were sampled by using the same areas of interest in both sets of images. As the first curve processing step, the background produced by the spillover of the first tracer in the pulse height analyzer (PHA) window of the second tracer was subtracted. Following that, the noise was removed. The noise in the input time activity curves and in the lung curve of the reference tracer was removed by curve fitting with gamma variates or Stewart-Hamilton functions. The noise in the lung curve of the test tracer was removed by fitting with a polynomial function of order 7. sz The IRF for the two tracers were calculated by deconvolution of the input and the lung time activity curves using general Fourier technique. The deconvolution was performed constraining the IRF to a predefined monoexponential model. This constraint was initially used because of its simplicity for software implementation. But,
TOUYA ET AL
because the monoexponential modeling of the IRF is less accurate than the non-predefined modeling of the IRF obtained by general Fourier technique, especially for systems with high differences between the input and the output time activity curves, it was abandoned. The pulmonary extraction function of the test tracer was calculated using equation 1, where HT(t) and HR(t) are the normalized IRFs for the test and the reference tracers, respectively. The extraction fraction of the test tracer was determined at the time that 90% of the reference tracer had left the lung compartment. Accuracy and reproducibility of this technique were previously demonstrated in a group of experiments in which a mixture consisting of 99mTc-MAA (macroaggregates of human serum albumin) and 99mTc-dextran in different known proportions were used as test tracers, and boluses of pure 99mTc-dextran as the reference tracers. 53 The IRF of the test tracers showed two components, a fast and a slow slope. The fast slope was due to the washout of 99mTc-dextran from the lung fields and the slow component was due to the trapping (extraction) of 99mTc-MAA within the lung capillary bed. The total number of amine endothelial receptors can be calculated knowing the binding isotherms of the test tracer amine for the specie in consideration. The binding isotherms can be computer generated, using as variables the amounts of injected amine and the percent of free binding sites, and as constants, the dissociation constant of the amine-receptor binding reaction and the total number of binding sites in the lung. 34 FUTURE STUDIES AND APPLICATIONS
The first-pass dual-indicator dilution technique using successive injection of two boluses has permitted in vivo assay of receptors in animals. Currently, this assay is done assuming that equilibrium in the ligand-receptor binding reaction is achieved during the short time interval the bolus is in the lung capillary vessels. Research is in progress on algorithms for computing the number of lung amine endothelial receptors during the dynamic stages of the ligand-receptor binding reaction. Additionally, further investigation is in progress for determining the sensitivity
THE LUNG AS A METABOLIC ORGAN
and specificity of the lung endothelial receptors for the different amines. For clinical applications, the use of two simultaneous radiotracers could be a major inconvenience. Studies designed to analyze the sensitivity and reproducibility of a modified technique in which the IRF of the reference tracer is assumed to be a constant are needed. If these studies demonstrate that the lung extraction of the test tracer can be estimated with reasonable precision by measuring only the IRF of the radioamine test tracer, clinical research on lung metabolic functions could be accelerated. On the contrary, if results of this research are negative, it would be necessary to modify the cameras in order to separately register photons with different energies. Separate photon registration would permit simultaneous injection of more than one radiotracer. Disorders of the lung metabolic functions are considered to be the basic physiopathological abnormality in a variety of unsuspected diseases. The noninvasive assessment of these functions would become important in the diagnosis, followup, and monitoring of the treatments of patients with these diseases. Among those diseases that may be considered to benefit most from the noninvasive evaluation of the metabolic lung functions are: systemic blood hypertension, neonatal and adult respiratory distress syndromes, cystic fibrosis, asthma, and several psychiatric disorders such as depression, schizophrenia, and drug addiction)'4z54'-56 Interest in the lung metabolic functions has increased with the realization
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that the injured lung might influence distant organ function. Thoracic surgeons have learned that systemic manifestations of altered lung metabolic functions are characteristically produced in acute injury and need to be properly treated. 57 In addition, preliminary results of research done in our laboratory on smoke inhalation injuries seem to demonstrate that I2~I-IMP can estimate the degree of endothelial lung injury and can quantify the effects of drugs such as cimetidine on the smoke-injured animal. Endothelial receptors, other than those in the lung, could be assessed in vivo, using a technique similar to the one discussed above. Yet, the lung is the organ where assessment of receptors can be accomplished with the greatest ease, because the radioligand can be injected IV and arrives as an intact bolus to the lungs. To measure receptors in other organs, it will be necessary to inject the radioligands by a catheter placed in the input artery of the organ or to develop a technique to compute the organ input following the IV injection of the tracers) 4 Finally, we predict that further investigation to refine the techniques and to determine specific clinical applications will unveil new knowledge about the lung as a metabolic organ. ACKNOWLEDGMENT
The authors thank Albert Santibanez, Robert Hooten, Ermelinda Holgrein, and Juan P. Medina for their expert technical assistance during the experiments, and Tami Jo McKnight for her assistance in preparing the manuscript. Special thanks to Dr R. Baldwin from Medi-Physic for the supply of radiopharmaceuticals and numerous consultations.
REFERENCES
1. Porter R, Whelen J (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New Series. Amsterdam, Excerpta Medica, 1980 2. Tierney DF: Lung metabolism and biochemistry. Ann Rev Physiol 36:209-23t, 1974 3. Mellins RB: Fleischner lecture: Metabolic functions of the lung and their clinical relevance. A JR 138:999-1009, 1982 4. Block ER, Stalcup SA: Today's practice of cardiopulmonary medicine, metabolic functions of the lung; of what clinical relevance? Chest 81:215-223, 1982 5. Said SI: Metabolic functions of the pulmonary circulation. Circ Res 50:325-333, 1982 (suppl) 6. Ben-Harari RR, Youdim MBH: Commentary, the lung as an endocrine organ. Biochem Pharmacol 32:189-197, 1983
7. Ferreira SH, Vane JR: The disappearance of bradykinin and eledoisin in the circulation and vascular beds of the cat. Br J Pharmacol Chemother 30:417-424, 1967 8. Ryan US, Ryan JW: Correlation between the fine structure of the alveolar-capillary unit and its metabolic activities, in Vane Jr, Bakhle YS (eds): Metabolic functions of the lung. Vol 4 in Series on Lung Biology in Health and Disease, Lenfant C (ed). New York, Marcel Derker, 1977, p 197-232 9. Gaddum JH, Hebb CO, Silver A, Swan AAB: 5Hydroxytryptamine. Pharmacological action and destruction in perfused lungs. Q J Exp Physiol 38:225-262, 1953 10. Nicholas TE, Strum JM, Angelo LS, Junod AF: Site and mechanism of uptake of 3H-l-Norepinephrine by isolated perfused rat lungs. Circ Res 35:670~80, 1974 11. McGiff JC, Terragno NA, Strand JC, Lee JB, Loni-
304
gro A J, Ng KKF: Selective passage of prostaglandins across the lung. Nature 216:762-766, 1969 12. Ng KKF, Vane JR: Fate of angiotension I in the circulation. Nature 218:144-150, 1968 13. Youdim MBH, Bakhle YS, Ben-Harari RR: Inactivation of monoamines by the lung, in Porter R, Whelen J (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New Series. Amsterdam, Excerpta Medica, 1980, pp 105-128 14. Vane JR: Introduction, in Porter R, Wheler j (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New Series. Amsterdam, Excerpta Medica, 1980, pp 1-10 15. Simionescu M: Ultrastructural organization of the alveolar-capillary unit, in Porter R, Whelon J (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New Series. Amsterdam, Excerpta Medica, 1980, pp 11-36 16. Armstrong JM, Lattimer N, Moncada S, Vane JR: Comparison of the vasodepressor effects of prostacyclin and 6-OXO-PGF~a with those PGE~ in rats and rabbits. Br J Pharmacol 62:125-130, 1978 17. Waldman HM, Alter I, Kot PA, Rose JC, Ramwell PW: Effect of lung transit on systemic depressor responses to arachidonic acid and prostaeyclin in dogs, J Phamacol Exp Ther 204:289-293, 1978 18. Dusting G J, Moncada S, Van R JR: Recirculation of prostacyclin (PGI~) in the dogs. Br J Phamacol 64:3 i 5-320, 1978 19. Krell RD, McCoy J, Christian P: Accumulation and metabolism of ['4C] histamine by rat lung in vivo. Biochem Pharmacol 27:820-821, 1978 20. Piper P J, Tippins JR, Samhoun MN, Morris HR, Taylor GW, Jones CM: SRS-A and its formation by the lung. Bull Eur Physiopathol Respir 17:571-583, 1981 21. Woods HF, Meredith A, Tucker GT, Shortland JR: Methods for the study of lung metabolism, in Porter R, Whelen J (eds): Metabolic Activities of the Lung. Ciba Foundation Symposium 78, New series. Amsterdam, Excerpta Medica, 1980, pp 61-83 22. Chinard FP: Estimation of extravascular lung water by indicator-dilution techniques. Circ Res 37:137-145, 1975 23. Fowler JS, Gallagher BM, Mac Gregor RR, Wolf AP, Ansari AN, Atkins HL, Slatkin DN: Radiopharmaceuticals XIX. HC-labeled octylamine, A potential diagnostic agent for lung structure and function. J Nucl Med 17:752-754, 1976 24. Motutsky H J, Insel PA: Adrenergic receptors in man. Direct identification, physiologic regulation, and clinical alterations. N Engl J Med 307:18-29, 1982 25. Gallagher BM, Christman D, Fowler JS, MacGregor RR, Wolf AP, Sam P, Ansari AN, Atkins H: Radioisotope scintigraphy for the study of dynamics of amine regulation by the human lung. Chest 71:282-284, 1977 (suppl 2) 26. Syrota A, Pascal O, Crouzel M, Kellershohn C: Pulmonary extraction of C-11 chlorpromazine, measured by residue detection in man. J NucI Med 22:145-148, 1981 27. Rahimian J, Bennett LR, Touya J J, Akber SF: Metabolic lung scanning: Demonstration of amine receptors with N-isopropyl-I-123-iodoamphetamine. Clin Nucl Med 6:453, 1981 (abstr)
T O U Y A ET AL
28. Mena I, Thompson C, Mason G, Glass EC, Bennett LR: Lung first-pass extraction of N-isopropyl-I123-pamphetamine in patients with chronic obstructive pulmonary disease. Clin Nucl Med 9:35, 1984 29. Pistolesi M, Miniati M, Ghelarducci L, Mazzuca N, Giuntini C, Taccini E, Renzoni G, Pellegrini N, Gilardi MC, Fazio F: Lung release of HIPDM: A new index of lung dysfunction for clinical and experimental studies. J Nucl Med 26:14, 1985 30. Rahimian J, Glass EC, Touya J J, Akber SF, Graham LS, Bennett LR: Measurement of metabolic extraction of tracers in the lung using a multiple indicator dilution technique. J Nucl Med 25:31-37, 1984 31. Guller B, Yipimtsoi T, Orvis AL, Bassingthwaighte JB: Myocardial sodium extraction at varied coronary flows in the dog. Estimation of capillary permeability by residue and outflows detection. Circ Res 37:359-378, 1975 32. Lassen NA, Perl W: Tracer Kinetic Methods in Medical Physiology. New York, Raven, 1979 33. Akber SF, Glass EC, Bennett LR: Scintigraphic demonstration and quantitation of pharmacologic inhibition of pulmonary uptake of N-isopropyl-I-123-p-iodoamphetamine by propranolol. J Nucl Med 26:14, 1985 34. Touya J J, Rahimian J, Grubbs DE, Corbus HF, Bennett LR: A noninvasive procedure for in vivo assay of a lung amine endothelial receptor. J Nucl Med 26:1302-1307, 1985 35. Ehrlich P: Chemotherapeutics: Scientific principles, methods, and results. Lancet 2:445, 1913 36. Langely JN: On the contraction of muscle, chiefly in relation to the presence of "receptive" substances. Part I. J Physiol (Lond) 36:347-384, 1907 37. Lefkowitz R J, Caron MG, Stiles GL: Mechanism of membrane receptor regulation, biochemical, physiological, and clinical insights derived from studies of the adrenergie receptors. N Engl J Med 310:1570-1579, 1984 38. Schafer DE: Measurement of receptor-ligand binding theory and practice, in Lambrecht RM, Rescingo A (eds): Tracer Kinetics and Physiological Modeling. New York, Springer-Verlag, 1983, pp 445-507 39. Lin MT, Chan HK, Chen CF, Teh GW: Involvement of both opiate and catecholaminergic receptors in the behavioral excitation provoked by thyrotropin-releasing hormone: Comparisons with amphetamine. Neuropharmacol 22:463469, 1983 40. Rahimian J, Bennett LR, Grubbs DE, Glass EC, Corbus HF, Touya J J: A noninvasive procedure to measure in vivo lung endothelial receptors. Clin Nucl Med 9:36, 1984 41. Oldendorf WH: The blood-brain barrier. Exp Eye Res 25:177-190, 1977 (suppl) 42. Wyatt R J, Murphy DL, et al: Reduced monoamine oxidase activity in platelets: A possible genetic marker for vulnerability to schizophrenia. Science 179:916-918, 1973 43. Starling EH, Verney EB: The secretion of urine as studied on the isolated kidney. Proc R Soc Lond B Biol Sci 97:321-363, 1925 44. Gillis CN, Cronau LH, Mandel S, Hammond GL: Indicator dilution measurement of 5-Hydroxytryptamine clearance by human lung. J Appl Physiol 46:1178-1183, I979
THE LUNG AS A METABOLIC ORGAN
45. Kung HF, Tramposch KM, Blau M: A new brain perfusion agent: [I-123] HIPDM: N, N, N'-trimethyl-N'[2-hydroxy-3-methyl-5-iodobenzyl]- 1,3-propanediamine. J Nucl Med 24:66-72, 1983 46. Holman BL, Lee RGL, Hill TC, Lovett RD, ListerJames J: A comparison of two cerebral perfusion tracers, N-isopropyl-I-123-p-iodoamphetamine and 1-123, HIPDM, in the human. J Nucl Med 25:25-30, 1984 47. Slosman D, Davidson D, Brill ABK, Alderson PO: Pulmonary accumulation of 1-131-MIBG in the isolated perfused rat lung. J Nucl Med 27:1076, 1986 48. Rahimian J, Glass EC, Touya J J, Akber SF, Graham LS, Bennett LR: Extraction fraction: Noninvasive determination by dual tracer deconvolution analysis. Clin Nucl Med 7:31, 1982 49. Chinard EP, Enns T: Transcapillary pulmonary exchange of water in the dog. Am J Physiol 178:197-202, 1954 50. Crone C: The permeability of capillaries in various organs as determined by use of the "indicator diffusion" method. Acta Physiol Scand 58:292-305, 1963
305
51. Fazio F, Jones T, Jones H, Rhodes CG, Hughes JMB: External counting for measuring pulmonary oodema. J Nucl Biol MOd 20:81-83, 1976 52. Rahimian J: Quantitative Assessment of Pulmonary Metabolic Extraction of Tracers Using Multiple Indicator Dilution Technique and Deconvolution Analysis. PhD Dissertation in Medical Physics, University of California, Los Angeles, 1983 53. Rahimian J, Grubbs DE, Gregory R, Santibanez A, Bennett LR, Glass EC, Corbus HF, Touya J J: Accuracy of noninvasive dual tracer technique for measuring pulmonary extraction of plasma substances. MOd Physics 12:673, 1985 54. Bockar J, Roth R, Heninger G: Increased human platelet monoamine-oxidase activity during lithium carbonate therapy. Life Sci 15:2109-2118, 1975 55. Sandier M, Youdim MB, et al: A phenylethylamine oxidizing defect in migraine. Nature 250:335-337, 1974 56. Axelrod J, Weinshilboum R: Catecholamines. N Engl J Med 287:237-242, 1972 57. Addonizio VP, Harken AH: The surgical implications of nonrespiratory lung function. J Surg Res 28:86-95, 1980