Neutrophil Lysosomal Elastase Activity in Normal Subjects and in Patients with Chronic Obstructive Pulmonary Disease" J. R. RODRIGUEZ, J. E. SEALS, A. RADIN, J. S. LIN, I. MANDL, and G. M. TURINO
SUMMARY Neutrophil lysosomal elastase activity was measured in a group of 31 asymptomatic adults (12 men, 19 women) with Pi M phenotype and normal lung function, 9 subjects (7 men, 2 women) with normal lung function and phenotypes other than Pi M, and a group of 42 patients (25 men, 17 women) with chronic obstructive pulmonary disease, 35 of whom had Pi M phenotype and 7 phenotypes associated with alpha^antitrypsin deficiency. Chronic obstructive pulmonary disease was diagnosed by history and radiologic, Spirometrie, and blood gas characteristics of chronic airway obstruction. Neutrophil lysosomal elastase activity was measured using oxalic-acid solubilized, ligamentum nuchae elastin substrate. The average 570 nm A absorbency per min per fig of protein for this activity in control subjects with M phenotype was 50 ± 15.6, corresponding to 793 ± 242 microunits of pancreatic elastase per fig of lysosomal protein as compared to A absorbency per min of 71 ±32.9, which corresponds to 1,125 ±522 microunits of pancreatic elastase equivalents per fig of lysosomal protein in patients with chronic obstructive pulmonary disease (P < 0.001). If the patients with chronic obstructive pulmonary disease who have phenotypes other than Pi M are excluded, the mean A absorbency per min of the patients with Pi M phenotype is 73.4 ± 33.7, which corresponds to a pancreatic elastase equivalent activity of 1,163 ± 5 3 4 microunits per fig of lysosomal protein. Reproducibility of neutrophil lysosomal elastase activity was determined by repeat tests in the same subjects at intervals of one to 12 months and showed a coefficient of variation of 21 per cent for control subjects and 38 per cent for patients with chronic obstructive pulmonary disease. No differences between smokers and nonsmokers were demonstrable in neutrophil lysosomal elastase activity in either normal subjects or patients. Significantly greater degrees of neutrophil lysosomal elastase activity in patients with Pi M phenotype and chronic obstructive pulmonary disease suggest a possible role for circulating elastase activity as a mechanism for damage to lung elastin in the pathogenesis of pulmonary emphysema.
Introduction In recent years, it has been demonstrated that pancreatic elastase induces changes in the mechanical characteristics of the lung (1-3) and the morphology of lung parenchyma that re-
(Received in original form April 14,1978 and in revised form December 4y 1978) i From the Departments and Medicine and Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York, N. Y. 2 Supported in part by Program Project Grant No.
semble those observed in human pulmonary emphysema (4, 5). Also, proteolytic enzymes with elastolytic activity have been isolated from human polymorphonuclear leukocytes (6), platelets (7), alveolar macrophages (8), and peritoneal macrophages (9). These cells may transgress or
HL 15832 from the U. S. Public Health Service and by grants from the Stony Wold-Herbert Fund and the Council for Tobacco Research. 3 Presented in part at the Annual Meeting of the American Thoracic Society, Montréal, Québec, May 1975.
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 119, 1979
409
410
RODRIGUEZ, SEALS, RADIN, LIN, MANDL, AND TURINO
reside in the lung. N o r m a l h u m a n circulating p o l y m o r p h o n u c l e a r leukocytes have been shown to have elastase-like esterase activity (10), a n d it has been suggested that persons with a deficiency of serum alpha 1 -antitrypsin whose l u n g function is n o r m a l have decreased concentrations of neutrophil elastase. Moreover, p r e p a r a t i o n s of neutrophils have b e e n shown to p r o d u c e m o r p h o logic destruction of l u n g p a r e n c h y m a (11). T h e present study was u n d e r t a k e n to determ i n e the degree of circulating p o l y m o r p h o n u clear leukocyte lysosomal elastase activity in a g r o u p of asymptomatic adults with n o r m a l l u n g function, in a g r o u p of patients with chronic obstructive p u l m o n a r y disease (COPD), a n d in a g r o u p of patients with a l p h a ^ a n t i t r y p s i n deficiency with a n d w i t h o u t evidence of C O P D . T h e d a t a were analyzed to d e t e r m i n e the relationship between the presence a n d severity of C O P D a n d concentrations of n e u t r o p h i l lysosomal elastase. R e p e a t d e t e r m i n a t i o n s of n e u t r o p h i l lysosomal elastase activity at intervals of o n e to 12 m o n t h s are also presented for a p o p u l a t i o n of n o r m a l subjects a n d a g r o u p of p a t i e n t s with C O P D .
Materials and Methods Subjects. Normal subjects were 31 asymptomatic persons who were demonstrated to have a normal spirogram and chest roentgenogram. In addition, all of these subjects were shown to have the Pi M alpha^antitrypsin phenotype and normal trypsin inhibitory capacity (mean and SD of 1.19 ±0.29 mg of trypsin inhibited per ml of undiluted serum). Most of the subjects were professional colleagues and laboratory personnel. Their mean age (± SD) was 43.3 ± 13.3 years; 12 subjects were women, and 19 were men. T h e patient group was comprised of 42 persons from the inpatient and outpatient departments of the Presbyterian Hospital. The mean age (± SD) was 56 ± 14 years; 17 patients were women and 25 were men. Of the 42 patients, 7 had phenotypes other than Pi M. Of those seven, 4 (3 men, one woman) had homozygous Pi Z phenotype, and 3 patients (2 men, one woman) had phenotypes MZ, MS, and FS, respectively. In addition, there were 9 subjects with no symptoms and normal lung function who had abnormal Pi phenotypes (7 men, 2 women). Of these nine, one had phenotype Pi Z, six had MS, and two had MX. The diagnosis of COPD was based on a history of shortness of breath, particularly on exertion, and physiologic evidence of airway obstruction. Criteria for inclusion of patients in the study were: (1) symptoms of shortness of breath for at least 5 years, and (2) evidence of airway obstruction that was not reversible; such evidence included pulmonary function tests that demonstrated vital capacity of 85 per
cent of predicted values or less, 1-sec forced expiratory volume (FEVX) of 60 per cent of predicted values or less, and ratio of residual volume to total lung capacity (RV/TLC) greater than 45 per cent and arterial Po 2 less than 75 mm Hg at rest. Patients were excluded who could be diagnosed as having allergic asthma by virtue of known sensitivities and the episodic nature of their dyspnea and disturbances in pulmonary function. Although common symptoms, cough and sputum were not considered criteria for inclusion. In 26 of the patients, the history of cough and sputum would meet the criteria for chronic bronchitis. However, in none of these patients was chronic bronchitis diagnosed alone. On the basis of decreased CO diffusing capacity (DLCO), increased RV/TLC, and increased TLC, emphysema was considered a concomitant abnormality. None of the patients could be classified as having episodic asthma, and there were no known allergies associated with the onset of shortness of breath. Shortness of breath on exertion was present in all subjects and was unremitting rather than episodic. The measurements of neutrophil lysosomal elastase activity, trypsin inhibitory capacity, and pulmonary function of patients were done at a time when each patient was considered free of exacerbations of pulmonary infection. However, most patients were considered to have some degree of chronic pulmonary infection related to chronic bronchitis. With respect to smoking history, only 2 of the 31 control subjects were smoking at the time of the study. Both were women and had smoked 25 and 35 pack-years, respectively. Other control subjects were nonsmokers. In the patient group, 8 of the 42 patients had never smoked. Thirty-four patients had smoked and had smoking histories that ranged from 20 to 85 pack-years (mean 53 ± 34 pack-years), but only two were still smoking at the time of study. These 2 patients smoked 40 and 35 pack-years. Thirty-two of the patients had stopped smoking at least 18 months before measurements of neutrophil elastase activity or pulmonary function testing. Spirometry was performed using a 13-liter spirometer (Warren E. Collins, Braintree, Mass.) with the patient in a sitting position. Arterial blood gas composition was measured by Po 2 , Pco 2 , and pH analyzer (Instrumentation Laboratories, Lexington, Mass.). Assay of neutrophil lysosomal elastase. T h e method of extraction and assay of neutrophil lysosomal elastase activity was the following: Fifty ml of venous blood were drawn by needle and syringe into a plastic tube containing 10 ml of acidcitrate-dextrose solution. Erythrocytes and lymphocytes were separated from leukocytes by sedimentation in 3 per cent dextran solution, after which the residual erythrocytes were lysed by suspension in hypotonic saline. T h e neutrophils and residual lymphocytes were
NEUTROPHIL LYSOSOMAL ELASTASE AND COPD
placed in 0.34 M sucrose solution for 30 min to weaken cell membranes. Cell counts at this stage yielded at least 92 per cent polymorphonuclear leukocytes. The solution was perfused through a Swinney apparatus and was centrifuged. The residual granules were exposed to freezing and thawing for 8 cycles to disrupt the granules. Centrifugation was done to separate the granular debris from the enzyme-containing supernatant. The supernatant was assayed immediately or was frozen at —70° C and stored. It has been demonstrated that storing at this temperature for 3 months did not result in any loss of elastolytic activity (Mandl, 1.: Unpublished observations). T h e elastase assay was carried out by a modification of the technique of Keller and Mandl (12), digesting 0.5 mg of oxalic-acid solubilized ligamentum nuchae elastin for 20 hours. T h e quantitation of the elastase activity for this substrate involves measurement by a colorimetric ninhydrin reaction (13) of the free amino groups released by scission of susceptible peptide bonds. Results are expressed as changes in the 570 nm absorbency at unit time per pg of lysosomal protein and in terms of pancreatic elastase equivalents as microunits (/¿u) per ¿ig. Pancreatic elastase equivalents are determined by comparing the observed change in absorbency with that obtained when a known amount of pancreatic elastase is used to digest the same substrate under the same conditions. Protein content was determined by the procedure of Lowry and associates (14). Results of neutrophil elastase activity expressed per unit leukocyte count of the patient's blood gave a wider range of variability of elastase activity and lower reproducibility. This was attributed to: (2) error in the blood leukocyte count per se, and (2) variability in the number of leukocytes represented in the final lysosomal pellet on which the assay for elastase activity was performed because of the several steps involved in the separation and isolation of the lysosomal fraction. An attempt to relate neutrophil elastase activity to markers of other neutrophilic enzyme activities such as /3-glucuronidase did not improve the limits of variability of elastase activity over the use of lysosomal protein per se. This observation suggests that elastase and ^-glucuronidase in neutrophils may be changing independently and that ßglucuronidase is not a suitable marker. Measurements of serum trypsin inhibitory capacity were made according to the method of Erlanger and associates (15). Phenotyping was done by the method of Fager hol and Laurell (16). RV was calculated from the Spirometrie expiratory volume and from the functional residual capacity measured by a steady-state helium dilution method (17). White blood cell counts and differential counts were done on each patient at the time blood was
411
drawn for measurements of neutrophil elastase activity. In selected cases, differential counts were also done after exposure to hypotonic saline and sucrose washing. Results T h e values for n e u t r o p h i l elastase activity a n d trypsin inhibitory capacity in each g r o u p are summarized in table 1. T h e average 570 n m A absorbency p e r m i n in the n o r m a l subjects with M p h e n o t y p e was 50 ± 15.3, c o r r e s p o n d i n g to 793 ± 242 ¡mu pancreatic elastase equivalents p e r /¿g of lysosomal p r o t e i n . T h e trypsin inhibitory capacity m e a s u r e m e n t in this g r o u p averaged 1.19 ± 0.30 m g of trypsin i n h i b i t e d per m l of undiluted serum. T h e n o r m a l average ( ± SD) trypsin inhibitory capacity in this laboratory is 1.08 ± 0.1 m g of trypsin i n h i b i t e d p e r m l of undiluted serum (18). T h e average value of n e u t r o p h i l lysosomal elastase activity in all the p a t i e n t s with C O P D , which includes those with Pi M p h e n o t y p e as well as those with other phenotypes, was significantly greater t h a n the m e a n values of the n o r m a l subjects with a m e a n A absorbency p e r m i n of 71 ± 32.9, corresponding to 1,125 ± 522 nu of pancreatic elastase per /xg of lysosomal protein (P < 0.001). T h e m e a n trypsin inhibitory capacity for this g r o u p was within n o r m a l limits, i.e., 1.19 ± 0.45 m g p e r m l of u n d i l u t e d serum. T h e n e u t r o p h i l elastase activity of C O P D patients with Pi M p h e n o t y p e demonstrates an even greater m e a n A absorbency per m i n of 73.4 ± 33.7, c o r r e s p o n d i n g to 1,163 ± 534 ¡m pancreatic elastase equivalent activity as shown in table 1. G r o u p i n g of all subjects with phenotypes other t h a n Pi M showed a m e a n A absorbency p e r m i n of 58.4 ± 27.4, c o r r e s p o n d i n g to pancreatic elastase activity of 925 ± 434 /m p e r fig, which was not significantly different from that of the control g r o u p . As expected, the trypsin inhibitory capacity of this g r o u p was significantly less at 0.62 ± 0.33 m g p e r ml. If all subjects with phenotypes other t h a n Pi M were divided further into those persons with C O P D a n d those w h o were asymptomatic a n d h a d n o r m a l Spirometrie a n d blood gas values, there was n o significant difference in m e a n values for n e u t r o p h i l lysosomal elastase activity within t h e subgroups. If the 9 patients with a b n o r m a l Pi p h e n o t y p e s b u t n o r m a l l u n g function are included in the control g r o u p , the m e a n 570 n m A absorbency per m i n is 53.3 ± 19.7, corresponding to 844 ±
4
1
3
8
Patients with COPO and ZZ phenotypes
Subject with normal lung function and ZZ phenotype
Patients with COPD and heterozygous phenotypes (MZ, MS, FS)
Subjects with normal lung function and heterozygous phenotypes (MZ, 6; MX,2)
Ch ron ic obstructive pu Imonary disease.
-*
7
16
All subjects with abnormal Pi phenotypes
Patients with COPD and abnormal Pi phenotypes
35
COPO patients with MM phenotype
9
42
All patients with CO PO *
Subjects with normal lung function and abnormal Pi phenotypes
31
(no.)
Control group
Group
Subjects
1
6
2
1
42-76 (61 ±17)
12-33 (17 ± 6)
1
2
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66.4 ±30.2
68.5
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51
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5
3
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12-51 (22±12)
58.4 ± 27.4
73.4 ± 33.7
71.0 ±32.9
50.1 ± 15.3
absorbency/ min)
12-76 (36 ±20)
27-80 (57 ± 14)
27-80 (56 ± 14)
18-67 (43 ± 13)
(range) (mean)
(,d
2
4
14
17
12
(F)
Age (years)
7
12
21
25
19
(M)
Sex
1,008 ±488
1,051 ± 479
1,085
624 ± 267
938 ±445
1,016 ± 457
925 ± 434
1,163 ± 534
1,125 ± 522
793 ± 242
(J.Lu pancreatic elastase equivalents)
Elastase ActivitY/J.L9 Protein
0.62 ± 0.33
0.67 ± 0.25
0.70 ± 0.62
t = 1.347 P < 0.20 0.10 t = 1.963 P < 0.10 0.05 t = 1.206 P < 0.30 0.20
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0.76 ± 0.31
0.89 ± 0.50
0.36
0.31 ± 0.07
1.28 ± 0.34
t = 3.64 P < 0.001
>
1.19 ± 0.45
1.19 ± 0.,30
(mg/ml serum)
Trypsin Inhibition
t = 3.53 P'< 0.001
t and P Values Compared to Controls
POLYMORPHONUCLEAR LEUKOCYTE LYSOSOMAL ELASTASE ACTIVITY AND TRYPSIN INHIBITORY CAPACITY IN CONTROL SUBJECTS, PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE, AND SUBJECTS WITH NORMAL LUNG FUNCTION AND NON-MM PHENOTYPES
TABLE 1
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413
NEUTROPHIL LYSOSOMAL ELASTASE AND COPD
312 fiu pancreatic elastase equivalents per /¿g of protein, which is still significantly different from the COPD patient group with mean A absorbency per min of 71 ± 32.9, corresponding to 1,125 ± 522 m per fig (P < 0.01). The mean A absorbency per min of 53.3 ± 19.7 (844 ± 312 fiu per fig) was not significantly different from the mean neutrophil elastase activity of the 31 control subjects with M phenotype and normal lung function shown in table 1 (A absorbency per min, 50 ± 15.3; pancreatic elastase equivalents, 793 ± 242 fiu per fig). Further subdivision of the patients with abnormal Pi phenotypes into those who had homozygous deficiency or heterozygous deficiency, either with or without evidence of COPD, did not yield significant differences in the mean values of elastase activity from the control subjects or within subgroups. However, as is shown in table 1, the number of persons in each subgroup was small, and in the important group of subjects with homozygous Pi Z phenotype but no evidence of COPD, there was only one such subject. As shown, in that person, neutrophil elastase activity was in the normal range. As expected, the trypsin inhibitory capacity of subjects with homozygous Pi Z phenotype or heterozygous subjects with Z or S alleles was significantly less than that of Pi M control subjects and averaged 0.31 ± 0.07 and 0.89 ± 0.50 mg per ml, respectively. The leukocyte count in the peripheral blood in the group of patients with COPD and Pi M phenotype averaged 6,140 ±1,265 cells per mm 3 . This value was not significantly different from the mean white blood cell count of 6,437 ± 1,889 cells per mm 3 in the control group or that of 6,769 ± 2,010 cells per mm 3 in the patients with alpha x -antitrypsin deficiency. The mean (± SD) per cent neutrophils of these groups were 58 ± 11 and 60 ± 14, respectively, with no significant difference between any of the groups. The results of pulmonary function tests are shown in table 2. Among the 42 patients studied, the mean value for vital capacity, RV/TLC, ¥EV1 as a per cent of vital capacity, resting arterial Po 2 and Pco 2 , and resting single-breath DLCO were all in the abnormal range. The decreased FEVj was consistent with moderate to severe airway obstruction. There were no significant differences in any of the measurements of pulmonary function between patients with Pi M phenotype and the 7 patients with other phenotypes.
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414
RODRIGUEZ, SEALS, RADIN, LIN, MANDL, AND TURINO
To determine whether the degree of neutrophil lysosomal elastase activity bore any relation to the degree of abnormality of results of pulmonary function tests, correlation coefficients were calculated for per cent of predicted values of vital capacity, total lung capacity, singlebreath DLCO> and for actual values of arterial Po 2 , Pco 2 , and RV/TLC. No significant correlations could be determined for neutrophil elastase activity or any of these physiologic variables of pulmonary dysfunction. The reproducibility of values for neutrophil elastase activity in the same subjects was studied by sampling blood at varying intervals from one to 12 months in 12 control subjects and 12 COPD patients all with Pi M phenotype. These results are shown in table 3. In the group of normal control adults, the mean values (± SD) of the.2 determinations were 56.4 ±11.4 A absorbency per min per fig, corresponding to 893 ±181 fiu pancreatic elastase equivalents per /¿g, and 60.9 ±14.9 A absorbency per min per fig, corresponding to 964 ± 237 Atu per fig of lysosomal protein. Among patients, the corresponding values were 94.8 ± 40.5 (1,502 ± 642 fiu per fig) and 90.2 ± 37.9 (1,429 ±600 fiu per fig). The differences between the mean values in each group of subjects were not significant. However, the mean values of the total number of 24 determinations in each group, i.e., 58.6 ± 12.7 versus 92.5 ± 38.5 or 928 ± 237 versus 1,465 ± 609, were significantly different (P < 0.01; t = 2.98). The values obtained in each group of subjects expressed as a coefficient of variation (SD/mean X 100) was 21 per cent for control subjects and 38 per cent for patients with COPD, which indicates a wider range of intersubject variation of the values in the latter group. Calculation of an intraclass coefficient of correlation (R) (19) permits an analysis of intrasubject variability. In both the control and the patient groups, the intrasubject variability is small in comparison to the intersubject variability. The intraclass coefficient of correlation was 0.85 for control subjects and 0.80 for the patients. By this analysis, a« intraclass coefficient of 0.0 would indicate perfect reproducibility (the range of 0 to 1) so that the calculated coefficient indicates a moderately high degree of intrasubject reproducibility. Discussion
The results of this study demonstrate that neutrophil lysosomal elastase activity was significantly increased in a population of patients with Pi M phenotype and obstructive pulmonary disease.
It indicates further that persons with alpha x antitrypsin deficiency had values for neutrophil lysosomal activity that were not significantly different from those of the population of normal control subjects. One other study has been reported in which neutrophil lysosomal elastase activity was measured using an elastin substrate in normal adult subjects (20). That study used 14C-labeled elastin as a substrate. The method of isolating neutrophil lysosomal fractions was similar to that used in the present study. The result of that study indicates a mean (± SD) of 0.93 ± 0.39 units of pancreatic elastase equivalents per mg of lysosomal protein (i.e., 930 ¡JLU per fig), which is in the same range as the data reported here. It is noteworthy that some relationship has been demonstrated recently between neutrophil elastase concentrations (determined by MBA esterase activity) and several tests of pulmonary function in patients with emphysema and Pi Z phenotype (21). The presence of a greater mean degree of neutrophil elastase activity in a population of patients with COPD has also been demonstrated by Galdston and associates (22). In that study, correlation was demonstrable between the severity of physiologic dysfunction of the lungs by pulmonary function testing and the degree of elastase increase. The difference between normal subjects and patients with COPD in mean elastase activity per fig of lysosomal protein was approximately 25 per cent, comparable to that in the present study. The lack of agreement in the correlation between neutrophil lysosomal elastase activity and the degree of severity of pulmonary dysfunction in these 2 studies may be related to differences in patient population or possibly to differences in the assay techniques. Galdston and co-workers (10, 22), Kidokoro and associates (21), and Harris and colleagues (23) used synthetic substrates for assay of neutrophil lysosomal elastase activity. Galdston used the substrate iV-t-butyloxycarbonyl-L-alanine pnitrophenyl ester (NBA), and results were expressed as units per 25 fig of protein extract. The normal values (mean ± SD) for a control group of 12 subjects reported in that study were 0.022 ± 0.004 per 25 fig of protein granule extract. Harris also reported elastase-like esterase activity of white blood cells using the same synthetic substrate (NBA), but used equivalent units per fig of porcine pancreatic elastase (fiu per fig X 107 cells). The value reported for both smokers and nonsmokers was approximately 15 fiu per fig
415
NEUTROPHIL LYSOSOMAL ELASTASE AND COPD
TABLE 3 I N T E R V A L REPEAT VALUES OF POLYMORPHONUCLEAR LEUKOCYTE LYSOSOMAL ELASTASE A C T I V I T Y (1 TO 12 MONTHS) Elastase Activity/jug Protein ßu Pancreatic Elastase Equivalents
AAbsorbency/min Subjects Controls* I.M. D.B. G.T. M.T. J.M. P.S. J.W. J.F. M.O. J.S. A.R. E.S. Mean ± S D Mean ± S D (I and II) t value (difference between I and II) Coeffient of variation (SD/mlean, '%) Patients* F.B. O.R. J.D. H.M. B.M. A.M. D.G. LP.
F.B. M.D. M.H. J.R. Mean ± S D
1 77.6 47.9 60.2 44.7 63.1 54.6 43.2 45.1 70.4 63.8 61.5 44.8 56.4 ± 1 1 . 4
II
81.5 53.1 58.3 57.8 63.8 58.4 39.7 53.5 77.4 78.1 74.6 34.5 60.9 ± 14.9
I
1,229
II
1,290
759
841
953
923
708
915
1,000
1,010
864
925
684 714
629
1,115 1,010 974
1,226 1,237 1,181
709
547
893 ± 1 8 1
964 ± 237
58.6 ± 1 2 . 7 NS**
847
928 ± 2 0 1 NS 21
135.7 71.9 130.1 35.6 64.8 77.1 90.9 104.2 104.2 89.5 50.5 183.1 94.8 ± 4 0 . 5
Mean ± S D (I and II) t value (difference between I and III) Coefficient of variation (SD/mean , %) t value (difference between controls•> and patients) P
104.2 87.2 112.8 50.5 117.4 75.1 57.1 74.9 97.4 66.8 53.0 186.2 90.2 ± 3 7 . 9
2,150 1,140 2,061
1,650 1,381 1,787
564
800
1,026 1,221 1,440 1,650 1,650 1,417
1,860 1,190
800
2,900 1,502 ± 6 4 2
904
1,186 1,543 1,058 840
2,950 1,429 ± 6 0 0 1,465 ± 609
92.5 ± 3 8 . 5
NS
NS 38
2.98
SUMMARY Neutrophil lysosomal elastase activity was measured in a group of 31 asymptomatic adults (12 men, 19 women) with Pi M phenotype and normal lung function, 9 subjects (7 men, 2 women) with normal lung function and phenotypes other than Pi M, and a group of 42 patients (25 men, 17 women) with chronic obstructive pulmonary disease, 35 of whom had Pi M phenotype and 7 phenotypes associated with alpha^antitrypsin deficiency. Chronic obstructive pulmonary disease was diagnosed by history and radiologic, Spirometrie, and blood gas characteristics of chronic airway obstruction. Neutrophil lysosomal elastase activity was measured using oxalic-acid solubilized, ligamentum nuchae elastin substrate. The average 570 nm A absorbency per min per fig of protein for this activity in control subjects with M phenotype was 50 ± 15.6, corresponding to 793 ± 242 microunits of pancreatic elastase per fig of lysosomal protein as compared to A absorbency per min of 71 ±32.9, which corresponds to 1,125 ±522 microunits of pancreatic elastase equivalents per fig of lysosomal protein in patients with chronic obstructive pulmonary disease (P < 0.001). If the patients with chronic obstructive pulmonary disease who have phenotypes other than Pi M are excluded, the mean A absorbency per min of the patients with Pi M phenotype is 73.4 ± 33.7, which corresponds to a pancreatic elastase equivalent activity of 1,163 ± 5 3 4 microunits per fig of lysosomal protein. Reproducibility of neutrophil lysosomal elastase activity was determined by repeat tests in the same subjects at intervals of one to 12 months and showed a coefficient of variation of 21 per cent for control subjects and 38 per cent for patients with chronic obstructive pulmonary disease. No differences between smokers and nonsmokers were demonstrable in neutrophil lysosomal elastase activity in either normal subjects or patients. Significantly greater degrees of neutrophil lysosomal elastase activity in patients with Pi M phenotype and chronic obstructive pulmonary disease suggest a possible role for circulating elastase activity as a mechanism for damage to lung elastin in the pathogenesis of pulmonary emphysema.
Introduction In recent years, it has been demonstrated that pancreatic elastase induces changes in the mechanical characteristics of the lung (1-3) and the morphology of lung parenchyma that re-
(Received in original form April 14,1978 and in revised form December 4y 1978) i From the Departments and Medicine and Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York, N. Y. 2 Supported in part by Program Project Grant No.
semble those observed in human pulmonary emphysema (4, 5). Also, proteolytic enzymes with elastolytic activity have been isolated from human polymorphonuclear leukocytes (6), platelets (7), alveolar macrophages (8), and peritoneal macrophages (9). These cells may transgress or
HL 15832 from the U. S. Public Health Service and by grants from the Stony Wold-Herbert Fund and the Council for Tobacco Research. 3 Presented in part at the Annual Meeting of the American Thoracic Society, Montréal, Québec, May 1975.
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 119, 1979
409
410
RODRIGUEZ, SEALS, RADIN, LIN, MANDL, AND TURINO
reside in the lung. N o r m a l h u m a n circulating p o l y m o r p h o n u c l e a r leukocytes have been shown to have elastase-like esterase activity (10), a n d it has been suggested that persons with a deficiency of serum alpha 1 -antitrypsin whose l u n g function is n o r m a l have decreased concentrations of neutrophil elastase. Moreover, p r e p a r a t i o n s of neutrophils have b e e n shown to p r o d u c e m o r p h o logic destruction of l u n g p a r e n c h y m a (11). T h e present study was u n d e r t a k e n to determ i n e the degree of circulating p o l y m o r p h o n u clear leukocyte lysosomal elastase activity in a g r o u p of asymptomatic adults with n o r m a l l u n g function, in a g r o u p of patients with chronic obstructive p u l m o n a r y disease (COPD), a n d in a g r o u p of patients with a l p h a ^ a n t i t r y p s i n deficiency with a n d w i t h o u t evidence of C O P D . T h e d a t a were analyzed to d e t e r m i n e the relationship between the presence a n d severity of C O P D a n d concentrations of n e u t r o p h i l lysosomal elastase. R e p e a t d e t e r m i n a t i o n s of n e u t r o p h i l lysosomal elastase activity at intervals of o n e to 12 m o n t h s are also presented for a p o p u l a t i o n of n o r m a l subjects a n d a g r o u p of p a t i e n t s with C O P D .
Materials and Methods Subjects. Normal subjects were 31 asymptomatic persons who were demonstrated to have a normal spirogram and chest roentgenogram. In addition, all of these subjects were shown to have the Pi M alpha^antitrypsin phenotype and normal trypsin inhibitory capacity (mean and SD of 1.19 ±0.29 mg of trypsin inhibited per ml of undiluted serum). Most of the subjects were professional colleagues and laboratory personnel. Their mean age (± SD) was 43.3 ± 13.3 years; 12 subjects were women, and 19 were men. T h e patient group was comprised of 42 persons from the inpatient and outpatient departments of the Presbyterian Hospital. The mean age (± SD) was 56 ± 14 years; 17 patients were women and 25 were men. Of the 42 patients, 7 had phenotypes other than Pi M. Of those seven, 4 (3 men, one woman) had homozygous Pi Z phenotype, and 3 patients (2 men, one woman) had phenotypes MZ, MS, and FS, respectively. In addition, there were 9 subjects with no symptoms and normal lung function who had abnormal Pi phenotypes (7 men, 2 women). Of these nine, one had phenotype Pi Z, six had MS, and two had MX. The diagnosis of COPD was based on a history of shortness of breath, particularly on exertion, and physiologic evidence of airway obstruction. Criteria for inclusion of patients in the study were: (1) symptoms of shortness of breath for at least 5 years, and (2) evidence of airway obstruction that was not reversible; such evidence included pulmonary function tests that demonstrated vital capacity of 85 per
cent of predicted values or less, 1-sec forced expiratory volume (FEVX) of 60 per cent of predicted values or less, and ratio of residual volume to total lung capacity (RV/TLC) greater than 45 per cent and arterial Po 2 less than 75 mm Hg at rest. Patients were excluded who could be diagnosed as having allergic asthma by virtue of known sensitivities and the episodic nature of their dyspnea and disturbances in pulmonary function. Although common symptoms, cough and sputum were not considered criteria for inclusion. In 26 of the patients, the history of cough and sputum would meet the criteria for chronic bronchitis. However, in none of these patients was chronic bronchitis diagnosed alone. On the basis of decreased CO diffusing capacity (DLCO), increased RV/TLC, and increased TLC, emphysema was considered a concomitant abnormality. None of the patients could be classified as having episodic asthma, and there were no known allergies associated with the onset of shortness of breath. Shortness of breath on exertion was present in all subjects and was unremitting rather than episodic. The measurements of neutrophil lysosomal elastase activity, trypsin inhibitory capacity, and pulmonary function of patients were done at a time when each patient was considered free of exacerbations of pulmonary infection. However, most patients were considered to have some degree of chronic pulmonary infection related to chronic bronchitis. With respect to smoking history, only 2 of the 31 control subjects were smoking at the time of the study. Both were women and had smoked 25 and 35 pack-years, respectively. Other control subjects were nonsmokers. In the patient group, 8 of the 42 patients had never smoked. Thirty-four patients had smoked and had smoking histories that ranged from 20 to 85 pack-years (mean 53 ± 34 pack-years), but only two were still smoking at the time of study. These 2 patients smoked 40 and 35 pack-years. Thirty-two of the patients had stopped smoking at least 18 months before measurements of neutrophil elastase activity or pulmonary function testing. Spirometry was performed using a 13-liter spirometer (Warren E. Collins, Braintree, Mass.) with the patient in a sitting position. Arterial blood gas composition was measured by Po 2 , Pco 2 , and pH analyzer (Instrumentation Laboratories, Lexington, Mass.). Assay of neutrophil lysosomal elastase. T h e method of extraction and assay of neutrophil lysosomal elastase activity was the following: Fifty ml of venous blood were drawn by needle and syringe into a plastic tube containing 10 ml of acidcitrate-dextrose solution. Erythrocytes and lymphocytes were separated from leukocytes by sedimentation in 3 per cent dextran solution, after which the residual erythrocytes were lysed by suspension in hypotonic saline. T h e neutrophils and residual lymphocytes were
NEUTROPHIL LYSOSOMAL ELASTASE AND COPD
placed in 0.34 M sucrose solution for 30 min to weaken cell membranes. Cell counts at this stage yielded at least 92 per cent polymorphonuclear leukocytes. The solution was perfused through a Swinney apparatus and was centrifuged. The residual granules were exposed to freezing and thawing for 8 cycles to disrupt the granules. Centrifugation was done to separate the granular debris from the enzyme-containing supernatant. The supernatant was assayed immediately or was frozen at —70° C and stored. It has been demonstrated that storing at this temperature for 3 months did not result in any loss of elastolytic activity (Mandl, 1.: Unpublished observations). T h e elastase assay was carried out by a modification of the technique of Keller and Mandl (12), digesting 0.5 mg of oxalic-acid solubilized ligamentum nuchae elastin for 20 hours. T h e quantitation of the elastase activity for this substrate involves measurement by a colorimetric ninhydrin reaction (13) of the free amino groups released by scission of susceptible peptide bonds. Results are expressed as changes in the 570 nm absorbency at unit time per pg of lysosomal protein and in terms of pancreatic elastase equivalents as microunits (/¿u) per ¿ig. Pancreatic elastase equivalents are determined by comparing the observed change in absorbency with that obtained when a known amount of pancreatic elastase is used to digest the same substrate under the same conditions. Protein content was determined by the procedure of Lowry and associates (14). Results of neutrophil elastase activity expressed per unit leukocyte count of the patient's blood gave a wider range of variability of elastase activity and lower reproducibility. This was attributed to: (2) error in the blood leukocyte count per se, and (2) variability in the number of leukocytes represented in the final lysosomal pellet on which the assay for elastase activity was performed because of the several steps involved in the separation and isolation of the lysosomal fraction. An attempt to relate neutrophil elastase activity to markers of other neutrophilic enzyme activities such as /3-glucuronidase did not improve the limits of variability of elastase activity over the use of lysosomal protein per se. This observation suggests that elastase and ^-glucuronidase in neutrophils may be changing independently and that ßglucuronidase is not a suitable marker. Measurements of serum trypsin inhibitory capacity were made according to the method of Erlanger and associates (15). Phenotyping was done by the method of Fager hol and Laurell (16). RV was calculated from the Spirometrie expiratory volume and from the functional residual capacity measured by a steady-state helium dilution method (17). White blood cell counts and differential counts were done on each patient at the time blood was
411
drawn for measurements of neutrophil elastase activity. In selected cases, differential counts were also done after exposure to hypotonic saline and sucrose washing. Results T h e values for n e u t r o p h i l elastase activity a n d trypsin inhibitory capacity in each g r o u p are summarized in table 1. T h e average 570 n m A absorbency p e r m i n in the n o r m a l subjects with M p h e n o t y p e was 50 ± 15.3, c o r r e s p o n d i n g to 793 ± 242 ¡mu pancreatic elastase equivalents p e r /¿g of lysosomal p r o t e i n . T h e trypsin inhibitory capacity m e a s u r e m e n t in this g r o u p averaged 1.19 ± 0.30 m g of trypsin i n h i b i t e d per m l of undiluted serum. T h e n o r m a l average ( ± SD) trypsin inhibitory capacity in this laboratory is 1.08 ± 0.1 m g of trypsin i n h i b i t e d p e r m l of undiluted serum (18). T h e average value of n e u t r o p h i l lysosomal elastase activity in all the p a t i e n t s with C O P D , which includes those with Pi M p h e n o t y p e as well as those with other phenotypes, was significantly greater t h a n the m e a n values of the n o r m a l subjects with a m e a n A absorbency p e r m i n of 71 ± 32.9, corresponding to 1,125 ± 522 nu of pancreatic elastase per /xg of lysosomal protein (P < 0.001). T h e m e a n trypsin inhibitory capacity for this g r o u p was within n o r m a l limits, i.e., 1.19 ± 0.45 m g p e r m l of u n d i l u t e d serum. T h e n e u t r o p h i l elastase activity of C O P D patients with Pi M p h e n o t y p e demonstrates an even greater m e a n A absorbency per m i n of 73.4 ± 33.7, c o r r e s p o n d i n g to 1,163 ± 534 ¡m pancreatic elastase equivalent activity as shown in table 1. G r o u p i n g of all subjects with phenotypes other t h a n Pi M showed a m e a n A absorbency p e r m i n of 58.4 ± 27.4, c o r r e s p o n d i n g to pancreatic elastase activity of 925 ± 434 /m p e r fig, which was not significantly different from that of the control g r o u p . As expected, the trypsin inhibitory capacity of this g r o u p was significantly less at 0.62 ± 0.33 m g p e r ml. If all subjects with phenotypes other t h a n Pi M were divided further into those persons with C O P D a n d those w h o were asymptomatic a n d h a d n o r m a l Spirometrie a n d blood gas values, there was n o significant difference in m e a n values for n e u t r o p h i l lysosomal elastase activity within t h e subgroups. If the 9 patients with a b n o r m a l Pi p h e n o t y p e s b u t n o r m a l l u n g function are included in the control g r o u p , the m e a n 570 n m A absorbency per m i n is 53.3 ± 19.7, corresponding to 844 ±
4
1
3
8
Patients with COPO and ZZ phenotypes
Subject with normal lung function and ZZ phenotype
Patients with COPD and heterozygous phenotypes (MZ, MS, FS)
Subjects with normal lung function and heterozygous phenotypes (MZ, 6; MX,2)
Ch ron ic obstructive pu Imonary disease.
-*
7
16
All subjects with abnormal Pi phenotypes
Patients with COPD and abnormal Pi phenotypes
35
COPO patients with MM phenotype
9
42
All patients with CO PO *
Subjects with normal lung function and abnormal Pi phenotypes
31
(no.)
Control group
Group
Subjects
1
6
2
1
42-76 (61 ±17)
12-33 (17 ± 6)
1
2
63.6 ±30.8
66.4 ±30.2
68.5
39.4..± 16.9
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2
51
59.2 ±28.1
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5
3
64.2 ±28.9
12-51 (22±12)
58.4 ± 27.4
73.4 ± 33.7
71.0 ±32.9
50.1 ± 15.3
absorbency/ min)
12-76 (36 ±20)
27-80 (57 ± 14)
27-80 (56 ± 14)
18-67 (43 ± 13)
(range) (mean)
(,d
2
4
14
17
12
(F)
Age (years)
7
12
21
25
19
(M)
Sex
1,008 ±488
1,051 ± 479
1,085
624 ± 267
938 ±445
1,016 ± 457
925 ± 434
1,163 ± 534
1,125 ± 522
793 ± 242
(J.Lu pancreatic elastase equivalents)
Elastase ActivitY/J.L9 Protein
0.62 ± 0.33
0.67 ± 0.25
0.70 ± 0.62
t = 1.347 P < 0.20 0.10 t = 1.963 P < 0.10 0.05 t = 1.206 P < 0.30 0.20
>
t = 1.48 P < 0.2 0.1
>
t = 1.62 P
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0.76 ± 0.31
0.89 ± 0.50
0.36
0.31 ± 0.07
1.28 ± 0.34
t = 3.64 P < 0.001
>
1.19 ± 0.45
1.19 ± 0.,30
(mg/ml serum)
Trypsin Inhibition
t = 3.53 P'< 0.001
t and P Values Compared to Controls
POLYMORPHONUCLEAR LEUKOCYTE LYSOSOMAL ELASTASE ACTIVITY AND TRYPSIN INHIBITORY CAPACITY IN CONTROL SUBJECTS, PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE, AND SUBJECTS WITH NORMAL LUNG FUNCTION AND NON-MM PHENOTYPES
TABLE 1
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413
NEUTROPHIL LYSOSOMAL ELASTASE AND COPD
312 fiu pancreatic elastase equivalents per /¿g of protein, which is still significantly different from the COPD patient group with mean A absorbency per min of 71 ± 32.9, corresponding to 1,125 ± 522 m per fig (P < 0.01). The mean A absorbency per min of 53.3 ± 19.7 (844 ± 312 fiu per fig) was not significantly different from the mean neutrophil elastase activity of the 31 control subjects with M phenotype and normal lung function shown in table 1 (A absorbency per min, 50 ± 15.3; pancreatic elastase equivalents, 793 ± 242 fiu per fig). Further subdivision of the patients with abnormal Pi phenotypes into those who had homozygous deficiency or heterozygous deficiency, either with or without evidence of COPD, did not yield significant differences in the mean values of elastase activity from the control subjects or within subgroups. However, as is shown in table 1, the number of persons in each subgroup was small, and in the important group of subjects with homozygous Pi Z phenotype but no evidence of COPD, there was only one such subject. As shown, in that person, neutrophil elastase activity was in the normal range. As expected, the trypsin inhibitory capacity of subjects with homozygous Pi Z phenotype or heterozygous subjects with Z or S alleles was significantly less than that of Pi M control subjects and averaged 0.31 ± 0.07 and 0.89 ± 0.50 mg per ml, respectively. The leukocyte count in the peripheral blood in the group of patients with COPD and Pi M phenotype averaged 6,140 ±1,265 cells per mm 3 . This value was not significantly different from the mean white blood cell count of 6,437 ± 1,889 cells per mm 3 in the control group or that of 6,769 ± 2,010 cells per mm 3 in the patients with alpha x -antitrypsin deficiency. The mean (± SD) per cent neutrophils of these groups were 58 ± 11 and 60 ± 14, respectively, with no significant difference between any of the groups. The results of pulmonary function tests are shown in table 2. Among the 42 patients studied, the mean value for vital capacity, RV/TLC, ¥EV1 as a per cent of vital capacity, resting arterial Po 2 and Pco 2 , and resting single-breath DLCO were all in the abnormal range. The decreased FEVj was consistent with moderate to severe airway obstruction. There were no significant differences in any of the measurements of pulmonary function between patients with Pi M phenotype and the 7 patients with other phenotypes.
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414
RODRIGUEZ, SEALS, RADIN, LIN, MANDL, AND TURINO
To determine whether the degree of neutrophil lysosomal elastase activity bore any relation to the degree of abnormality of results of pulmonary function tests, correlation coefficients were calculated for per cent of predicted values of vital capacity, total lung capacity, singlebreath DLCO> and for actual values of arterial Po 2 , Pco 2 , and RV/TLC. No significant correlations could be determined for neutrophil elastase activity or any of these physiologic variables of pulmonary dysfunction. The reproducibility of values for neutrophil elastase activity in the same subjects was studied by sampling blood at varying intervals from one to 12 months in 12 control subjects and 12 COPD patients all with Pi M phenotype. These results are shown in table 3. In the group of normal control adults, the mean values (± SD) of the.2 determinations were 56.4 ±11.4 A absorbency per min per fig, corresponding to 893 ±181 fiu pancreatic elastase equivalents per /¿g, and 60.9 ±14.9 A absorbency per min per fig, corresponding to 964 ± 237 Atu per fig of lysosomal protein. Among patients, the corresponding values were 94.8 ± 40.5 (1,502 ± 642 fiu per fig) and 90.2 ± 37.9 (1,429 ±600 fiu per fig). The differences between the mean values in each group of subjects were not significant. However, the mean values of the total number of 24 determinations in each group, i.e., 58.6 ± 12.7 versus 92.5 ± 38.5 or 928 ± 237 versus 1,465 ± 609, were significantly different (P < 0.01; t = 2.98). The values obtained in each group of subjects expressed as a coefficient of variation (SD/mean X 100) was 21 per cent for control subjects and 38 per cent for patients with COPD, which indicates a wider range of intersubject variation of the values in the latter group. Calculation of an intraclass coefficient of correlation (R) (19) permits an analysis of intrasubject variability. In both the control and the patient groups, the intrasubject variability is small in comparison to the intersubject variability. The intraclass coefficient of correlation was 0.85 for control subjects and 0.80 for the patients. By this analysis, a« intraclass coefficient of 0.0 would indicate perfect reproducibility (the range of 0 to 1) so that the calculated coefficient indicates a moderately high degree of intrasubject reproducibility. Discussion
The results of this study demonstrate that neutrophil lysosomal elastase activity was significantly increased in a population of patients with Pi M phenotype and obstructive pulmonary disease.
It indicates further that persons with alpha x antitrypsin deficiency had values for neutrophil lysosomal activity that were not significantly different from those of the population of normal control subjects. One other study has been reported in which neutrophil lysosomal elastase activity was measured using an elastin substrate in normal adult subjects (20). That study used 14C-labeled elastin as a substrate. The method of isolating neutrophil lysosomal fractions was similar to that used in the present study. The result of that study indicates a mean (± SD) of 0.93 ± 0.39 units of pancreatic elastase equivalents per mg of lysosomal protein (i.e., 930 ¡JLU per fig), which is in the same range as the data reported here. It is noteworthy that some relationship has been demonstrated recently between neutrophil elastase concentrations (determined by MBA esterase activity) and several tests of pulmonary function in patients with emphysema and Pi Z phenotype (21). The presence of a greater mean degree of neutrophil elastase activity in a population of patients with COPD has also been demonstrated by Galdston and associates (22). In that study, correlation was demonstrable between the severity of physiologic dysfunction of the lungs by pulmonary function testing and the degree of elastase increase. The difference between normal subjects and patients with COPD in mean elastase activity per fig of lysosomal protein was approximately 25 per cent, comparable to that in the present study. The lack of agreement in the correlation between neutrophil lysosomal elastase activity and the degree of severity of pulmonary dysfunction in these 2 studies may be related to differences in patient population or possibly to differences in the assay techniques. Galdston and co-workers (10, 22), Kidokoro and associates (21), and Harris and colleagues (23) used synthetic substrates for assay of neutrophil lysosomal elastase activity. Galdston used the substrate iV-t-butyloxycarbonyl-L-alanine pnitrophenyl ester (NBA), and results were expressed as units per 25 fig of protein extract. The normal values (mean ± SD) for a control group of 12 subjects reported in that study were 0.022 ± 0.004 per 25 fig of protein granule extract. Harris also reported elastase-like esterase activity of white blood cells using the same synthetic substrate (NBA), but used equivalent units per fig of porcine pancreatic elastase (fiu per fig X 107 cells). The value reported for both smokers and nonsmokers was approximately 15 fiu per fig
415
NEUTROPHIL LYSOSOMAL ELASTASE AND COPD
TABLE 3 I N T E R V A L REPEAT VALUES OF POLYMORPHONUCLEAR LEUKOCYTE LYSOSOMAL ELASTASE A C T I V I T Y (1 TO 12 MONTHS) Elastase Activity/jug Protein ßu Pancreatic Elastase Equivalents
AAbsorbency/min Subjects Controls* I.M. D.B. G.T. M.T. J.M. P.S. J.W. J.F. M.O. J.S. A.R. E.S. Mean ± S D Mean ± S D (I and II) t value (difference between I and II) Coeffient of variation (SD/mlean, '%) Patients* F.B. O.R. J.D. H.M. B.M. A.M. D.G. LP.
F.B. M.D. M.H. J.R. Mean ± S D
1 77.6 47.9 60.2 44.7 63.1 54.6 43.2 45.1 70.4 63.8 61.5 44.8 56.4 ± 1 1 . 4
II
81.5 53.1 58.3 57.8 63.8 58.4 39.7 53.5 77.4 78.1 74.6 34.5 60.9 ± 14.9
I
1,229
II
1,290
759
841
953
923
708
915
1,000
1,010
864
925
684 714
629
1,115 1,010 974
1,226 1,237 1,181
709
547
893 ± 1 8 1
964 ± 237
58.6 ± 1 2 . 7 NS**
847
928 ± 2 0 1 NS 21
135.7 71.9 130.1 35.6 64.8 77.1 90.9 104.2 104.2 89.5 50.5 183.1 94.8 ± 4 0 . 5
Mean ± S D (I and II) t value (difference between I and III) Coefficient of variation (SD/mean , %) t value (difference between controls•> and patients) P
104.2 87.2 112.8 50.5 117.4 75.1 57.1 74.9 97.4 66.8 53.0 186.2 90.2 ± 3 7 . 9
2,150 1,140 2,061
1,650 1,381 1,787
564
800
1,026 1,221 1,440 1,650 1,650 1,417
1,860 1,190
800
2,900 1,502 ± 6 4 2
904
1,186 1,543 1,058 840
2,950 1,429 ± 6 0 0 1,465 ± 609
92.5 ± 3 8 . 5
NS
NS 38
2.98
