Angiotensin-1-Converting Enzyme Activity As Index of Pulmonary Damage in Thermal Injury with or without Smoke Inhalation

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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 22, No. 1 Copyright 1992, Institute for Clinical Science, Inc. Angiotensin-1-Converting Enzyme Activity As Index of Pulmonary Damage in Thermal Injury with or without LOREDANA BRIZIO-M OLTENI, M.D., F.A.C.S.,t GIANCARLO PIANO, M.D.,t RAYMOND L. WARPEHA, M.D., Ph.D.. NORMAL H. SOLLIDAY, M.D.," AGOSTINO M OLTENI, M.D.,t Ph.D., JUAN ANGELATS, M.D., NOLAN LEW IS, M.D.,f and HALINA PATEJAK-RADWANSKI, D.D.S. fdepartm ent of Pathology Northwestern University Medical School Chicago, IL and tu niversity o f Illinois at Chicago, Chicago, IL and Loyola University Stritch School o f Medicine, M aywood, IL and 1Christ Hospital and Medical Center, Oak Lawn, IL and f Gottlieb Memorial Hospital, Melrose Park, IL ABSTRACT Serum angiotensin-converting enzym e (ACE) activity, plasm a renin activity (PRA), and serum aldosterone levels were m easured up to four weeks in a population of adults exposed to thermal injury, with or without concomitant exposure to smoke inhalation. In 10 patients, plasma levels of angiotensin-2 and ACE activity in bronchial lavage w ere also evaluated. Patients with severe burn injury had a significant decline of serum ACE activity while the concentrations of aldosterone and PRA were m arkedly elevated. Smoke inhalation seem ed to counterbalance the decline of serum ACE activity, and, in the last group of patients, ACE concentrations were higher than those recorded in patients suffering only from cutaneous burn. The ACE activity was evidenced in bronchial lavage of patients exposed to smoke inhalation with the highest values present in the first day after the injury. The same patients had also very elevated levels of plasma angiotensin 2. In conclusion, serum ACE activity decreases in bum patients according to the severity of the cutaneous burn; smoke inhalation influences serum /92/ $01.50 Institute for Clinical Science, Inc.

2 2 BRIZIO-MOLTENI, PIANO, WARPEHA, SOLLIDAY, MOLTENI, ET AL levels of the enzyme with concentration values opposite to the low ones present in cutaneous bum. Finally, the enzyme activity has an independent pattern from that of the other components of the renin angiotensin aldosterone system. The evaluation of ACE activity may be a m arker of pulm onary dam age in smoke inhalation. Introduction The lung is the site of numerous m etabolic processes. To its known role in gas exchanges has to b e added its contribution to the regulation of systemic homeostasis by controlling the secretion of substances such as angiotensin converting enzym e (ACE), endothelin, serotonin, plasm inogen activator (PA), prostaglandins, and thromboxane, which all play an essen tial role as vasodilators and vasoconstrictors ,33,38 Lung injury m ay s ig n ific a n tly c h a n g e th e p u l m onary en d o c rin e balance and alter the hom eostasis. Acute smoke inhalation is one of the m ost severe lung injuries and increasingly so w hen associated with a serious cutaneous burn, affecting the synthesis of many of the substances secreted by the lung parenchyma. This altered secretion may indicate the extension and severity of the pulmonary damage and could be considered a m arker of it. In the past few years we devoted our a tte n tio n to o n e o f th e su b sta n c e s secreted by the lung: the angiotensin converting enzym e. C hem ically id en tified as a dipeptylpeptidase this enzyme is localized on the luminal face of the pulm onary endothelium w here it converts approxim ately 90 percent of the angiotensin entering the pulm onary capillary bed to the powerful vasoconstrictor ang io ten sin-2, and also inactivates 30 * Send reprint requests to: Agostino Molteni, M.D., Ph.D., Northwestern Memorial Hospital, Wesley Pavilion, Room 409, 250 East Superior Street, Chicago, Illinois percent of the bradykinin. Lung, serum, and bronchial lavage ACE enzym e activity are markedly changed in several models of experimental lung injury: exposure to ra d ia tio n,41 ch ro n ic h y p o x ia,36,43 administration of bleom ycin,26,27 monocrotaline,32,33 paraquat,22 and thiourea.23 In at least tw o of th ese co n d itio n s, c h ro n ic hypoxia and m o n o cro talin e administration, ACE plays a relevant role in the developm ent of post-injury pulmonary hypertension. Serum ACE levels are also elevated in several conditions of lung damage in hum ans such as sarcoidosis,18,28 new born respiratory distress syndrom e,30 and cardiopulm onary bypass.9 A ngiotensin-converting enzym e activity has been studied in two models of smoke inhalation in dogs. In one group, the induced injury was lim ited to one lung, w hile the contralateral was m aintained in condition of acute hypoxia.8 In th e second group, b o th lungs w ere e x p o sed to sm o k e.10 A s ig n ific a n t in c re a se in lu n g ACE a c tiv ity was observed in the first group (the experim ents w ith only one lung exposed to smoke), w hile a decline in lung ACE activity was observed when both lungs were involved. In this last model, despite a fall in lung ACE activity, a significant increase of peripheral plasma angiotensin-2 levels was also observed, indicating a systemic metabolic response in these animals. Furtherm ore, in rats exposed to sm oke in h a la tio n for ap p ro x im ately seven m inutes, w ith the anim als kept w ithout restrain t in a glass cham ber where the fumes were produced by burning household material, it was observed that one hour and three hours after smoke

3 e x p o su re se ru m A C E a c tiv ity w as in c re a s e d w h ile lu n g A CE a c tiv ity was decreased.39 T he current authors report the observation in serum and bronchial lavage ACE activity as well as angiotensin-2 levels in a population of patients exposed to thermal injury with or without concomitant smoke inhalation. Materials and Methods SERUM ACE ACTIVITY IN HUMAN THERMAL INJURY 3 T h e stu d y c o n s is te d of 30 a d u lt patients who participated upon signature of the inform ed consent. Tw enty-three were Caucasians, six were Blacks, and one was O riental. T he average age was 42.5 ± 5.3 years, ranging from 18 to 92 years. Average b urn surface was 32.2 ± 6.0 burn of total bum area (TBA) (range 1 to 90 percent). Eleven subjects had only cutaneous burn, 17 had cutaneous burn and smoke inhalation, and two had only smoke inhalation (cutaneous burns were of unaccountable degree: approximately one percent). T he R inger s lactate solution (4 ml per Kg per body weight percent of bum ) was used for resuscitation. No steroids w ere used. The diagnosis of smoke inhalation was made on medical history, clinical examination, laboratory, a n d ra d io lo g ic a l e v a lu a tio n. T h re e patients with 90 percent, 60 percent, and 50 percent bum died. Their deaths were attributed to generalized sepsis and renal failure. In 20 patients, serum ACE activity was determ ined four tim es a day (at 00.00, 06.00, 12.00, and hours) every day during the first w eek of the study and then twice a week to the completion of the study (four weeks). In 10 of these patients, all with smoke inhalation, ACE activity was m easured twice a day in the serum to post burn day 5 (at and hours) and once a day in the bronchial lavage. In all patients plasm a renin activity (PRA) and serum aldosterone levels were also m easured tw ice a w eek betw een and hours. D aily m easurem ent of plasm a angiotensin-2 was also performed in the 10 patients with smoke inhalation w here ACE activity of bronchial lavage was evaluated. The m ethod of Cushman and C heung13 was used for m easuring ACE. Samples were run in batches of 15 to 30 determ i nations using a series of patient sera of know n ACE activity as in te rn a l stan dards. Specimens were stored at -20 C. Serum and bronchial lavage ACE values w ere ex p ressed as units p e r ml p er m inute (a unit of ACE is the am ount of enzyme able to produce one mm of hipp u ric acid from a h ip p u ril-h is tid illeucine substrate). For serum determ ination, 0.2 ml was used; for the ACE m easurem ents in bronchial lavage, one ml of fluid was used and the am ount was calculated in relation to the volume of fluid collected into the tube. Serum aldosterone was m easured by a radioimmunoassay kit;* PRA and angiotensin-2 were m e a s u re d b y a ra d io im m u n o a s s a y m ethod with other kits.t Standard deviation and standard errors of the m ean (s.e.m.) w ere calculated at each in te r val time. The M an-w hitney test was used for com paring the enzym e values of the patients w ith smoke inhalation versus those of the patients with cutaneous burn only. The Kruska-Wallis test for several independent samples was used for comp a rin g th e e n z y m e d e te rm in a tio n s within each group of patients at different days.12 C orrelation coefficients w ere also studied. Results In table I are reported the results of all serum ACE activity determ inations of the patients studied for four weeks. Values of burn days 3, 5, 6, 11, 16 and 29 after ther- * Supplied by Damon Diagnostic, Needham, MA. t Supplied by New England Nuclear, Boston, MA.

4 4 BRIZIO-MOLTENI, PIANO, WARPEHA, SOLLIDAY, MOLTENI, ET AL TABLE I Serum Angiotensin-converting Enzyme and Plasma Renin Activity and Serum Aldosterone Concentrations in Population of Thermally Injured Adults Suffering from Cutaneous Burn With or from Cutaneous Burn Only Angiotensin-converting Enzyme (mu/ml/minute) and Cutaneous Burn * 18 ± 2 16 ± 2 21 ± 3 24 ± 2 32 ± 3 Cutaneous Burn Only 16 ± 2 14 ± 3 13 ± 2 15 ± 2 14 ± 3-16 ± 2 Significance (P) NS NS NS <0.05 <0.05 <0.01 Normal range: 17 to 25 mu/ml/minute Plasma Renin Activity (ng/mi/hr incubation) and Cutaneous Burn 12± ± 5 10 ± 3 9 ± 5 9 ± 2 Cutaneous Burn Only ± 3 10± ±4 10±3 Significance (P) NS NS NS NS NS NS Normal range: 1 to 6 ng/mi/hr incubation Serum Aldosterone (pg/ml) and Cutaneous Burn 168 ± ± ±3 145 ± ± ± 3 Cutaneous Burn Only 166 ± ± ± ± ± 3 Significance (P) NS NS NS NS NS NS Normal range: 20 to 85 pg/ml ' One standard error of the mean. NS = not significant. mal injury were chosen as the most representative ones. The ACE activity levels were within normal limits at every interval time in the group of patients exposed to sm oke in h alatio n reach in g values slightly above the normal limits at day 29, even if a trend to decline at days 5 and 7 was apparent. In the patients suffering from cutaneous burn only, the value of serum ACE activity was already low at day 3, declined at days 5 and 7, and showed a slight but not significant rise at days 11, 16, and 29. From day 11 to day 29, th e d iffe re n c e in A C E a c tiv ity betw een the two groups was statistically significant. Plasm a renin activity and serum aldosterone concentrations were elevated in both groups of patients, especially at days 3 and 5 with the hormone levels persisting above normal limits for the whole study. Smoke inhalation did not influence in a significant way either PRA or aldosterone levels. Evaluation of patients with extensive cutaneous burn (40 percent TBA and above) with or without smoke inhalation confirmed again the difference of ACE activity betw een the two groups as can be

5 SERUM ACE ACTIVITY IN HUMAN THERMAL INJURY 5 TABLE II Serum Angiotensin-converting Enzyme and Plasma Renin Activity and Serum Aldosterone Concentrations in Population of Severely Burned Adults With or Without Angiotensin-converting Enzyme (mu/mi/minute) and Cutaneous Burn 18 ± 2 14 ± 1 14 ± 1 19 ± ± 2 34 ± 2 Cutaneous Burn Only 12 ± 1 9 ± 1 10± 1 12 ± 1 12 ± 1 16 ± 1 Significance (P) <0.05 <0.05 NS <0.05 <0.05 <0.01 Normal range: 17to25mU/ml/minute Plasma Renin Activity (ng/ml/hr incubation) and Cutaneous Burn 18 ± 2 19±5 14 ± 3 13 ± ± 2 Cutaneous Burn Only 16 ±3 21 ± 4 16±3 12 ± 3 15 ± 3 13 ± 2 Significance (P) NS NS NS NS NS NS Normal range: 1 to 6 ng/ml/hr incubation Serum Aldosterone (pg/ml) and Cutaneous Burn 175 ± ± ± ±4 150 ± ± 3 Cutaneous Burn Only 190 ± ± ± ± ± Significance (P) NS NS NS NS NS NS Normal range: 20 to 85 pg/ml aforty percent total burn or more. " One standard error of the mean. seen in table II. Patients w ith severe cutaneous b u rn had a m ore m arked decline of serum ACE activity than those with less extensive injury. For all determinations the values rem ained below the normal range with the decline specially evident at days 5 and 7. Exposure to smoke inhalation seem ed to counter balance this m arked fall in ACE activity, and the difference betw een the two groups was statistically significant for almost all the interval points studied. Plasma renin activity a n d serum ald o stero n e levels were elevated in both groups of patients, even more than the values seen for the w hole burn population, b u t no statistically sig n ifican t differen ce was e v i denced betw een the two groups. T he two patients with smoke inhalation only and negligible cutaneous burns had values of ACE activity ranging from 32 to 39 mu per ml per minute, which are well above the upper normal limit. No significant variations w ere o b serv ed among the ACE values collected during the different hours of the day, thus fail

6 6 BRIZIO-MOLTENI, PIANO, WARPEHA, SOLLIDAY, MOLTENI, ET AL ing to evidence the existence of a circadian rhythm in therm al injury. No statistic a lly sig n ific a n t c o rrelatio n s w ere found betw een age, sex, race, systolic and diastolic blood pressure, and ACE activity. A c e Ac t iv it y D e t e r m in a t io n s in B r o n c h ia l L a v a g e The enzyme m easured in samples from bronchial lavage show ed its highest concentrations on days 1 and 2, then gradually decreased until day 5 (table III). After this day, the collection of bronchial fluid becam e more difficult. The values of ACE activity in the serum had a parallel pattern with that of the enzyme measured from the bronchial lavage. In the same patient, the highest concentration from day 1 gradually decreased to the lowest values on day 5, with a statistically significant difference. Plasm a a n g io te n sin -2 lev els w ere found to be extrem ely elevated during the first five days follow ing the injury (table III), showing a trend opposite to the ACE concentrations in the serum and in the bronchial lavage. Discussion From these prelim inary studies, the following information was derived: (1) serum ACE activity changes according to the severity of cutaneous b u rn s; (2) smoke inhalation influences serum levels of the enzyme, with concentration values opposite to the low ones present in cutaneous bum s; and (3) the enzyme activity has an independent pattern other than that of other com ponents of the renin angiotensin aldosterone (RAA) system. Renin, an ACE precursor, and aldosterone, an ACE successor, in the chain of the renin-angiotensin aldosterone system are very elevated in cutaneous burn, especially in patients w ith extensive injury.4,5,6 Serum ACE decreases and reaches its lowest values at days 5 and 7 post burn. On these days, PRA and aldosterone concentrations are still quite ele- TABLE III Serum and Bronchial Lavage Angiotensin-converting Enzyme Activity and Plasma Angiotensin-2 Levels in Patients Suffering From Thermal Injury and First Five Days of Admission in 10 Patients Serum (ACE) mil/ml 30 ± 9 ' 26 ± 6 22 ± 4 18 ± 5 16 ± 6 Significance Day 1 vs. day 5 p < 0.05 paired couples T test Bronchial lavage (mll/ml) 3 ± ± ±0.25 I0.20 ± ±0.05 Significance Day 1 vs. day 5 p < 0.05 paired couples T test Plasma Angiotensin-2 pg/ml ±180 ±78 ±175 ±96 ±120 Significance Not significant differences One standard error of the mean. Normal adult serum angiotensin-converting enzyme levels: 17 to 25 mu/ml/minute. Normal angiotensin-converting enzyme bronchial lavage concentrations: unknown. Normal adult plasma angiotensin-2 levels: 0 to 200 pg per ml.

7 SERUM ACE ACTIVITY IN HUMAN THERMAL INJURY 7 vated6,35 as well as the concentrations of se v e ra l o th e r h o rm o n es, e s p e c ia lly angiotensin-2,15,16 cortisol,2,35,40 corticotropin, and catecholam ines.1,2 A lthough th e lo w est lev els of the enzym e were observed since the first 48 hours in patients with the most severe bum, its declining trend was also evident in patients with small bum surface. This rules out that either the postburn hemoconcen tratio n, or iatrogenic in d u ced hem odilution (fluid adm inistration) or hypoproteinem ia have any prom inent role on this matter. The decreasing serum ACE activity could be a defense response in order to control through the conversion of A 1 to A2 an excessive (and perhaps harm ful) production of angiotensin-2. Drugs blocking the serum converting enzyme activity were found to decrease or interrupt the secretion of renin and aldosterone.14,19*25>37 H ow ever, it has been dem onstrated that change of renin secretion by high or low sodium chloride diets is not associated with variation of ACE activity in the serum and lungs of ex p erim ental an i m als.24,31 In patients w ith cutaneous burn, the enzyme decreases in the serum concomitantly to high levels of plasma renin and serum aldosterone6,35 which m ight last for several days up to the third or fourth week post injury, according to the patient s conditions. It is interesting that in chronic ACE blockade, (adm inistration of ACE in h ib ito rs C aptopril) aldosterone concentrations return to norm al values after an in itia l p h ase of depression.29,42 This return to a normal secretion of aldosterone by the zona glom erulosa of the adrenal cortex following captopril adm inistration has been interpreted as a possible synthesis of angiotensin-2 through pathw ays other than angiotensin-1 and ACE. In fact the presence of another type of converting enzym e, the B -converting enzym e or tonin, also capable of synthesis A2 has been reported.3 Furtherm ore, the stimulation and regulation of the zona glomerulosa, aldosterone secreting, can be caused not only by angiotensin-2 but by hormones of the zona fasciculata.42 This reveals the possibility of a vicarious function of the different zones o f the adrenal cortex upon a metabolic demand. It is known that in thermal injury serum cortisol is elevated, as well as ACTH.15,40 Another relevant observation provided by our study was th at no circ ad ia n rhythm of serum ACE could be identified in burned patients. There is no information know n to us concerning the circadian rhythm of ACE in physiologic conditions. The fact, however, that in burned patients a m odified daily rhythm w ith p a rallel tre n d exists b e tw e en ren in, angiotensin-2, and aldosterone m ay sugg est th a t sim ilar circad ian v ariatio n occurs for ACE. This alteration is present in burns for several hormones,6,35 and it may reflect the extensive changes in their metabolism and emphasize the complexity of the response of the RAA system in these conditions. S tatistically significant d ifferences w ere ev id en t in serum ACE activity w hen the enzyme levels of patient suffering from sm oke inhalation w ere com pared to those of patients suffering only from cutaneous burn. H igher activity of the enzym e was observed in two patients who w ere classified as smoke inhalation having cutaneous injury of u n a c co u n ta b le d eg re e. T h e enzym e levels were also found higher w hen the blood for ACE activity determ ination was draw n b e tw e e n six to 12 hours post injury. In the same patients, the ACE activity was m easured in the bronchial lavage. The variations of serum and bronchial lavage of ACE activity in smoke inhalation show marked similarities w ith the enzym e variations reported after radiatio n,41 ad m in istratio n of m onocrotaline,32,33,34 or bleom ycin.26,27 T hese variations are opposite to th o se found in

8 8 BRIZLO-MOLTENI, PIANO, WARPEHA, SOLLIDAY, MOLTENI, ET AL adults resp irato ry d istress syndrom e underlying that the serum and bronchial lavage ACE response is an effect of the smoke related injury and not related to the pattern typical of the respiratory distress syndrome.7 The findings in humans confirm the results of our study of smoke inhalation in dogs w here preferential exposure of one lung to smoke caused, w ithin m inutes, a marked increase of the enzyme levels in its parenchyma.8 The phases of increase, decrease, and return to high values of serum ACE in burned patients may reflect and parallel similar events in the lung. An interesting report is the autoptic finding20 of the presence of giant endothelial cells, capillary proliferation, interstitial fibrosis, intraalveolar fibrosis as early as 24 to 48 hours post therm al injury w ith smoke inhalation. These giant endothelial cells could b e a new source of ACE productio n. I t can also b e c o n s id e re d an increased response of the endothelium to a noxious stimulus, with an alternation of a phase of hyperactivity, exaustion, and then recovery of the secretion of the converting enzym e. In bleom ycin-treated mice, when the drug is w ithdraw n w ithin a time limit including only a m oderate injury of the pulm onary parenchym a (characterized by parvicellular infiltration, early and m ild fibrosis and low ACE lung and serum activity), the serum and lung enzym e levels rise again to normal or slig h tly above norm al w ith in a few days.27 A sim ilar process m ay occur in h u m a n s fo llo w in g e x p o s u re to smoke inhalation. Conclusions of the present study show that cutaneous thermal injury and smoke inhalation do not have the same influence on the serum ACE activity. The enzyme, although a primary component of the RAA system, does not duplicate in this type of injury the changes of the other components of the same system: renin and aldosterone. M onitoring of the converting enzym e activity w ould be useful for the evaluation of the pulm o nary damage caused by smoke inhalation. Such damage could later lead to chronic complications like pulm onary hypertension and pulm onary fibrosis. Acknowledgment Thanks are extended to Ms. Peggy Adams for her secretarial assistance. References 1. B e c k e r, R. A., Va u g h a n, G. M., G o o d w i n, C. W., et al.: Plasma norepinephrine, epinephrine and thyroid hormone interaction in severely burned patients. Arch. Surg. 115: , Bir k e, G., Bu n e r, H., L il i h e d a h l, S. O., et al.: Histamine, catecholamines and adrenocortical steroids in burns. Acta Chir. Scand. 114:87-92, B o u c h e r, R., As s e l in, J., and G e n e s t, J.: A new enzyme leading to the direct formation of angiotensin II. Circ. Res. (Suppl. l):i: , Bozovic, L., C a s t e n f o r s, J., E k l u n d, J., et al.: Studies on burns: XIV plasma renin activity correlated to renal function in burned patients. Scand. J. Urol. Nephrol. 6: , B r iz io -M o l t e n i, L., M o l t e n i, A., C l o u t ie r, L. C., and R a in e y, S.: Incidence of post-burn hypertensive crisis in patients admitted to two burn centers and a community hospital in the United States. Scand. J. Plastic. Reconst. Surg. i 3:21 28, B r iz io -M o l t e n i, L., W a r p e h a, R. L., A n g e - LATS, J., and L e w is, N.: Endocrine aspects of thermal injury. Cont. Surg. 18:47 64, C a s e y, L., Kr ie g e r, B., Ko h l e r, J., R i c e, C., O p a r il, S., and SziDON, P.: Decreased serum angiotensin converting enzyme in adult respiratory distress syndrome associated with sepsis: A preliminary report. Critical Care Med. 9: , Br iz i o -M o l t e n i, L., P ia n o, G., R i c e, P. L., W a r p e h a, R., F r e s c o, R., S o l l id a y, N. H., and M o l t e n i, A. Effect of wood combustion smoke inhalation on angiotensin-l-ce in the dog. Ann. Clin. Lab. Sci. 14: , C l a r e m o n t, D. J. and Br a n t h w a i t e, M. A. Metabolic indices of pulmonary damage changes in angiotensin converting enzyme and alpha! antitrypsin activity after cardiopulmonary bypass. Anesthesia 35: , C l a r k, W. R., M o l t e n i, A., N e i m a n, G., Br iz io -M o l t e n i, L., and So l l id a y, N. Effect of acute smoke inhalation on angiotensin con

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