Influences of nonpulsatile pulmonary flow on pulmonary function

General Thoracic Surgery

Influences of nonpulsatile pulmonary flow on pulmonary function Evaluation in a chronic animal model To clarify the influences of nonpulsatile blood flow on the physiologic function of the lung, we established nonpulsatile pulmonary circulation with a centrifugal pump in a chronic animal model (adult goats, n = 6). As the initial phase, a pulsatile right ventricular assist device was implanted to bypass the whole blood supply from both the right atrium and right ventricle to the pulmonary artery. After 2 weeks of pumping, the pulsatile pump was replaced with a centrifugal pump without anesthesia, and nonpulsatile pulmonary circulation was instituted. In this experimental model, no significant change was observed in either mean pulmonary arterial pressure or pulmonary vascular resistance index during the pulsatile pumping compared with that on the fourteenth day of nonpulsatile pumping. Blood gas data, extravascular lung water content, and serum level of angiotensin-converting enzyme were maintained within normal ranges. There was also no significant change in the ventral to dorsal blood perfusion ratio of the lower lobe of the right lung. These results indicate that pulmonary functions are not affected by nonpulsatile pulmonary circulation for a period of 14 days in this animal model. (J THORAC CARDIOVASC SURG 1994;108:495-502)

Masayuki Sakaki, MD, Yoshiyuki Taenaka, MD, Eisuke Tatsumi, MD, Takeshi Nakatani, MD, and Hisateru Takano, MD, Osaka, Japan

Recently, the centrifugal pump has been widely applied in assisted circulation for severe heart failure as the pump for right or left heart bypass. In particular, right heart bypass with a centrifugal pump is increasingly applied in the treatment of patients with right heart failure.' Because centrifugal pumps may become more commonly applied for long-term use, such as From the Department of Artificial Organs, National Cardiovascular Center Research Institute, Osaka, Japan. Received for publication Nov. 8, 1993. Accepted for publication March 9, 1994. Address for reprints: Masayuki Sakaki, MD, Department of Artificial Organs, National Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita, Osaka, 565, Japan. Copyright © 1994 by Mosby-Year Book, Inc. 0022-5223/94 $3.00

+ 0 12/1/55992

for bridging to heart transplantation or for an implantable total artificial heart, the physiologic effects of nonpulsatile pulmonary blood flowon body or organ perfusion may be a major concern in these applications. Although its effect on systemic circulation has been extensively examined, the influence of nonpulsatile pulmonary circulation on the lung has not yet been fully elucidated. Previous experiments in acute animal models have revealed elevation of pulmonary vascular resistance-" or increase in lung water content's 7 in nonpulsatile pulmonary circulation. Furthermore, in patients, abnormal distribution of pulmonary blood flowhas been observed after the Glenn or Fontan operation.I"? Such results suggest that nonpulsatile blood flow may affect the pulmonary microvascular circulation. However, experiments in chronic animal models have demonstrated survival for 495

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Lung water catheter

PAP

Fig. 1. Schematic view of the right heart bypass circuit. Blood was diverted from the right atrium and right ventricle and sent to the pulmonary artery. After 2 weeks, the VAD was replaced with a centrifugal pump (Blood pump). Right atrial pressure (RAP), left atrial pressure (LAP), pulmonary artery pressure (PAP), and right ventricular pressure (RVP) were continuously monitored. A lung-water catheter was inserted from the left carotid artery into the descending aorta for measurement of extravascular lung water.

more than 3 months with nonpulsatile pulmonary circulation.!"!" Because the need for prolonged use of such bypass support in patients may increase, a chronic model for the study of nonpulsatile pulmonary blood flow should be established. In view of the foregoing, we established a chronic animal model to clarify the influence of nonpulsatile blood flow on pulmonary hemodynamics, gas exchange, metabolic functions, and the distribution of lung tissue perfusion in awake animals.

Materials and methods Six healthy adult goats weighing from 50 to 63 kg (57.5 ± 1.9kg, mean ± standard error of the mean) were used. Sterile techniques were enforced throughout the experiments. The experiment was carried out in two stages as follows to analyze the effect of the depulsation itself rather than the effects of the surgical intervention and general anesthesia. Initially, the goats were premedicated intramuscularly with ketamine hydrochloride 10 rug/kg and atropine sulfate 0.01 rug/kg. After intubation of the goats, anesthesia was maintained with halothane (0.5% to 1.5%) with a mixture of nitrous oxide and oxygen. Left thoracotomy was performed through the fourth costal bed. An inflow cannula with multiple side holes was inserted into the right atrium and ventricle through the right

atrial appendage, and an outflow cannula was sutured onto the main pulmonary artery to bypass the wholevenous return blood. A diaphragm-type ventricular assist device!" (VAD, Toyobo Co., Ltd., Osaka, Japan) was installed between these two cannulas. Right heart bypass was started at a rate of 70 to 80 beats/min in a fixed rate mode, and the bypass flow was maintained the same as the aortic flow. We did not ligate the pulmonary artery to occlude the bypass circuit for a few minutes during switching of the pump. After the chest was closed, the introducer for the lung-water catheter was inserted via the left carotid artery (Fig. I). Pulmonary circulation was maintained as pulsatile flow with the VAD for 2 weeks, after which the effects of the operation and anesthesia had become negligible. The puisatile pump was then quickly replaced with a centrifugal pump (modified MD-IO, Iwaki Pump Co. Ltd., Tokyo, Japan) without anesthesia. The bypass flow through the centrifugal pump was set to maintain the nonpuisatile pulmonary arterial pressure wave form and to maintain the same mean aortic pressure as before the replacement. Systemic anticoagulation was attained with continuous infusion of heparin to maintain the activated coagulation time between 200 and 300 seconds. Measurements Hemodynamic parameters. Fluid-filled pressure-monitoring catheters were inserted into the main pulmonary artery, internal thoracic artery and vein, left atrium, and right ventricle. An electromagnetic flow probe (MFV-2100, Nihon Koden Co., Tokyo, Japan) was placed on the aortic root to measure the cardiac output. Pulmonary vascular resistance (PVR) and PVR index (PVRI) were calculated by the following equations: PVR = (PAP - LAP)/Flow X 79.9 dyne. sec . cm ? PVRI = PVR X Body weight dyne. sec . cm- 5 . kg where PAP is pulmonary artery pressure and LAP is left atrial pressure. Blood gases. Arterial and mixed venous blood samples were obtained from the pressure-monitoring lines for the aorta and pulmonary artery, and the partial pressures of oxygen and carbon dioxide were measured with a blood gas analyzer (Radiometer ABL-2, Copenhagen, Denmark). Extravascular lung water. A lung-water catheter for measuring extravascular lung water, (HE-2900, 5F, Electro-Catheter Corp., Rahway, N.J.) was inserted via the introducer in the left carotid artery into the descending aorta 3 days before pump replacement. As the index of extravascular lung water, extravascular thermal volume was measured with a lung water computer by means of a heat-sodium double indicator dilution method'" (MTV-IIOO, Nihon Koden Co., Tokyo, Japan). Then, 10 ml of ice-cold 5% saline solution was rapidly injected into the right atrium through the catheter, and the thermal dilution and electrical conductivity curves in the aortic arch were obtained. Each measurement of extravascular thermal volume was carried out three times, and the mean value was used for statistical analysis. Angiotensin-converting enzyme (ACE). Blood samples were taken from the aortic line for the measurement of angiotensinconverting enzyme (ACE). The serum ACE levelwas measured by the Kasahara method.!? Pulmonary blood distribution (ventral/dorsal perfusion ratio). Pulmonary blood flow distribution was evaluated by a colored microsphere method.!" Microspheres (diameter 20 Jim, 3 million, E-Z Trac, Los Angeles, Calif.) were injected into the

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ECG mmHg

RVAD PAP

2°f ° 10

ECG Centrifugal pump

mmHg

PAP

2°f 10

°

II(

..

1 second

Fig. 2. Wave forms of electrocardiogram (ECG) and pulmonary artery pressure (PAP) during right ventricular assist device (RVAD) pumping and centrifugal pumping. The wave forms of PAP became almost flat when the centrifugal pump was installed.

right atrium through the pressure-monitoring line just before the depulsation and on the first and fourteenth days after depulsation. Microspheres of different colors were used for each measurement. They were distributed to the lung tissue and lodged in the microvasculature. After the goats were killed, the microspheres were extracted from tissue samples (2 to 3 gm) of the lower lobe of the right lung, which was contralateral to the side of the thoracotomy and was not affected, and the number of microspheres of each color was counted. Seven samples from the ventral portion of the right lower lobe and seven from the dorsal portion were obtained in each goat to calculate the blood perfusion ratio of the mean values of the ventral to dorsal lung portions. Pathologic assessment. At the time of chest closure and at autopsy, small tissue fragments were excised from the left lower lobe of the lung. All samples were fixed in 10% formalin solution and stained with hematoxylin and eosin. Control data and statistical analysis. Fluid-filled pressuremonitoring catheters and an electromagnetic flow probe were inserted, and the ventral/dorsal blood flow ratio was measured by the colored microsphere method in three adult goats to obtain control data. The serum ACE level was also measured in 10 control goats to determine the normal range. All data were expressed as mean value and standard error of the mean and analyzed by paired t test or analysis of variance for repeated measures. A probability value ofless than 0.05 was considered statistically significant. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).

Results All of the goats tolerated the operation well.They could be extubated within I hour after the operation and could stand on the day of the operation. After 2 weeks, the conversion from pulsatile YAD to nonpulsatile pump was performed smoothly, and the behavior of the goats, including respiratory pattern, activity, and appetite, remained unchanged after the replacement. Two of the six goats died suddenly between 10 and 14 days after the operation. Hemodynamics. Fig. 2 shows the wave forms of PAP during YAD pumping and centrifugal pumping. The pulse pressure of PAP ranged from 8 to 12 mm Hg during YAD pumping. On the other hand, pulsatility was successfully obliterated by centrifugal pumping except for periodic fluctuations corresponding to the respiratory cycle, and nonpulsatile pulmonary blood flow was considered to have been obtained. The cardiac index during YAD and centrifugal pumping was maintained at 109 ± 4.7 and 115 ± 7.6 ml/kg per minute, respectively, and no significant difference was observed. Changes in mean PAP and calculated PYRI are shown in Figs. 3 and 4, respectively. The mean PAP and PYRI during pulsatile pumping were 14.0 ± 0.7 mm Hg and 6800 ± 140 dyne· sec· cm- s . kg, respectively, and those on the fourteenth day of nonpulsatile pumping were 13.5 ± 0.3 mm Hg and 6000 ± 1600 dyne· sec· cm" . kg, respectively. Although PYRI decreased

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Table I. Effect of nonpulsatile flow on gas exchange and extravascular lung water Nonpulsatile flow Pulsatile flow Pal (rnm Hg) PC02 (rnm Hg) V0 2 (nil/min) EVTV (ml/kg)

93.7 37.3 252 3.8

± ± ± ±

I day

2.4 0.8 \6 0.7

102.6 35.6 258 3.8

± ± ± ±

3 days 2.7 1.0 33 0.8

98.2 35.5 257 3.6

± ± ± ±

4.\ l.I 26 0.4

7 days IOl.I 37.4 247 3.9

± ± ± ±

4.5 3.0 36 0.5

14 days

91.5 42.3 228 4.2

± ± ± ±

3.1* 3.9* 19* 0.9t

Po, and Pco-, Oxygen tension and carbon dioxide tension of arterial blood; Va,. oxygen transfer rate; EVTV, extravascular thermal volume. Values are mean ± standard error of the mean. *n = 4. tValue at 10 days (n = 3).

Table II. Effect of nonpulsatile flow on serum ACE level Nonpulsatile flow Pulsatile flow

SACE

1 day

2 days

7 days

6.6 ± 1.1 6.9 ± 1.2 7.2 ± 1.2 7.\ ± l.I

14

days 7.\ ± 1.7*

(lUjL) SACE, Serum ACE levels. *n = 4. Values are mean ± standard error of the mean.

slightly after conversion, all values were considered to be within the normal range of physiologicfluctuation, and no significant change was observed in either mean PAP or PVRI. Gas exchange data and extravascular thermal volume. The values of arterial oxygen tension, carbon dioxide tension, oxygen transfer rate, and extravascular thermal volume are shown in Table I. Because of occlusion of the introducer and unreliability of long-term electrode measurement with the lung-water catheter, extravascular thermal volume could be measured until the tenth day after depulsation in only three goats. Although arterial carbon dioxide tension and extravascular thermal volume were slightly increased on the fourteenth and tenth days, respectively, no significant difference from the value observed during pulsatile flow was observed during nonpulsatile circulation. Serum level of ACE. The serum level of ACE remained within the normal range (8.5 ± 2.6 IV /L, n = 10) during both pulsatile and nonpulsatile pulmonary circulation (Table 11). Blood flow distribution. In five of the six goats, blood flow distribution could be measured by the colored microsphere method; the microspheres could not be extracted in the remaining goat. Fig. 5 shows the blood perfusion ratio of the ventral to dorsal lung segments of the right lower lobe during pulsatile pumping and on the first and fourteenth days of nonpulsatile pumping in each goat. All values were within the normal range, and ven-

tral/dorsal perfusion ratio during pulsatile pumping (1.8 ± 0.3) was not significantly different from that (1.5 ± 0.1) on the fourteenth day of nonpulsatile pulmonary circulation. Histologic findings. Microscopic evaluation of the samples obtained on the fourteenth day of nonpulsatile pumping revealed no significant changes in the thickness of the alveolar wall compared with that at the time of chest closure, and no perivascular interstitial edema was noted (Fig. 6). Discussion Several types of centrifugal pumps have recently been developed for assisted circulation.l? These pumps have been widely applied in not only left but also right heart bypass. 1 On the other hand, since Lee and DuBois2o demonstrated in 1955 that the pulmonary capillary bed has pulsatile flowunder normal conditions, several studies in which the effects of pulsatile and nonpulsatile pulmonary blood flow were compared have been reported.i''' However, the question of whether the lung requires pulsatile blood flow has not yet been resolved. One reason for the difficulty in concluding the meaning of pulsatile flow in these studies is that they were performed under unphysiologic conditions. Reactions of the pulmonary vasomoter system to the autonomic nervous system 2 1- 24 and to vasoactive agents" in the isolated perfused lung or in an animal model under general anesthesia may not be comparable with those under normal physiologic conditions. To resolve this issue, we designed a two-stage experimental model. By using a VAD as the pulsatile pump and a centrifugal pump as the nonpulsatile pump in the right heart bypass circuit, we could immediately convert the pulmonary blood flow pattern from pulsatile to nonpulsatile in an awake animal. Regarding the influence of surgical intervention, we26 have previously observed in a similar model that the systemic influence of surgery can be ignored after about 2 weeks after the operation. Influence of depulsation on hemodynamics. Many

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mmHg 18 16 0.. 14
I

dyne-sec-cml-kg

10000

-

8000

*

I I

0::: 6000 ;>

I I

.. P ...,' .

0..

I

I I

C

I I

NP

.,

2000

I

C

..

I

i

Conversion

7

14 days

Fig. 3. Mean pulmonary arterial pressure (mPAP) during pulsatile and nonpulsatile pulmonary circulation. No significant change in mPAP wasobserved after depulsation (dotted line). P, Pulsatile pulmonary circulation with a VAD on each of the last 3 daysbefore conversion; NP, nonpulsatile pulmonary circulation with a centrifugal pump; C, control data from three adult goats. *n = 4. investigators.have found an increase in pulmonary arterial pressure and vascular resistance with nonpulsatile blood flow in ex vivo or acute animal experiments.v'" 27 However, we speculated that the behavior of the regulatory system of pulmonary vascular resistance in an ex vivo or acute animal model would differ from that in a chronic awake animal model. The Cleveland Clinic group 11-14 has performed long-term evaluations of the nonpulsatile systemic and pulmonary circulations with centrifugal pumps in awake animals during ventricular fibrillation. They reported that PAP and PVR in nonpulsatile pulmonary circulation were stable during the experiment, but that both parameters were higher than those observed in normal circulation. We considered that these changes might have been caused by deleterious effects derived from the surgical intervention or from the four conduits placed in the thoracic cavity for the biventricular bypass, including possible atelectasis and effusion of the lung. In our experimental model, we could observe the reaction of the pulmonary vascular system in the absence of the influence of these factors, and we observed no significant changes in PAP and PVRI after depulsation. The results may indicate that the pulmonary vasomotor system in an awake animal can accommodate the immediate change of pulsatile blood flow to nonpulsatile flow, with stable maintenance of pulmonary circulation comparable with that in the pulsatile state. Influence of depulsation on pulmonary microvascular circulation. The results of several studies of the effects

.,

NP i

i

14days

7 Conversion pumping time

I

pumping time

P

t

0

I

I

4000

I

Fig. 4. Pulmonary vascular resistance index (PVRJ) during pulsatile and nonpulsatile pulmonary circulation. No significant changein PVRI wasobserved after depulsation (dotted line). P, Pulsatile pulmonary circulation witha VAD on eachof the last 3 days before conversion; NP, nonpulsatile pulmonary circulationwitha centrifugal pump; C. controldata from three goats. *n = 4 (79.9 dyne. sec . cm- 5 = I unit).

o

3.0

•

o '"0

~ a

2.0

1.0

I C

o

•

o ~

.. P

..

.,

t

Conversion

NP

i

., 14 days

pumping time Fig. 5. Ventral/dorsalblood perfusion ratio of the lower lobe of the right lung (Qv/Qd) during pulsatile pumpingand on the first and fourteenth daysof the nonpulsatile pumping. The differentsymbols correspond to individual goats. P, Pulsatilepulmonarycirculation with a VAD; NP, nonpulsatile pulmonary circulation witha centrifugal pump;C,controldata from three goats.

of

pulsatile

and

nonpulsatile

perfusion

on

gas

exchanger- 7, 28-30 suggest that nonpulsatile pulmonary blood flow does not affect gas exchange. Our results are consistent with this observation. However, Taguchi and colleagues7 observed a tendency for lung water to accu-

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The Journal of Thoracic and Cardiovascular Surgery September 1994

Fig. 6. Microscopic appearance of the lung on the fourteenth day of nonpulsatile pumping. No perivascular interstitial edema or alveolar wall thickening was evident. (Hematoxylin and eosin stain;original magnification X200.) mulate when nonpulsatile pulmonary circulation with right heart bypass was maintained for 10 hours. Wilkens, Regelson, and Hoffmeister 3 l suggested that the pulsation of blood flow has an important effect on lymphatic return from isolated organs. In addition, they indicated the possibility of relatively mild edematous changes that do not affect gas exchange. These reports suggest that nonpulsatile pulmonary circulation may change the permeability of the pulmonary capillary wall. In contrast with these results, however, we found no accumulation of extravascular lung water or evidence of perivascular or interstitial edema in histologic examinations performed after 2 weeks of nonpulsatile pulmonary circulation in the present study. Although the reason for this discrepancy between our results and those obtained in previous studies is not clear, we consider that anesthesia or surgical manipulation of the lung tissue or unbalanced intravenous fluid infusion may have affected the permeability of the lung in the acute models. ACE, which converts angiotensin I to angiotensin II, is located on the luminal surface of endothelial cells, especially those of the pulmonary capillaries. To our knowledge, no study has been conducted concerning the relation between pulsatility and release of cellular ACE. Several studies 32. 35 have demonstrated changes in serum ACE activity when pulmonary vascular endothelial cell injury or lung disease, including bronchial asthma or chronic lung disease, is present. Although the causes of such change are still unclear, we consider that the change in serum ACE level may be influenced under nonpulsatile pulmonary circulation, in which an insufficiency of

microvascular blood flow can be present. However, our data indicate that the serum level of ACE remains stable and within the normal range during both pulsatile and nonpulsatile pulmonary circulation. We therefore consider the microvascular pulmonary blood flow to be well maintained in nonpulsatile pulmonary circulation. Distribution of pulmonary blood flow. Clinical studiess.lo of the distribution of regional blood flow in lung tissue after the Glenn or Fontan procedure have suggested that nonpulsatile pulmonary blood flow is one of the factors associated with abnormal distribution of pulmonary blood flow. The abnormal distribution of pulmonary blood flow observed in these clinical studies may have been magnified by the compromised condition of the patients. We considered that the design of the present experiment would clarify the influence of nonpulsatile flow on pulmonary blood flow distribution. Rather than the upper/lower perfusion ratio, we measured the ventral/dorsal perfusion ratio to examine the effect of gravitation on the blood distribution.F'-'? We observed no significant difference in this ratio during pulsatile compared with nonpulsatile pulmonary blood flow. In the present study, a centrifugal pump was used to provide pulmonary blood flow, and the experiment did not last long enough to allow comparison with the Fontan/Glenn circulation. However, these data suggest that nonpulsatile pulmonary flow with a right ventricular assist device does not acutely affect the blood distribution of the lung. The limitations of our experiment were the absence of a control group, the small number of animals, which reduces the power of analysis, and the short experimental

The Journal of Thoracic and Cardiovascular Surgery Volume 108, Number 3

period. With regard to the lack of a control group, we assumed that the measurements would not change as a result of pulsatile assistance during the remainder of the support period. The other limitations cannot presently be resolved, because the present experimental method is technically demanding and the centrifugal pump cannot be driven for a longer period. The development of a centrifugal pump with the capacity for long-term use would be of use in performing more detailed studies. In summary, we established non pulsatile pulmonary flow with a centrifugal pump in a chronic animal model. No significant effects on basic hemodynamics, gas exchange performance, extravascular lung water content, serum levels of vasoactive agents, or the microscopic appearance or blood flow distribution of the lung were observed during 14 days of pumping of pulmonary blood flow under the non pulsatile condition. These results suggest that nonpulsatile flow can maintain pulmonary circulation within physiologic limits without appreciable adverse effects on lung function in awake animals. We thank Dr. T. Kasugai, the Department of Pathology, Osaka University, for pathologic comments, and the other members of the Department of Artificial Organs, National Cardiovascular Center Research Institute, for their assistance. We also acknowledge Professor H. Matsuda, the First Department of Surgery, Osaka University, for relevant discussions and reviewing the manuscript. REFERENCES I. Millar AC, Pae WE, Pierce WS. Combined registry for the clinical use of mechanical ventricular assist devices: post cardiogenic shock. Trans Am Soc Artif Intern Organs 1990;36:43-6. 2. Mandelbaum I, Burns WHo Pulsatile and nonpulsatile blood flow. JAMA 1965;191:657-60. 3. Raj JU, Kaapa P, Anderson J. Effect of pulsatile flow on microvascular resistance in adult rabbit lung. J Appl Physiol 1992;72:73-81. 4. Clarke PC, Kahn DR, Dufek JH, Sloan H. The effects of nonpulsatile blood flow on canine lungs. Ann Thorac Surg 1968;6:450-7. 5. Furuse A, Brawley RK, Gott VL. Pulsatile cava-pulmonary artery shunt: surgical technique and hemodynamic characteristics. J THORAC CARDIOVASC SURG 1972;63: 495-500. 6. Richenbacher WE, Pierce WS, Jurmann M, et al. Pulmonary vascular effects of pulsatile and nonpulsatile mechanical right ventricular assistance. Surg Forum 1989;40:254-

5. 7. Taguchi S, Yozu R, Iseki H, Soma Y, Inoue T. Effects of nonpulsatile and pulsatile right ventricular bypass on pulmonary circulation. Trans Am Soc Artif Intern Organs 1988;34:213-21.

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32. Hollinger MA, Giri SN, Patwell S, Zuckerman JE, Gorin A, Parsons G. Effect of acute lung injury on angiotensin converting enzyme in serum, lung lavage, and effusate. Am Rev Respir Dis 1980;121:373-6. 33. Krulewitz AH, Fanburg BL. The effect of oxygen tension on the in vitro production and release of angiotensin converting enzyme by bovine pulmonary artery endothelial cells. Am Rev Respir Dis 1984;130:866-9. 34. Lieberman J. Elevation of serum angiotensin-converting enzyme (ACE) level in sarcoidosis. Am J Med 1975; 59:365-72. 35. Suetsugu M, Takahashi M, Ohmi T, et al. Serum angiotensin converting enzyme level in bronchial asthma. Ann Allergy 1978;40:51-7. 36. Nicolaysen G, Shepard J, Onizuka M, Tanita T, Hattner RS, Staub NC. No gravity-independent gradient of blood flow distribution in dog lung. J Appl PhysioI1987;63:5405. 37. Johansen JK, FlateboT, Melsom MN, Iversen PO, Muller C, Nicolaysen G. Regional distribution of ventilation-perfusion ratio (V /Q ratio) in the goat. J Physiol 1992;452: 28-9.