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Bronchial Aspirate Alkaline Phosphatase: a Sensitive Marker Enzyme to Evaluate Bronchial Cell Damage

Bronchial Aspirate Alkaline Phosphatase: a Sensitive Marker Enzyme to Evaluate Bronchial Cell Damage Respiration 37: 36-41 (1979) Bronchial Aspirate Alkaline Phosphatase: a Sensitive Marker Enzyme to Evaluate Bronchial Cell Damage S. H. Kühn, M. A . De Kock and W. Gevers Diffuse Obstructive Pulmonary Syndrome Research Unit, South African Medical Research Council and Molecular and Cellular Cardiology Research Unit, Department of Medical Biochemistry, Medical School, University of Stellenbosch, Tygerberg Key Words. Bronchial aspirate • Alkaline phosphatase • Acid phosphatase ■ Cathepsin D Abstract. A correlation between the number of bronchial cells and alkaline phospha­ tase levels in bronchial aspirates obtained from patients was demonstrated. study [6] showed that elevated alkaline Introduction phosphatase activities were also present in bronchial aspirates of patients with lung The alkaline phosphatases are a group of cancer and bronchitis. These results sug­ enzymes which hydrolyse a variety of ester gested a possible relationship between orthophosphates under alkaline conditions bronchial cell destruction and high levels of (pH 9-11). The enzymes have a restricted alkaline phosphatase in the aspirates [6]. It tissue distribution, and although they are appears further that alkaline phosphatase is present in most tissues, high activities are a cell membrane enzyme [3]. found only in a few; particularly the small On the assumption that analysis of fluids intestine, kidney, bone and placenta. They obtained directly from a diseased organ is differ from acid phosphatases which are ac­ more revealing than that of circulating se­ tive at low pH only and from 5'-nucleoti- rum, enzymological analyses of aspirate sam­ dase and glucose 6-phosphatase which are ples have been initiated [6]. The early re­ optimally active under neutral conditions. sults, though based on a relatively small se­ Alkaline phosphatase has been prepared ries of patients suggested that the procedure in crystalline form from Escherichia coli may have diagnostic value. [8], from rat intestinal mucosa [10], and also from human placentas [5]. A consider­ able amount of information about the struc­ Materials and Methods ture and active centres of such alkaline phosphatases has been obtained [9]. All aspirate samples obtained and treated as Progressive elevation of serum alkaline reported [6]. The assays for acid and alkaline phosphatase activities has been described in phosphatase, cathepsin D and the method of pro­ numerous patients with metastatic broncho­ tein determination were all performed as de­ genic carcinoma [2, 4, 11]. A preliminary scribed [6]. Aspirate Alkaline Phosphatase The following procedures were used to deter­ The specific activities obtained in the gel mine the distribution of the enzyme activities in and filtrate fractions differed considerably, an aspirate sample (table I). The aspirate was fil­ as illustrated in table III. The filtrates were tered through a nylon net (No. 6 mesh) and centri­ consistently used in this screening test, for fuged at 500 g for 10 min to remove unbroken cells (e.g. macrophages, polymorphonuclear cells they were more homogeneous, mixed more and erythrocytes) in a Sorvall RC-5 refrigerated readily with water and the buffer solutions, centrifuge. The supernatant was again centrifuged and did not need any further preparation. at 22,000 g for 15 min, to remove all lysosomes The gel fraction, on the other hand, had to and mitochondria. The 22,000 g supernatant was finally centrifuged at 120,000 g for 120 min (Sorv­ be homogenized and did not mix readily all Ultra T-865 Rotor) in a Sorvall OTD-2 Ultra- with the buffer solutions, tending to form Centrifuge to collect the microsomal pellet. The emulsions. latter, as well as the previous pellets, was resus­ The correlation coefficients (r) obtained pended, using 10 passes of a loose-fitting Douncc in comparing the enzyme activities of acid homogenizer, in 2 ml deionized water. The ‘gel­ phosphatase, /J-glycerophosphatase, ^-glu­ like’ fraction collected on the nylon mesh was dried on five layers of Whatman No. 1 filter paper. curonidase and cathepsin D with each other, A 0.2-gram dried aliquot was resuspended in 5 ml suggested that there was a significant corre­ deionized water, sequentially using a loose- and a lation between these enzymes indicating a tight-fitting Dounce homogenizer. The suspension uniform release of these enzymes from the was then divided into five 1-m! fractions (1-5) and same lysosomal vesicle [7]. No correlation individually treated as follows: (1) no further treatment, (2) 30 sec; (3) 60 sec; (4) 90 sec, and (5) was apparent between these acid hydrolases 120 sec of sonication with an MSE 150-Watt Ul­ and alkaline phosphatase [7], indicating trasonic Disintegrator, at an amplitude of 20 /tm again that alkaline phosphatase is either not (peak to peak), in a 5-ml test tube below 4 °C. associated with the same particle or that it Triton X-100 was added to fraction 5 at a final originated from a different region of the concentration of 0.1%. particle, e.g. the membranes. The cytological analysis used in this in­ vestigation was based on a system where the Results visible cell density in bronchial aspirates was graded on a scale 1-4, and thus the cor­ Even at high speeds - 120,000 g for relation coefficients obtained (illustrated in 120 min (table I), 86.4% of the total protein table IV) were approximations, since one of aspirate samples was unsedimentable (ta­ was comparing continuous with ranked ble II). The distribution of sedimentable data. It nevertheless appears that a signifi­ and unsedimentable acid hydrolases, acid cant correlation probably did exist between phosphatase (87.2%) and cathepsin D certain cell types and specific enzyme activi­ (94.8%) activities was similar to that of the ties, e.g. between p-nitrophenylpliosphatase total protein. In contrast, a larger propor­ and polymorphonuclear cells, and also be­ tion of alkaline phosphatase was sedimenta­ tween /J-glycerophosphatase and poly­ ble (58.3%; table III) suggesting a correla­ morphs as well as dust cells, emphasizing tion between cell membranes and alkaline the role of these cells in infections. High ac­ phosphatase. This supports the conclusion tivities of these two enzymes were demon­ of Daems el al. [3], that alkaline phospha­ strated in diagnosed lung infections [6]. tase is a cell membrane enzyme. Kühn/de Kock/Gcvers Table I. Centrifugation procedure of an aspirate sample A f\s sp piira rau te Filtered through nylon mesh Gel (on mesh) Filtrate 1 ! Centrifuged at 500 g x 10 min Supernatant 1 Pellet I (cells and nuclei) Centrifuged at 22,000 g x 15 min h — LPellet II (lysosomes and mitochondria) Supernatant I Centrifuged 120,000 £ x 120 min Supernatant III — '— Pellet III (microsomal) Table II. Distribution of total protein in an aspirate sample Distribution, % Fraction Protein, mg/ml Volume, ml Total protein, mg Cel 1 1.784 1 1.784 1.5 1.760 2 1.760 1 1.5 1.638 1 1.638 1.4 7.4 1.5 4 1.711 1 1.711 1.778 1.5 5 1.778 1 Filtrate 106.468 92.6 Filtrate (A) 2.476 43 4.041 3.8 Pellet I 2.021 2 2 3.9 2.063 4.126 ß = Pellet II 99.7 of (A) 86.4 Supernatant III 2.130 43 91.584 6.299 5.9 Pellet III 3.149 phosphatase (p-nitrophenylphosphatase) and Furthermore, a significant correlation exists cathepsin D activities tended to be high in between alkaline phosphatase and the pres­ ence of normal bronchial cells in the aspir­ aspirates obtained from patients with lung ate (table IV). Since elevated alkaline phos­ infections at the time of bronchoscopy, il­ phatase aspirate activities also correlated lustrated in figure lb. In patients with chronic infections, however, there are well-recogniz­ significantly with diagnosed lung cell de­ struction [7], these data point to a probable ed anatomical features that connote destruc­ relation between cell membranes and alka­ tion of lung tissue, e.g. an increase in the line phosphatase which appears therefore, numbers of bronchial glands and conse­ to be a suitable marker enzyme to evaluate quently an increase in the thickness of the lung cell membrane damage or shedding. glandular layer of the bronchial wall. Goblet Low values for the three enzyme activi­ cells are characteristically increased and ties were found in lung-infection-free pa­ there is an associated increase in mucus in tients (at the time of bronchoscopy) as illus­ the bronchial lumen. The probable release trated in figure la. On the other hand, acid of alkaline phosphatase from destroyed Aspirate Alkaline Phosphatase Table III. Distribution of enzyme activities in an aspirate sample Fraction Activity Distribution, % specific total Acid phosphatase1 Gel 1 14.8 27 x 5 = ......... 7.2 2 9.9 18 8.9 15 4 8.2 14 5 5.7 10 Filtrate (A) 16.3 1,741 92.8 Pellet I 11.9 48 3.2 Pellet II 20.3 84 5.6 = 85.8 of (A) Supernatant III 14.3 1,309 Tree’ 87.7 Pellet III 8.2 52 3.5 Alkaline phosphatase1 2 Gel 1 3.6 6.9 3.6 6.3 3 3.8 6.2 4 3.6 6.2 4.1 7.3 x 5 — ......... 20 Filtrate (A) 1.6 166.6 80 Pellet I 3.2 12.8 8.3 Pellet II 4.8 19.9 12.9 = 92.3 of (A) Supernatant III 0.3 31.5 ‘free’ 20.5 Pellet 111 14.2 89.6 58.3 Cathcpsin D- Gel 1 1.5 2.7 x 5 = ......... 2 1.5 2.7 1.4 2.3 4 1.4 2.3 5 1.3 2.3 Filtrate (A) 2.6 272.2 88 Pellet 1 1.0 4.0 1.6 Pellet 11 2.5 4.7 1.8 = 94 of (A) Supernatant III 1.1 247.7 ‘free’ 94.8 Pellet II 2.7 4.7 1.8 1 Specific activity: nmol/mg prot./min; total activity: nmol. 2 Specific activity: zfA/mg prot./min x 103; total activity: A A x 103. bronchial cell membranes in patients with No significant correlation between aspir­ chronic infected lungs is illustrated in figure ate and serum alkaline phosphatase values lc by the elevated activities in aspirates of was evident, as was also found in a previous all three enzymes studied. study [6]. 40 Kühn/de Kock/Gevers Tabic IV. Correlation coefficients obtained from simple linear regression analysis, comparing the enzyme activities of p-nitrophenylphosphatase, /?-glyccrophosphatasc, alkaline phosphatase, cathepsin D and ^-glucu­ ronidase, with cytological bronchial aspirate cell ranking Cells demonstrated in bronchial aspirates polymorphs histiocytes lymphocytes dust cells bronchial cells p-Nitrophenylphosphatase 0.287 0.061 0.035 0.057 0.017 /?-Glycerophosphatase 0.213 0.156 0.063 0.294 0.019 Alkaline phosphatase 0.078 0.012 -0.152 0.049 0.435 Cathepsin D 0.241 0.014 0.006 0.090 0.045 /(-Glucuronidase 0.027 0.033 0.037 0.108 0.024 Fig. 1. A histogram illustrating the mean and standard deviation of acid and alkaline phospha­ b tase (nmol/mg prot./min.) and cathepsin D (ZlA/mg prot./min.) activities in bronchial aspi­ rates obtained from ‘normal’ (lung) patients (a), pa­ tients with lung infections at the time of broncho­ scopy (b) and patients with chronic bronchitis and other infections lung diseases, with resultant lung cell necrosis (c). Discussion In line with previous reports [6, 7], this study demonstrates that acid and alkaline phosphatase as well as cathepsin D aspirate values are low in normal patients. Acid phosphatase and cathepsin D aspirate val- Aspirate Alkaline Phosphatase 41 ues are high in patients with infection at the 2 Byers, D. A.; Fernley, N., and Walker, P. G.: Studies on alkaline phosphatase. Eur. J. Bio- time of bronchoscopy. These results relate chem. 29: 205 (1972). to the findings [1] that proteolytic enzymes 3 Daems, W.; Wisse, E., and Brederoo, P.: Elec­ are released on phagocytic stimulation from tron microscopy of the vacuolar system; in polymorphs and macrophages. These cells Dingle, Lysosomes a laboratory handbook, normally engulf particulate matter that pas­ vol. 1, p. 161 (North-Holland, Amsterdam 1972). ses the mucociliary escalator v/hich lines the 4 Fishman, W. H.; Inglis, N. I.; Stolbach, L. L., upper respiratory passages and release a and Krant, M. J.: Serum alkaline phosphatase portion (15-25°/o) of their enzyme load [1]. iso-enzyme of human neoplastic cell origin. The enzyme activities demonstrated in pa­ Cancer Res. 28: 150 (1968). tients’ aspirates, therefore represent the 5 Gosh, N. and Fishman, W. H.: Purification and properties of molecular weight variants of phagocytic response of these cells. Under human placental alkaline phosphatase. Bio- normal conditions, the a,-antitrypsin and chem. J. 108: 779 (1968). other proteolytic enzyme inhibitors found in 6 Kühn, S. H. and De Kock, M. A.: A prelimi­ lungs would be expected to inactivate the nary study of elevated alkaline phosphatase enzymes. and cathepsin in bronchial aspirates of patients with lung cancer and bronchitis. Chest 68: 326 In the absence of cq-antitrypsin as in (1975). patients with a genetic homozygous defi­ 7 Kühn, S. H.; De Kock, M. A., and Gevers, ciency or when the phagocytic response is W.: Screening with the new biochemical-en­ excessive, sufficiently high levels of proteo­ zymatic method for early detection of lung lytic enzymes may be free to damage the cancer. Chest (in press). 8 Malamy, M. H. and Horecker, B. L.: Purifica­ bronchial cells. tion and crystallization of the alkaline phos­ It was also demonstrated that a signifi­ phatase of Escherichia coli. Biochemistry, N. cant correlation probably exists between Y. 3: 1893 (1964). bronchial cell destruction and high alkaline 9 Milstein, C.: The amino sequence around the phosphatase values. Elevated activities of reactive serine residue in alkaline phosphatase from Escherichia coli. Biochem. J. 92: 410 acid and alkaline phosphatase and cathepsin (1964). D were found in cases of chronic bronchitis 10 Portmann, P.; Rossier, R. und Chardenners, (patients with infection at the time of bron­ H.: Zur Kenntnis der alkalischen Darmphos- choscopy), pneumonia, lung abscess and phatase. Helv. physiol, pharmacol. Acta 18: tuberculosis [6, 7], 414 (1960). These results imply that the extent of 11 Stolbach, L. L.; Krant, M. J., and Fishman, W. H.: Ectopic production of an alkaline phospha­ bronchial cell destruction can be evaluated tase iso-enzyme in patients with cancer. New by means of this biochemical test and that Engl. 1.281:151 (1969). treatment of such diseases can be monitored afterwards in the same way. Received: August 19, 1977 Accepted: December 14,1977 References Dr. S. H. Kühn, 1 Ackerman, N. R. and Beebe, J. R.: Release of Medical School, lysosomal enzymes by alveolar cells. Nature, University of Stellenbosch, Lond. 247: 475 (1974). PO box 63, Tygerberg 7505, South Africa http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Respiration Karger

Bronchial Aspirate Alkaline Phosphatase: a Sensitive Marker Enzyme to Evaluate Bronchial Cell Damage

Kühn, S.H.; De Kock, M.A.; Gevers, W.
Respiration , Volume 37 (1): 6 – Jan 1, 1979
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Publisher
Karger
Copyright
© 1979 S. Karger AG, Basel
ISSN
0025-7931
eISSN
1423-0356
DOI
10.1159/000194005
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Abstract

Respiration 37: 36-41 (1979) Bronchial Aspirate Alkaline Phosphatase: a Sensitive Marker Enzyme to Evaluate Bronchial Cell Damage S. H. Kühn, M. A . De Kock and W. Gevers Diffuse Obstructive Pulmonary Syndrome Research Unit, South African Medical Research Council and Molecular and Cellular Cardiology Research Unit, Department of Medical Biochemistry, Medical School, University of Stellenbosch, Tygerberg Key Words. Bronchial aspirate • Alkaline phosphatase • Acid phosphatase ■ Cathepsin D Abstract. A correlation between the number of bronchial cells and alkaline phospha­ tase levels in bronchial aspirates obtained from patients was demonstrated. study [6] showed that elevated alkaline Introduction phosphatase activities were also present in bronchial aspirates of patients with lung The alkaline phosphatases are a group of cancer and bronchitis. These results sug­ enzymes which hydrolyse a variety of ester gested a possible relationship between orthophosphates under alkaline conditions bronchial cell destruction and high levels of (pH 9-11). The enzymes have a restricted alkaline phosphatase in the aspirates [6]. It tissue distribution, and although they are appears further that alkaline phosphatase is present in most tissues, high activities are a cell membrane enzyme [3]. found only in a few; particularly the small On the assumption that analysis of fluids intestine, kidney, bone and placenta. They obtained directly from a diseased organ is differ from acid phosphatases which are ac­ more revealing than that of circulating se­ tive at low pH only and from 5'-nucleoti- rum, enzymological analyses of aspirate sam­ dase and glucose 6-phosphatase which are ples have been initiated [6]. The early re­ optimally active under neutral conditions. sults, though based on a relatively small se­ Alkaline phosphatase has been prepared ries of patients suggested that the procedure in crystalline form from Escherichia coli may have diagnostic value. [8], from rat intestinal mucosa [10], and also from human placentas [5]. A consider­ able amount of information about the struc­ Materials and Methods ture and active centres of such alkaline phosphatases has been obtained [9]. All aspirate samples obtained and treated as Progressive elevation of serum alkaline reported [6]. The assays for acid and alkaline phosphatase activities has been described in phosphatase, cathepsin D and the method of pro­ numerous patients with metastatic broncho­ tein determination were all performed as de­ genic carcinoma [2, 4, 11]. A preliminary scribed [6]. Aspirate Alkaline Phosphatase The following procedures were used to deter­ The specific activities obtained in the gel mine the distribution of the enzyme activities in and filtrate fractions differed considerably, an aspirate sample (table I). The aspirate was fil­ as illustrated in table III. The filtrates were tered through a nylon net (No. 6 mesh) and centri­ consistently used in this screening test, for fuged at 500 g for 10 min to remove unbroken cells (e.g. macrophages, polymorphonuclear cells they were more homogeneous, mixed more and erythrocytes) in a Sorvall RC-5 refrigerated readily with water and the buffer solutions, centrifuge. The supernatant was again centrifuged and did not need any further preparation. at 22,000 g for 15 min, to remove all lysosomes The gel fraction, on the other hand, had to and mitochondria. The 22,000 g supernatant was finally centrifuged at 120,000 g for 120 min (Sorv­ be homogenized and did not mix readily all Ultra T-865 Rotor) in a Sorvall OTD-2 Ultra- with the buffer solutions, tending to form Centrifuge to collect the microsomal pellet. The emulsions. latter, as well as the previous pellets, was resus­ The correlation coefficients (r) obtained pended, using 10 passes of a loose-fitting Douncc in comparing the enzyme activities of acid homogenizer, in 2 ml deionized water. The ‘gel­ phosphatase, /J-glycerophosphatase, ^-glu­ like’ fraction collected on the nylon mesh was dried on five layers of Whatman No. 1 filter paper. curonidase and cathepsin D with each other, A 0.2-gram dried aliquot was resuspended in 5 ml suggested that there was a significant corre­ deionized water, sequentially using a loose- and a lation between these enzymes indicating a tight-fitting Dounce homogenizer. The suspension uniform release of these enzymes from the was then divided into five 1-m! fractions (1-5) and same lysosomal vesicle [7]. No correlation individually treated as follows: (1) no further treatment, (2) 30 sec; (3) 60 sec; (4) 90 sec, and (5) was apparent between these acid hydrolases 120 sec of sonication with an MSE 150-Watt Ul­ and alkaline phosphatase [7], indicating trasonic Disintegrator, at an amplitude of 20 /tm again that alkaline phosphatase is either not (peak to peak), in a 5-ml test tube below 4 °C. associated with the same particle or that it Triton X-100 was added to fraction 5 at a final originated from a different region of the concentration of 0.1%. particle, e.g. the membranes. The cytological analysis used in this in­ vestigation was based on a system where the Results visible cell density in bronchial aspirates was graded on a scale 1-4, and thus the cor­ Even at high speeds - 120,000 g for relation coefficients obtained (illustrated in 120 min (table I), 86.4% of the total protein table IV) were approximations, since one of aspirate samples was unsedimentable (ta­ was comparing continuous with ranked ble II). The distribution of sedimentable data. It nevertheless appears that a signifi­ and unsedimentable acid hydrolases, acid cant correlation probably did exist between phosphatase (87.2%) and cathepsin D certain cell types and specific enzyme activi­ (94.8%) activities was similar to that of the ties, e.g. between p-nitrophenylpliosphatase total protein. In contrast, a larger propor­ and polymorphonuclear cells, and also be­ tion of alkaline phosphatase was sedimenta­ tween /J-glycerophosphatase and poly­ ble (58.3%; table III) suggesting a correla­ morphs as well as dust cells, emphasizing tion between cell membranes and alkaline the role of these cells in infections. High ac­ phosphatase. This supports the conclusion tivities of these two enzymes were demon­ of Daems el al. [3], that alkaline phospha­ strated in diagnosed lung infections [6]. tase is a cell membrane enzyme. Kühn/de Kock/Gcvers Table I. Centrifugation procedure of an aspirate sample A f\s sp piira rau te Filtered through nylon mesh Gel (on mesh) Filtrate 1 ! Centrifuged at 500 g x 10 min Supernatant 1 Pellet I (cells and nuclei) Centrifuged at 22,000 g x 15 min h — LPellet II (lysosomes and mitochondria) Supernatant I Centrifuged 120,000 £ x 120 min Supernatant III — '— Pellet III (microsomal) Table II. Distribution of total protein in an aspirate sample Distribution, % Fraction Protein, mg/ml Volume, ml Total protein, mg Cel 1 1.784 1 1.784 1.5 1.760 2 1.760 1 1.5 1.638 1 1.638 1.4 7.4 1.5 4 1.711 1 1.711 1.778 1.5 5 1.778 1 Filtrate 106.468 92.6 Filtrate (A) 2.476 43 4.041 3.8 Pellet I 2.021 2 2 3.9 2.063 4.126 ß = Pellet II 99.7 of (A) 86.4 Supernatant III 2.130 43 91.584 6.299 5.9 Pellet III 3.149 phosphatase (p-nitrophenylphosphatase) and Furthermore, a significant correlation exists cathepsin D activities tended to be high in between alkaline phosphatase and the pres­ ence of normal bronchial cells in the aspir­ aspirates obtained from patients with lung ate (table IV). Since elevated alkaline phos­ infections at the time of bronchoscopy, il­ phatase aspirate activities also correlated lustrated in figure lb. In patients with chronic infections, however, there are well-recogniz­ significantly with diagnosed lung cell de­ struction [7], these data point to a probable ed anatomical features that connote destruc­ relation between cell membranes and alka­ tion of lung tissue, e.g. an increase in the line phosphatase which appears therefore, numbers of bronchial glands and conse­ to be a suitable marker enzyme to evaluate quently an increase in the thickness of the lung cell membrane damage or shedding. glandular layer of the bronchial wall. Goblet Low values for the three enzyme activi­ cells are characteristically increased and ties were found in lung-infection-free pa­ there is an associated increase in mucus in tients (at the time of bronchoscopy) as illus­ the bronchial lumen. The probable release trated in figure la. On the other hand, acid of alkaline phosphatase from destroyed Aspirate Alkaline Phosphatase Table III. Distribution of enzyme activities in an aspirate sample Fraction Activity Distribution, % specific total Acid phosphatase1 Gel 1 14.8 27 x 5 = ......... 7.2 2 9.9 18 8.9 15 4 8.2 14 5 5.7 10 Filtrate (A) 16.3 1,741 92.8 Pellet I 11.9 48 3.2 Pellet II 20.3 84 5.6 = 85.8 of (A) Supernatant III 14.3 1,309 Tree’ 87.7 Pellet III 8.2 52 3.5 Alkaline phosphatase1 2 Gel 1 3.6 6.9 3.6 6.3 3 3.8 6.2 4 3.6 6.2 4.1 7.3 x 5 — ......... 20 Filtrate (A) 1.6 166.6 80 Pellet I 3.2 12.8 8.3 Pellet II 4.8 19.9 12.9 = 92.3 of (A) Supernatant III 0.3 31.5 ‘free’ 20.5 Pellet 111 14.2 89.6 58.3 Cathcpsin D- Gel 1 1.5 2.7 x 5 = ......... 2 1.5 2.7 1.4 2.3 4 1.4 2.3 5 1.3 2.3 Filtrate (A) 2.6 272.2 88 Pellet 1 1.0 4.0 1.6 Pellet 11 2.5 4.7 1.8 = 94 of (A) Supernatant III 1.1 247.7 ‘free’ 94.8 Pellet II 2.7 4.7 1.8 1 Specific activity: nmol/mg prot./min; total activity: nmol. 2 Specific activity: zfA/mg prot./min x 103; total activity: A A x 103. bronchial cell membranes in patients with No significant correlation between aspir­ chronic infected lungs is illustrated in figure ate and serum alkaline phosphatase values lc by the elevated activities in aspirates of was evident, as was also found in a previous all three enzymes studied. study [6]. 40 Kühn/de Kock/Gevers Tabic IV. Correlation coefficients obtained from simple linear regression analysis, comparing the enzyme activities of p-nitrophenylphosphatase, /?-glyccrophosphatasc, alkaline phosphatase, cathepsin D and ^-glucu­ ronidase, with cytological bronchial aspirate cell ranking Cells demonstrated in bronchial aspirates polymorphs histiocytes lymphocytes dust cells bronchial cells p-Nitrophenylphosphatase 0.287 0.061 0.035 0.057 0.017 /?-Glycerophosphatase 0.213 0.156 0.063 0.294 0.019 Alkaline phosphatase 0.078 0.012 -0.152 0.049 0.435 Cathepsin D 0.241 0.014 0.006 0.090 0.045 /(-Glucuronidase 0.027 0.033 0.037 0.108 0.024 Fig. 1. A histogram illustrating the mean and standard deviation of acid and alkaline phospha­ b tase (nmol/mg prot./min.) and cathepsin D (ZlA/mg prot./min.) activities in bronchial aspi­ rates obtained from ‘normal’ (lung) patients (a), pa­ tients with lung infections at the time of broncho­ scopy (b) and patients with chronic bronchitis and other infections lung diseases, with resultant lung cell necrosis (c). Discussion In line with previous reports [6, 7], this study demonstrates that acid and alkaline phosphatase as well as cathepsin D aspirate values are low in normal patients. Acid phosphatase and cathepsin D aspirate val- Aspirate Alkaline Phosphatase 41 ues are high in patients with infection at the 2 Byers, D. A.; Fernley, N., and Walker, P. G.: Studies on alkaline phosphatase. Eur. J. Bio- time of bronchoscopy. These results relate chem. 29: 205 (1972). to the findings [1] that proteolytic enzymes 3 Daems, W.; Wisse, E., and Brederoo, P.: Elec­ are released on phagocytic stimulation from tron microscopy of the vacuolar system; in polymorphs and macrophages. These cells Dingle, Lysosomes a laboratory handbook, normally engulf particulate matter that pas­ vol. 1, p. 161 (North-Holland, Amsterdam 1972). ses the mucociliary escalator v/hich lines the 4 Fishman, W. H.; Inglis, N. I.; Stolbach, L. L., upper respiratory passages and release a and Krant, M. J.: Serum alkaline phosphatase portion (15-25°/o) of their enzyme load [1]. iso-enzyme of human neoplastic cell origin. The enzyme activities demonstrated in pa­ Cancer Res. 28: 150 (1968). tients’ aspirates, therefore represent the 5 Gosh, N. and Fishman, W. H.: Purification and properties of molecular weight variants of phagocytic response of these cells. Under human placental alkaline phosphatase. Bio- normal conditions, the a,-antitrypsin and chem. J. 108: 779 (1968). other proteolytic enzyme inhibitors found in 6 Kühn, S. H. and De Kock, M. A.: A prelimi­ lungs would be expected to inactivate the nary study of elevated alkaline phosphatase enzymes. and cathepsin in bronchial aspirates of patients with lung cancer and bronchitis. Chest 68: 326 In the absence of cq-antitrypsin as in (1975). patients with a genetic homozygous defi­ 7 Kühn, S. H.; De Kock, M. A., and Gevers, ciency or when the phagocytic response is W.: Screening with the new biochemical-en­ excessive, sufficiently high levels of proteo­ zymatic method for early detection of lung lytic enzymes may be free to damage the cancer. Chest (in press). 8 Malamy, M. H. and Horecker, B. L.: Purifica­ bronchial cells. tion and crystallization of the alkaline phos­ It was also demonstrated that a signifi­ phatase of Escherichia coli. Biochemistry, N. cant correlation probably exists between Y. 3: 1893 (1964). bronchial cell destruction and high alkaline 9 Milstein, C.: The amino sequence around the phosphatase values. Elevated activities of reactive serine residue in alkaline phosphatase from Escherichia coli. Biochem. J. 92: 410 acid and alkaline phosphatase and cathepsin (1964). D were found in cases of chronic bronchitis 10 Portmann, P.; Rossier, R. und Chardenners, (patients with infection at the time of bron­ H.: Zur Kenntnis der alkalischen Darmphos- choscopy), pneumonia, lung abscess and phatase. Helv. physiol, pharmacol. Acta 18: tuberculosis [6, 7], 414 (1960). These results imply that the extent of 11 Stolbach, L. L.; Krant, M. J., and Fishman, W. H.: Ectopic production of an alkaline phospha­ bronchial cell destruction can be evaluated tase iso-enzyme in patients with cancer. New by means of this biochemical test and that Engl. 1.281:151 (1969). treatment of such diseases can be monitored afterwards in the same way. Received: August 19, 1977 Accepted: December 14,1977 References Dr. S. H. Kühn, 1 Ackerman, N. R. and Beebe, J. R.: Release of Medical School, lysosomal enzymes by alveolar cells. Nature, University of Stellenbosch, Lond. 247: 475 (1974). PO box 63, Tygerberg 7505, South Africa

Journal

Respiration – Karger

Published: Jan 1, 1979

Keywords: Cathepsin D; Bronchial aspirate; Alkaline phosphatase; Acid phosphatase

There are no references for this article.