Elastin content of normal and emphysematous lung parenchyma

Elastin Content of Normal and Emphysematous Lung Parenchyma

P. CHRZANOWSKI, M.D. S. KELLER, Ph.D. J. CERRETA. Ph.D. I. MANDL, Ph.D. G. M. TURINO. M.D. New York, New York

From the Departments of Medicine and Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York New York. This work was supported by Grants from the National Institutes of Health NHLBI HL15832, The New York Lung Association and the Stony Weld-Herbert Fund. Requests for reprints should be addressed to Dr. G. M. Turino. Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. Manuscript accepted March 24, 1980.

Elastin destruction has been recognized as a crucial factor in the development of experimental pulmonary emphysema. However, no consistent alterations in lung elastin have as yet been demonstrated in human subjects with diffuse panacinar emphysema. In this study elastin content was measured in pulmonary parenchyma obtained by surgical biopsy and postmortem from eight patients with clinical, physiologic and morphologic criteria for panacinar emphysema, and postmortem from six normal adults. Concentrations of the elastin-specific amino acids, desmosine and isodesmosine, were determined both in the crude connective tissue of lung parenchyma and separately in elastin isolated by a modification of the Lansing method. Elastin proportions were then expressed as percent of lung parenchymal crude connective tissue. In lungs of six normal control subjects mean elastin was 30.5 percent standard deviation (SD) f 3.69 (range 25.1 to 35.0 percent) In five patients the proportions of lung elastin were all below control: mean 12.1 percent SD f 3.7 (range 9.0 to 17.5 percent). Three patients of this group had Pi phenotype MM; one had ZZ and one had MZ. In three additional patients (one with Pi Mz; two with Pi MM), all of whom had received glucocorticoid therapy (21 months to nine years), elastin proportions were similar to those observed in normal subjects: mean 36.6 percent SD f 5.96 (range 31.7 to 43.4 percent). The amino acid composition of isolated elastin did not differ significantly between any of the groups. Results demonstrate reduced proportions of lung elastin in pulmonary emphysema except in patients treated with glucocorticoids which may decrease collagen synthesis and increase the relative proportions of elastin. Elastin plays a major role in the structure and function of the parenchyma of mammalian lung. However, estimates of elastin content in lung have varied widely (1.3 to 47 percent of dry lung weight), probably as a result of different methods of analysis, nonuniform sampling of lung and variable ages of subjects [l-7]. Wright [8] has indicated that elastic fibers in patients with emphysema, diagnosed clinically and morphologically, appear abnormal and reduced in amount. Also, studies of experimental emphysema have shown that only enzymes with elastolytic properties will produce the physiologic and morphologic changes of emphysema [g-13]. Both in this laboratory in rats and dogs [l4], and in the laboratory of Kuhn et al. in rats and hamsters [IS], the elastin content of lung parenchyma in emphysema induced experimentally was decreased. However, previously published reports [16-191 show no decrease in the content of elastin in lungs from patients with emphysema, with the exception of walls of lung bullae [ZO]. Quantitation of lung elastin has usually been attempted by gravimetric methods. However, small amounts of elastin measured by these

September 1980

The American Journal of Medicine

Volume 89

351

ELASTIN

CONTENT

TABLE I

Case No.

OF LUNG

PARENCHYMA-CHRZANOWSKI

Clinical Data and Lung Morphology in Patients with Pulmonary Emphysema

Age (yr) and Sex 17.F

Dyspnea Mild

Sputum None

1 yr

38.F

Severe

None

3 yr 71,F

Severe 12yr

Minimal

Moderate

Minimal

2 yr 55,F

Severe 12 yr

62.F

Severe

Copious

9 yr

77,M

Severe

None

6 yr 72,F

Severe

None

7 yr

l

ET AL.

ChestFilm Hyperinflation low diaphragms increased PA diameter Advanced bullous emphysema Extensive bilateral bullae Extensive lateral bullae Prominent pulmonary artery, low diaphragms, increased basilar markings Low diaphragms, hyperlucency, increased PA diameter Low diaphragms, bullae in lower lobes Low diaphragms, hyperlucency, increased PA

Biopsy Moderate to severe panacinar emphysema Panacinar emphysema

SerumTrypsin Inhibitory ct’, Antitrypsin Smoking Capacity Pi Phenotype (mglml serum) History’

Steroid Therapy

MZ

0.72

None

MM

1.01

None

None

Extensive panacinar emphysema, acute bronchitis Wall of bulla

MM

2.46

None

None

MM

0.81

40-O

None

Panacinar emphysema

22

0.25

None

None

Panacinar emphysema

MM

1.30

80-7

Chronic

Moderate to severe panacinar emphysema Moderate to severe emphysema

MZ

1.14

130-7

Chronic

MM

2.54

95-10

Chronic

Signifies pack-years smoked and years before lung tissue analysis when smoking was stopped.

methods may be affected by the presence of insoluble inorganic materials and losses in handling. In the present investigation we utilized a nongravimetric method in which lung elastin content was determined on the basis of the proportions of crosslinking amino acids specific to elastin, desmosine and isodesmosine, in total connective tissue of human lung. Elastin proportions in normal lung are compared with those of patients with pulmonary emphysema with both MM and non-MM alpha*-antitrypsin phenotypes. METHODS

AND

MATERIALS

Six normal lungs from three adult men, aged 39, 50 and 55 years, and from three adult women, aged 26,45 and 69 years, were obtained from the New York Medical Examiner and the Columbia Presbyterian Medical Center Department of Pathology. Each subject had an accidental or sudden death, and none had histories of previous cardiorespiratory disease. Pulmonary morphology was normal, grossly and microscopically. Lung tissue for analysis was obtained also from eight patients from the Columbia Presbyterian Medical Center with the clinical and morphologic diagnosis of pulmonary emphysema. They were a 37 year old woman (Case 2) and a 47 year old man 352

September 1980

The American Journal of Medicine

[Case 4) who underwent surgical resection for bullae, a 71 year old woman [Case 3) from whom tissue was obtained both during lung surgery and postmortem, a 17 year old woman [Case 1) who had a diagnostic lung biopsy and was subsequently found to have MZ alphal-antitrypsin phenotype, a 55 year old woman (Case 5) with known ZZ alphal-antitrypsin phenotype, a 72 year old woman (Case 8) with known MM phenotype and long-standing pulmonary emphysema, a 77 year old man (Case 7) with MZ alphal-antitrypsin phenotype and a 62 year old woman (Case 6) with MM phenotype. In each patient emphysema was classified as panacinar and was generalized over the whole specimen. None of the specimens could be classified as centrilobular emphysema. Brief clinical summaries are presented in Table I. Three patients (Cases 6, 7 and 8) had received glucocorticoids in, the form of oral prednisolone for airway obstruction and generalized debility. The ranges and duration of doses were 20 to 40 mg/day for three years (Case 6); 40 mg every other day for 21 months (Case 7) and 15 to 30 mg/day for nine years (Case 8). Pulmonary function tests included spirometry by a 13 liter Collins Spirometer (Warren E. Collins, Braintree, MA) with the patient in a sitting position, measurement of residual volume by helium dilution [21], arterial blood gas composition by an Instrumentation Laboratories oxygen tension (POz],carbon dioxide tension (Pco,) and pH analyzer (Instrumentation Volume 88

ELASTIN CONTENT OF LUNG PARENCHYMA-CHRZANOWSKI

IIL 3

RANGE

25-35

NUMBER OF SAMPLES

-.

.

-.

-

5.4-9.0 2

I

.

4

16-21

-

3

I

5

14-19 3

6 35-40 2

7 32-51 6

ET AL.

6 36-43 4

_

Figure 1. tlastin content of lung parenchyma expressed as a percentage of crude connective tissue in six normal subjects and eight patients with pulmonary emphysema. Three patients (Cases 6,7 and 6) (dotted bar) received regular doses of glucocorticoids as part of their therapy. The height of the bar for the control subjects is the mean value. The height of the bar for each patient is the mean value from analysis of the number of samples indicated.

Laboratories, Lexington MA] and single breath diffusing capacity for carbon monoxide by the method of Forster et al. [22] using a Beckman Instruments carbon monoxide analyzer. Morphologic Appearance and Lung Tissue Sampling. Emphysema was diagnosed from the gross and microscopic appearance of lungs [23,24] which was consistent with clinical, physiologic and roentgenologic manifestation. Multiple samples were analyzed from each lung except in two instances (Cases 1 and 4) in which tissue samples were small [Figure 1). Since all tissue samples were obtained either at biopsy during surgery or as portions of whole lung obtained at autopsy, standard inflation procedures prior to fixation could not be carried out. Thus, data on point counting, mean linear intercept or internal surface area were not obtained. Descriptions of microscopic and gross appearance are from lungs fixed in the collapsed state at atmospheric pressure. Grossly, in all sections, there were areas of destruction of alveolar-containing portions of lung which resulted in loss of tissue and some bulla formation. A method of grading was adopted to describe areas from which samples were obtained. Thus, specimens classified as severe demonstrated loss of tissue with large cystic areas usually over 3 cm in diameter. There was absence of bronchial and vascular tissue in these regions. Specimens classified as moderate contained cystic areas averaging 0.5 to 2.5 cm in diameter. Specimens classified as mild showed general widening of air spaces with occasionally small cystic areas 1 to 5 mm in diameter. This classification is not applied as a descriptive term for the amount of over-all emphysema in whole lungs, or even in the region of the lung per se, since standard inflation could not be carried out. All microscopic sections taken from the lung showed alveolar septal destruction, thinning of alveolar septa and wid-

ening of the alveolar lumen consistent with pulmonary emphysema. Determination of Elastin Content. Lung elastin was isolated by the method of Lansing [25] as modified in this laboratory. Lung parenchyma is mechanically separated from the pleura, large airways and blood vessels. It is minced, homogenized in an all-glass apparatus and stirred for 24 hours in 1 M sodium chloride at 5OC to remove all salt-soluble proteins. The residue was washed three times with water to remove the salt, freeze-dried and then extracted three times with n-butanol at 5% and three times with acetone at 5% to remove lipids. The air-dried product, designated “crude connective tissue,” was extracted with hot alkali to remove collagen, glycosaminoglycans and microfibrils. Fifty milligrams of crude connective tissue were placed in a centrifuge tube along with 5 ml of 0.1 N sodium hydroxide. This tube was placed in a boiling water bath for 45 minutes then chilled to 5’C and centrifuged for 10 minutes at 1,100 X g. The supernatant was discarded, and the residue washed twice with 10 ml water. The final residue of elastin was suspended in 1 ml water and freeze-dried. For amino acid analysis, weighed samples of crude connective tissue (3 mg each] and purified elastin (1.5 mg each] were hydrolyzed in vacua in 2 ml 6 normal hydrochloric acid for 24 hours at 11O’C and dried in a desiccator in the presence of sodium hydroxide. Their amino acid composition was determined on a Beckman No. 120 Amino Acid Analyzer with type PA 35 resin in the short column and type AA 15 resin in the long column. Two modifications were made in the analyzer program in order to resolve all the amino acids present in connective tissue. To allow separation of hydroxyproline from the aspartic acid in the long column at 55’C the column was

September 1990

The American Journal of Medicine

Volume 69

353

ELASTIN CONTENT OF LUNG PARENCHYMA-CHRZANOWSKI

TABLE If

ET AL

Pulmonary Function in Patients with Emphysema’

De Age(yr), Case No.

TLC

Race and Sex 17,W,F 38,W,F 71,W,F 47,B,M 55,W,F 62,W,F, 77,W,M 72,W,F

3.3 1.7 0.7 3.3 1.0 1.3 2.Q 2.0

3.3 3.7 2.3 4.5 2.6 2.6 4.0 3.0

Pred

FEY,

FEV,lVC x100

(lters)

(ItIers)

b-W

5.5 6.0 2.3 9.0

4.4 5.5 4.1 6.8

52

3:;

5:;

5:;

1.5 0.6 0.5 1.6 0.5 0.4 0.5 0.5

45 35 71 48 50 30 25 25

RV Ph

Pace,

(ton)

(twr1

UtW

ohs

NW

83 68 62 85 71 64 41 48

35 38 43 35 34 46 57 42

2.2 4.8 1.6 5.6

1.1 2.0 1.9 2.3

15 12

23 23

... ...

... ...

39

G

4*’

‘16

319

2:;

1::

1::

Pred

~~~,

m HaI

~~

mmtW

NOTEz VC = vital capacity; TLC = total lung capacity; FEV, = forced expiratory volume in 1 second; Pao, = arterial PO*; Pace? = arterial Pco,; RV = residual volume; Df$ = single breath diffusing capacity for carbon monoxide; Obs = observed; Pred = predicted. l Sieady state.

eluted with 0.2 M citrate buffer pH 3.08 containing 2 percent propanol for the first 111 minutes. The usual 0.2 M citrate buffer pH 4.25 was used from 111 to 162 minutes. From 162 to 360 minutes a 0.35 M citrate buffer was used to elute desmosine and isodesmosine. Desmosine and isodesmosine residues are expressed in lysine equivalents divided by a factor of 4 ac-

cording to standard amino acid analyzer procedure. The elastin proportions of the crude connective tissue fraction are calculated from the proportions of desmosines determined in the crude connective tissues and in the purified elastin. The concentration of desmosine and isodesmosine(in residues per 1,000) found in crude connective tissue is divided by the concentration of desmosine plus isodesmosine (in residues per 1,000) found in the purified elastin from the same specimen. Since desmosine and isodesmosine are elastin specific, this ratio times 100 expresses the percentage of elastin in the crude connective tissue of lung parenchyma. Proteinase Inhibitor Studies. Patients were Pi phenotyped by acid-starch gel and antigen-antibody electrophoresis [26]. In addition, serum trypsin inhibitory capacity was determined using the benzoyl arginine p-nitroanilide substrate of Erlanger et al. 1271.

RESULTS The clinical and morpholo~c data from the eight patients studied are summarized in Table I. Patients range from 17 to 77 years. Dyspnea ranged from mild and of one year’s duration to severe of 12 years’ duration. One subject (Case 5) complained of significant sputum production. Chest roentgenograms were all consistent with emphysema as demonstrated by hyperlucency, low flat diaphragm and increased anterior posterior diameter of the chest. Biopsy and autopsy sections of lungs demonstrated moderate to severe panacinar emphysema as already described. The serum trypsin inhibitory capacity was in the intermediate low range (0.72 mg/ml serum) in one patient (Case 1) with phenotype MZ and severely decreased (0.25 mg/ml serum) in one patient [Case 5) with phenotype ZZ. One patient (Case 7) had a trypsin inhibitory capacity in the normal range (1.14 mg/ml serum) despite

354

September 1980 The American Journal of Medicine

an MZ phenotype possibly due to chronic treatment with glucocorticosteroids. The pulmonary function data on the eight patients with emphysema are summarized in Table II. All patients had moderate to severe airway obstruction with a mean 1 second forced expiratory volume (FEV1) of 1 liter and mean FEV*:FEV of 41 percent. With the exception of one patient (Case 4). all patients had reduced arterial oxygen tension, PaOa, and two patients had abnormally high arterial carbon dioxide tension (PaCO& In each of the four subjects in whom residual volume was measured, it was abnormally high, The reduction in total lung capacity in one patient (Case 3) was the result of bullous disease which was extensive and gave a low residual volume on the basis of the helium washout technique. In each of three patients in whom diffusing capacity was measured, it was abnormally low. As can be seen from Table III and Figure 1, the mean elastin proportion expressed as a percentage of crude connective tissue of lung parenchyma is 30.5 percent SD f 3.69 (range 25.1 to 35.0) in the group of six normal lungs. The elastin proportions of the eight patients with pulmonary emphysema, also shown in Table III and Figure 1, fell into two groups: Group 1 is composed of five subjects in all of whom the relative elastin contents were statistically significantly below the range in normal subjects, with no overlap; group 2 is composed of three patients who had been treated with corticosteraids with elastin percentage in the same range as in the normal subjects. In group 1,the mean proportion of elastin was 12.1 percent SD fi 3.70 (range 9.0 to 17.5). In group 2, the mean percent elastin was 36.8 SD f 5.98 (range 31.7 to 43.4). The major differences in clinical course which distinguished the patients of group 1 from the ones in group 2 was the usa of oral corticosteroids for several years in the patients of group 2. This is discussed herein. The amino acid ~om~sition of total crude connective tissue of lung parenchyma is shown in Table III for all control subjects and patients. It can be seen that the desmosine and isodesmosine content in the five patients

Volume 69

37.4 67.4 46.6

68.0 111.4

56.3 20.3 45.5 12.3

37.9 53.1 42.9 23.4 32.9 63.4 101.7 294.0 139.6 59.7 21.9 49.8 15.0 20.8

36.8

65.1

45.3

22.2

33.2

67.2

111.2

284.3

132.7

60.5

20.4

47.0

12.4

19.7

Arginine

Hydroxyproline

Aspartic acid

Threonine

Serine

Glutamic acid

Proline

Glycine

Alanine

Valine

lsoleucine

Leucine

Tyrosine

Phenylalanine

25.1

l

Results expressed in amino acid residues/1000 residues. t NP:P ratio = nonpolar to polar amino acid ratio.

27.4

30.9

3.8

3.2 10.3

3.8 30.8

9.0

3.5

17.5

3.2

6.8

9.9

3.3

6.7

4.2 0.9 5.3 4.0 43.4

2.8 3.1 31.7

3.6 3.4

3.8 2.7

3.8 3.4 7.0

35.4

35.0

33.5

3.4

5.6

3.6

5.9

0.19

13.6

Percent elastin

3.54

4.3

7.3

1.1

2.7 1.9

1.6

4.7

0.32

0.16 4.6

0.24 6.0

3.0

3.9

3.7

6.9

8.3

6.3

4.3

0.56

4.2

NP:P ratio+

Methionine sulfoxide

7.2

1.9

4.96

0.50

hkthionine

6.5

6.8

2.3

5.6

0.46

4.2

5.1

2.1

Cysteic acid

5.0

0.43

4.9

3.3

4.5

Hydroxylysine

Cystine

0.48

0.59

Deskcdesmosine

0.62

22.3 22.1 16.9

4.9

16.6 21.4

16.1 17.2

12.0

0.39

51.5 53.1 54.6

36.6

4.2

14.4

22.2

22.9 22.7 18.4

3.2

19.5

14.2

6.5

31.1

70.9

59.9 64.1 35.8

2.0

12.2

43.6

6.9

29.4

14.5

139.8

123.0

3.0

13.4

5.2

28.1

19.3

13.5

32.6

273.4 255.5 130.4 114.4

257.3

290.4 113.4

326.5

63.0 109.2

114.2 112.0

112.7

31.9 71.3 75.4

71.1

107.5

23.2 37.7 35.8 35.1

34.8

75.8

41.5 26.9 26.6

21.8

18.1

53.4 52.2

55.2 46.5

86.9

53.1

38.7 38.5

50.1 46.4

5.4 34.1

7.1 7.4

6.5 79.8

20.6 25.2

24.5

25.3

48.2

44.1

6.1

23.4

4.2

21.5

14.9

51.1

13.1

48.9

28.1

115.8

282.3

346.3 113.7

112.3

108.1

75.3

39.8

33.5 64.6

24.3

51.7

17.9

72.5

41.1

39.5

47.2 90.9

6.9

23.3

7.0

24.4

Corticosteroid Therapy Case 6 Case 7 Case a

0.56

20.3

12.6

46.2

21.8

67.0

57.8 20.6

117.4

138.4 27.4

319.5

273.6

118.9

70.1

63.7 110.3

35.3

17.1

33.1

23.8

46.8

98.7

57.5 45.2

45.5

6.1

6.8 34.4

25.0

23.2

131.8

286.8

112.1

63.5

31.8

22.1

44.2

65.0

35.8

6.7

22.6

Case 1

6

5.7

19.1

11.4

45.0

19.8

54.3

126.4

285.3

109.3

69.7

35.2

23.3

46.3

69.9.

38.1

6.9

22.8

5

Patientswith Emphysema No Corticosteroid Therapy Case 2 Case 3 Case 4 Case 5

0.23

19.3

129.6

254.4

33.8

22.2

6.8

tr

24.2

28.3

6.2

Lysine

21.8

2

1

NormalSubjects 3 4

Amino Acid Composition of the Crude Connective Tissue Fraction of Lungs of Normal Subjects and Patients with Pulmonary Emphysema+

Histidine

TABLE III

ELASTIN CONTENT OF LUNG PARENCHYMA-CHRZANOWSKI

in group 1 (mean 0.22 residues/l,000 residues f SD 0.045) is significantly below that in control subjects (mean 0.52 f SD 0.075) (p
356

September 1960 The American Journal of Medicine

ET AL.

and Case 4) in group 1 of the present study who showed significantly decreased elastin proportions. These analyses indicated also that neither elastolytic damage nor age affected the susceptibility of the pulmonary elastin to alkali degradation and are consistent with the findings of essentially unchanged amino acid composition in the purified elastin (Table IV]. The separation of elastin from the crude connective tissue by the Lansing technique and quantitation of amino acid residues from the isolated elastin in each specimen (rather than assuming a constant desmosine-isodesmosine content on the basis of a single elastin standard [28] increases the accuracy of the method, particularly in disease states in which elastin structure may be altered. The method can be performed on small masses of tissue in the range of 1 g or even less with no loss in accuracy. It is, therefore, suitable for analysis of lung biopsy specimens. The reduced proportion of lung parenchymal elastin in the five patients of group 1 did not overlap the range of values in the controls. Also, in two patients (Cases 3 and 5) in whom multiple samples were Qbtained from autopsy lungs there was a good correlation between the gross appearance of emphysematous destruction of lung parenchyma and the reduction of the proportion of lung elastin with the wall of bullae being lowest. The reduced elastin proportions in the five patients of this study are consistent with morphologic data from human lungs which show reduced and disrupted elastin in human subjects [8] and with data from animals in which emphysema was induced by intratracheal and intravenous [14,29] administration of elastases and papain. The results in these five patients are also consistent with data obtained in experimental animals indicating that elastin is a primary target in the pathogenesis of emphysema whereas collagen alteration does not lead to alveolar wall destruction characteristic of morphologic pulmonary emphysema [9,11]. Although it is possible that reduced elastin could result from inordinate increases in lung collagen, this seems unlikely in the absence of increased collagen morphologically and the reduction in elastin that occurs with acute induction of experimental emphysema [l&29]. Thus, Kuhn et al. [l5] showed a significant although transient destruction of elastin after intratracheal administration of a single dose of elastase. As the animals recovered, elastin was resynthesized and normal levels were restored. In human emphysema, unlike this model, the insult continues over many years and degradation of lung elastin by endogenous enzymes may exceed its biosynthesis resulting in depletion of elastin. In the three patients whose elastin proportions were in the upper limits of the normal values the role of corticosteroids in altering collagen and elastin synthesis must be considered. Several studies have demonstrated suppression of collagen synthesis when whole animals or cell cultures have been exposed to clinical doses of

Volume 69

9.8

16.0

17.5

0

0

0

1.57

22.5

20.6

61.5

24.4

131.3

234.6

293.2

128.0

23.0

8.4

9.4

8.3

14.5

7.9

1.7

9.1

3

21.1

0

0

0

1.83

22.9

20.9

62.4

24.8

136.8

235.6

294.3

126.7

20.6

8.4

9.4

6.7

14.3

6.7

1.1

6.9

4

l

Results expressed in amino acid residues/ 1,000 residues. t NP:P ratio = nonpolar to polar amino acids.

21.9

0

Methionine sulfoxide

NPP ratio+

0

0

Cysteic acid 0

0

0

1.36

22.6

Hydroxylysine

22.7

Phenylalanine

21.2

1.76

21.1

Tyrosine

59.1

23.7

118.6

236.9

312.2

Deskodesmosine

62.5

Leucine

135.4

Valine

24.9

239.0

Alanine

lsoleucine

125.8

295.8

Proline

Glycine

117.2

8.2

Serine

11.1 23.1

9.0

Threonine

9.5

12.2

9.3

1.9

10.5

19.4

6.3

Glutamic acid

l-3.6

6.7

Arginine

Aspar-tic acid

1.2

Hydroxyproline

6.7

Histidine

NormalSubjects 1 2

18.7

0

0

0

1.62

23.1

22.8

21.6

0

0

0

1.82

23.3

21.7

62.2

24.5

24.3 60.8

136.9

236.3

295.3

126.0

20.0

8.1

6.8

6.0

14.1

6.4

1.1

7.2

6

132.3

233.1

294.7

127.4

21.0

9.0

10.4

8.0

13.8

7.5

1.4

8.9

5

23.9

0

0

0

2.34

22.0

11.6

55.1

22.4

121.2

239.2

327.8

127.0

19.1

9.6

9.5

5.9

15.1

6.0

0.9

5.3

Case 1

21.8

0

0

0

1.79

19.7

14.5

57.6

24.9

117.0

236.9

331.3

119.9

19.6

9.2

9.1

6.1

17.3

6.8

1.0

7.2

16.9

0

0

0

1.83

23.2

22.9

62.6

25.1

134.3

230.8

282.9

132.9

23.9

9.5

10.5

8.8

12.7

8.7

1.7

8.4

17.9

0

0

0

1.92

23.6

20.2

61.9

25.7

131.6

225.5

303.3

121.2

23.9

9.4

10.9

8.6

16.2

7.9

1.5

6.6

16.5

0

0

0

1.69

21.7

23.7

52.9

23.0

107.8

228.6

319.6

135.7

23.3

9.4

10.2

8.3

13.3

8.9

1.9

10.1

Patientswith Emphysema No Corticosteroid Therapy Case 5 Case 2 Case 3 Case 4

Amino Acid Composition of the Elastin Fraction in Normal Subjects and Patients with Pulmonary Emphysema*

Lysh-te

TABLE IV

14.2

0

0

0

1.58

23.1

24.2

62.8

23.8

125.7

222.1

288.2

131.6

25.7

9.8

11.1

10.3

15.8

10.4

2.4

11.4

12.0

0

0

0

1.23

23.6

24.9

63.6

25.4

128.2

220.8

275.4

127.9

29.3

12.3

12.7

14.1

13.9

12.1

2.5

12.0

13.6

0

0

0

1.43

23.7

23.8

62.4

25.1

129.7

225.5

283.4

126.3

27.1

11.1

11.6

12.6

13.2

10.8

1.6

10.9

Corticosteroid Therapy Case 6 Case 7 Case 6

ELASTIN CONTENT

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corticosteroids [30-331. Recently Kruse et al. [32] demonstrated that corticoids inhibited collagen synthesis in mouse sponge granulomas both by decreasing the number of fibroblasts and by diminishing collagen synthesis per cell. On the other hand, Eichner and Rosenbloom [33] observed that the corticosteroids depress collagen synthesis but enhance elastin biosynthesis in chick embryo aorta. Since, in the three patients presented here, corticosteroids in the form of oral prednisone had been administered for at least 21 months uninterruptedly, it is suggested that the increased proportion of elastin could result from suppressed collagen synthesis whereas elastin synthesis remained relatively normal. The low hydroxyproline content in the crude connective tissue fraction of the three patients in group 2 is suggestive of reduced collagen synthesis in the lung parenchyma of these patients. It is noteworthy that lung connective tissue from this group contains fewer hydroxyproline residues than tissue from normal subjects with similar relative elastin proportions. There is little direct evidence from previous studies as to the effect of corticosteroids on elastin synthesis in any organ system. The substantial increase in elastin biosynthesis observed in corticosteroid-treated chick aorta [33] with concomitant reduction in collagen synthesis lends support to one possible mechanism by which normal proportions may have been restored in the treated patients. The present data, however, do indicate an inability of elastin synthesis to reconstitute a normal proportion of elastin of approximately 30 percent of crude connective tissue in the patients with emphysema of group 1. The possibility exists that continuous breakdown of elastin in situ exceeds the capacity of cells to resynthesize elastin or, possibly, cells responsible for elastin synthesis may be functionally abnormal. In this regard the precise cell types in lung parenchyma responsible for elastin synthesis remain unknown. Since vascular smooth muscle cells have this capability, as demonstrated in aortic wall smooth muscle [34,35], it is conceivable that the general reduction of capillaries, arterioles and venules in emphysematous lung is a potential mechanism. However, decreased elastin synthesis may be only one mechanism resulting in decreased elastin proportions. The possibility exists

ET AL.

also that elastases may be elaborated in greater concentration or elastase inhibitors in decreased concentration in the lungs of patients with emphysema. Adrenal corticosteroids could alter this balance between tissue elastases and inhibitors rather than enhance net elastin synthesis and suppress collagen synthesis. It is of interest that in two patients with abnormal alphal-antitrypsin phenotypes, a 17 year old girl with Pi MZ (Case 1) and a 55 year old woman with Pi ZZ (Case 5), elastin proportions are reduced to a similar extent as in the other three patients who had MM Pi phenotypes. Similarly in the one patient of group 2 with an abnormal phenotype, a 77 year old man with Pi MZ (Case 71, the elastin proportions were not significantly different from those of the other two patients who received glucocorticoid therapy. This suggests that the end result of destruction of lung elastin in patients who are predisposed to emphysema because of abnormalities in alphal-antitrypsin phenotype is similar to that in patients with emphysema who have the Pi MM phenotype. In this regard, it is relevant that the patient (Case 1) who had a very low proportion of elastin is a young nonsmoking heterozygote with advanced emphysema morphologically, suggesting that additional variables are operating in the pathogenesis of emphysema in at least some heterozygotes. The amino acid composition and the nonpolar to polar ratios of the purified elastin from the lungs of patients with pulmonary emphysema were not significantly different from control lungs. This result is consistent with the concept that people with MM or non-MM phenotype in whom pulmonary emphysema develops, do not have an abnormal pulmonary elastin structure as a predisposing factor. This observation is also consistent with the concept that emphysema develops because of interruptions in the continuity of parenchymal lung elastin in locations such as the alveolar duct or the openings of alveoli from the alveolar duct in which elastin may have a critical structural role. ACKNOWLEDGMENT We acknowledge with thanks the helpful advice of Dr. Stephen F. Ryan in the preparation of this manuscript.

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