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Elevated serum galactosylhydroxylysyl glucosyltransferase, a collagen synthesis marker, in fibrosing lung diseases
Clinica Chimica Acta, 3 (1985) 3-8
3
Elsevier CCA 03152
Elevated serum galactosylhydroxylysyl glucosyltransferase, a collagen synthesis marker, in fibrosing lung diseases Henrik Anttinen
a**, Erkki 0. Terho b, Pekka M. Jarvensivu Eeva-Riitta Savolainen d
’ and
a Department of Medicine, University of Turku, Turku, b Department of Pulmonary Diseases, Kuopio University Central Hospital, Tarinaharju, and’ Department of Medicine and * Collagen Research Unit, University of Ouly Oulu (Finland) (Received September 4th, 1984; revision January 3rd, 1985) Key words: Pulmonary fibrosis; Farmer’s lung; Collagen synthesis; Galactosylhydroxytjsyl glucosyltransferase
summtuy The activity of galactosylhydroxylysyl glucosyltransferase, an enzyme catalyzing collagen biosynthesis, was measured in the sera of 101 patients with various pulmonary diseases to study whether detectable enzyme amounts are liberated into the serum from the lung tissue, and whether this is associated with the development of lung fibrosis. Increased serum galactosylhydroxylysyl glucosyltransferase activity was found in all the patients with progressive pulmonary fibrosis and in about half of the patients with acute stages of farmer’s lung and infectious pneumonia. In one third of the patients with stage I sarcoidosis the serum enzyme activity was slightly increased, whereas in bronchial asthma and chronic bronchitis the values were mostly within the normal range. In conclusion, elevated serum enzyme activity was demonstrated in connection with those respiratory diseases in which pulmonary fibrosis was already verifiable or relatively often develops later. Measurements of serum galactosylhydroxylysyl glucosyltransferase may, thus, be useful in evaluating actual lung collagen synthesis in human pulmonary diseases.
Introduction
No serum parameter has hitherto been reported which reflects changes in the rate of collagen biosynthesis in the lung tissue. Measurement of such a parameter would * Correspondence to: Dr. Hemik Anttinen, Department of Medicine, University of Turku, Kiinamyllynkatu 4-8, SF-20520 Turku 52, Finland. 0009-8981/85/$03.30
0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
be valuable in connection with those pulmonary diseases which lead to fibrosis in which excessive collagen deposition is a central pathological feature. Galactosylhydroxylysyl glucosyltransferase (GGT, EC 2.4.1.66) is an intracellular enzyme required in one of the unique post-translational modifications in collagen biosynthesis which catalyzes no significant transfer of glucose to glycoproteins other than collagens [l]. This enzyme activity can reliably be determined in human serum (S-GGT) by a relatively simple procedure feasible for large series of samples [2-51, the activity being very stable during the storage of the serum [4]. Previous reports indicate that S-GGT reflects the actual collagen synthesis in certain liver [4,6-91 and neuromuscular [lo] diseases and is even sensitive enough to detect collagen scar formation following acute myocardial infarction [5]. Furthermore, this measurement can be used in the evaluation of treatment in fibrosing liver diseases [7]. However, S-GGT is normal in the great majority of localized skin diseases [ll] and in many malignant diseases not involving liver [6]. At the moment, no information is available on S-GGT in human lung diseases. The present work sets out to study whether detectable amounts of GGT are liberated into the serum from the lung tissue in human pulmonary disorders, and whether elevated S-GGT values can be demonstrated in those pulmonary diseases in which lung collagen synthesis is increased and local or generalized pulmonary fibrosis develops. Patients and methods Patients
Serum samples were obtained from 101 patients (35 males and 66 females, aged TABLE I Patient characteristics and mean activity of serum gaIactosyIhydroxylysy1 ghmosyltransferase (S-GGT) in various groups of pulmonary diseases Diagnostic group
Bronchial asthma chronic bronchitis Sarcoidosis Infectious pneumonia Farmer’s lung Pulmonary fibrosis
No
Sex
Mean age (range) yr
Stage of disease
Mean S-GGT f (U/I)
SD
p (compared with reference mean ” )
15
3M,12F
56 (28-78)
Severe
0.49 f 0.06
< 0.01
8 6
8M 3 M, 3 F
68 (52-77) 41 (29-67)
Exacerbated Stage I
0.51 f0.06 0.5OkO.12
< 0.01 < 0.05
22
12 M, 10 F
56 (15-93)
Acute
0.58kO.18
< 0.001
45
7M,38F
46 (21-69)
Acute
0.63 f 0.17
< 0.001
5
2M,3F
53 (35-64)
Progressive
0.71 f 0.06
< 0.001
a S-GGT reference mean f
SD
is 0.43 f 0.07 U/l and the upper normal Iimit 0.57 U/I (71.
5
15-93 yr, mean age 51 yr). Fifty-six of the patients were admitted to hospital for diverse pulmonary disorders and 45 with an acute stage of farmer’s lung but without any detectable pulmonary fibrosis. The subjects fulfilled the criterion of not suffering from any other disease known or assumed to affect tissue collagen synthesis or S-GGT activity [1,3-7,9-111, but were otherwise consecutive and unselected. The patient characteristics are shown in Table I. Progressive pulmonary fibrosis was documented by follow-up of the chest X-ray findings and in all but one of the patients, also by lung biopsies. This group consisted of five patients, two having fibrosing alveolitis and the other also pleural fibrosis. Both were treated with corticosteroids. In one patient the fibrosis resulted from farmer’s lung and in two from recurrent infectious pneumonias. These three patients were receiving no anti-inflammatory or antifibrotic treatment. Methoh The blood samples were allowed to clot for 1.5 h at room temperature before the separation of the sera. S-GGT activity was assayed in duplicate with 40-~1 samples of the serum by determining the amount of radioactive glucosylgalactosylhydroxylysine formed from uridine diphosphate-[‘4C]glucose in a gelatinized collagen substrate [2,3,12]. The gelatin substrate was prepared from insoluble calf skin collagen as described in detail previously [2]. One unit of GGT activity was defined as the activity required to synthesize an amount of radioactive glucosylgalactosylhydroxylysine in dpm corresponding to 1 pmol in 1 h at 37°C under conditions in which a saturating concentration of denatured purified citrate-soluble rat skin collagen is used as a substrate [12]. The reference S-GGT value for 77 healthy control subjects (aged 14-61 yr) determined in our laboratory is 0.43 + 0.07 U/l (mean f SD) [7], and values exceeding 0.57 U/l (mean + 2 SD) were considered abnormal. No changes are found in this serum enzyme activity with age or with sex in subjects aged > 12 yr [3]. Samples of three control sera were assayed together with the sera of the patients in each enzyme series. The statistical significance of the differences between the means was calculated using the two-tailed Student’s t test. Results
Individual S-GGT values in various diagnostic groups are shown in Fig. 1. Elevated values were recorded most frequently in progressive pulmonary fibrosis (all 5 patients) and in the acute stages of farmer’s lung (23 out of 45, 51%) and infectious pneumonia (10 out of 22, 45%). One third (2 out of 6) of the patients with stage I sarcoidosis had slightly increased S-GGT. In bronchial asthma and chronic bronchitis S-GGT was mostly within the normal range. The highest mean S-GGT was found in progressive pulmonary fibrosis, but the mean S-GGT also exceeded the upper normal limit in the acute stages of farmer’s lung and infectious pneumonia (Table I). In stage I sarcoidosis, bronchial asthma and chronic bronchitis the mean S-GGT, although being slightly higher than the reference mean, was within the normal range (Table I).
6
1.1
c
I
0.9 -
5
v
0 _____
v)
0.5
0-e .*& 0
0
0 I____
l
G0
L
0 00 0.3
---_,
__--
_-_
-.
---_
OL
Fig. 1. Individual values of S-GGT activity in various groups of pulmonary diseases. The horizontal solid and dashed lines indicate the mean and limits of the mean f 2 SD for the reference material.
Very high individual S-GGT values exceeding the reference mean +4 SD were found in 60% (3 out of 5) of patients with progressive pulmonary fibrosis, 29% (13 out of 45) of those with the acute stage of farmer’s lung and 23% (5 out of 22) of those with the acute stage of infectious pneumonia, but they were lower than this value in all the patients in the other groups. Discussion Human S-GGT measurements have previously been used for evaluating fibrotic processes and treatment in certain liver diseases [4,6,7,9] and diseases affecting muscular tissue [5,10]. The present results clearly show that detectable amounts of GGT are also liberated into the serum from the affected tissue in human pulmonary diseases.
7
Recent animal studies with experimentally induced lung collagen accumulation demonstrate increased lung GGT activities which correlate in a statistically significant manner with increased lung collagen content, and that S-GGT parallels these changes (H. Anttinen, U. Puistola, A. Oikarinen, L. Ryhanen, unpubl. obs.). Thus, it seems likely that S-GGT reflects the actual changes in tissue collagen synthesis occurring in human pulmonary diseases. The elevated S-GGT values were highest and most frequently encountered in progressive pulmonary fibrosis and the acute stages of farmer’s lung and infectious pneumonia. Concerning the present results it is significant, that the rate of collagen synthesis usually increases in inflammation of the lung [13,14] and in the early inflammatory stages preceding fibrosis, even though no evident collagen accumulation can yet be observed [13,14]. In the sarcoidosis group the chest X-ray findings and pulmonary function tests of the patient with the highest S-GGT value revealed progressive impairment in 6 mth, whereas in the other sarcoidosis patients no progression of the disease was found during this follow-up period. According to our clinical experience, roentgenologically verifiable pulmonary fibrosis seldom develops in farmer’s lung, but pulmonary function tests far more often remain permanently impaired, suggesting the development of irreversible pulmonary fibrosis. In the pathogenesis of farmer’s lung both immune complex-mediated (Type III) and cell-mediated (Type IV) immune reactions seem to be involved [15], which both tend to lead to fibrotic tissue changes. The high frequency of elevated S-GGT values found here in the acute stage of farmer’s lung led us to start a follow-up study on this disease. The results, although still preliminary, suggest that the S-GGT values at the acute stage can be used to predict irreversible pulmonary impairment due to fibrosis (H. Anttinen, EO. Terho, R. Myllyl$ E-R. Savolainen, unpubl. obs.). Monitoring of a serum indicator of changes in lung collagen synthesis would be a convenient way of gaining useful information for evaluating the fibrotic process and the need for, or efficiency of, therapeutic intervention. S-GGT may prove to be such an indicator, even in early stages of fibrosing pulmonary diseases. Acknowledgements This study was supported by grants from the Sigrid Juselius Foundation, Finland, and the Medical Research Council of the Academy of Finland. We thank Professor Kari I. Kivirikko for his valuable suggestions and comments, and Mrs. Ritva Pihkakoski and Miss ArJa Oja for their expert technical assistance. References 1 Kivirikko KI, Myllyla R. Collagen glycosyltrausferases. Int Rev Connect Tissue Res 1979; 8: 23-72. 2 Myllylil R, Risteli L, Kivirikko KI. Assay of collagen-galactosyltransferase and collagen-glucosyltransfemse activities and preliminary characterization of enzymic reactions with transferases from chick-embryo cartilage. Eur J Biochem 1975; 52: 401-410. 3 Anttinen H. Collagen glucosyltransferase activity in human serum. Clin Chim Acta 1977; 77: 323-330.
8 4 Kuutti-Savolainen E-R, Anttinen H, Miettinen TA, Kivirikko KI. Collagen biosynthesis enzymes in serum and hepatic tissue in liver disease. II. Galactosylbydroxylysyl glucosyltransferase. Eur J Clin Invest 1979; 9: 97-101. 5 Anttinen H, Jarvensivu PM, Savolainen E-R. Serum galactosylbydroxylysyl glucosyltransferase in acute myocardial infarction and during subsequent collagen scar formation. Eur J Clin Invest 1981; 11: 375-379. 6 Bolarin DM, Savolainen E-R, Kivirikko KI. Enzymes of collagen synthesis and type III procollagen amino-propeptide in serum from Nigerians with hepatocellular carcinoma and other malignant diseases. Int J Cancer 1982; 29: 401-405. 7 Savolainen E-R Miettinen TA, Pikkarainen P, Salaspuro MP, Kivirikko KI. Enzymes of collagen synthesis and type III procollagen aminopropeptide in the evaluation of D-penicillamine and medroxyprogesterone treatments of primary bihary cirrhosis. Gut 1983; 24: 136-142. 8 Anttinen H, Ryhanen L, Puistola U, Arrant0 A, Gikarinen A. Decrease in liver collagen accumulation in carbon tetrachloride-injured and normal growing rats upon administration of zinc. Gastroenterology 1984; 86: 532-539. 9 Boiarin DM, Savolainen E-R, Kivirikko KI. Three serum markers of collagen biosynthesis in Nigerians with cirrhosis and various infectious diseases. Em J Clin Invest 1984; 14: 90-95. 10 Myllyla R, Myllyla W, Tolonen U, Kivirikko KI. Changes in collagen metabolism in diseased muscle. I. Biochemical studies. Arch Neurol 1982; 39: 752-755. 11 Kuutti-Savolainen E-R. Enzymes of collagen biosynthesis in skin and serum in dermatological diseases. II. Serum enzymes. Clin Chim Acta 1979; 93: 53-58. 12 Myllyla R, Risteli L, Kivirikko KI. Collagen ghtcosyltransferase. Partial purification and characterization of the enzyme from whole chick embryos and chick-embryo cartilage. Eur J Biochem 1976; 61: 59-67. 13 Karlinsky JB, Goldstein RH. Editorial. Fibrotic lung disease - a perspective. J Lab Clin Med 1980; 96: 939-942. 14 Laurent GJ, McAnulty RJ. Protein metabolism during bleomycin-induced pulmonary fibrosis in rabbits. In vivo evidence for collagen accumulation because of increased synthesis and decreased degradation of the newly synthesized collagen. Am Rev Respir Dis 1983; 128: 82-88. 15 Terho EO. Extrinsic allergic alveolitis - the state of the art. Eur J Respir Dis 1982; 63 (suppl 124): 10-26.
3
Elsevier CCA 03152
Elevated serum galactosylhydroxylysyl glucosyltransferase, a collagen synthesis marker, in fibrosing lung diseases Henrik Anttinen
a**, Erkki 0. Terho b, Pekka M. Jarvensivu Eeva-Riitta Savolainen d
’ and
a Department of Medicine, University of Turku, Turku, b Department of Pulmonary Diseases, Kuopio University Central Hospital, Tarinaharju, and’ Department of Medicine and * Collagen Research Unit, University of Ouly Oulu (Finland) (Received September 4th, 1984; revision January 3rd, 1985) Key words: Pulmonary fibrosis; Farmer’s lung; Collagen synthesis; Galactosylhydroxytjsyl glucosyltransferase
summtuy The activity of galactosylhydroxylysyl glucosyltransferase, an enzyme catalyzing collagen biosynthesis, was measured in the sera of 101 patients with various pulmonary diseases to study whether detectable enzyme amounts are liberated into the serum from the lung tissue, and whether this is associated with the development of lung fibrosis. Increased serum galactosylhydroxylysyl glucosyltransferase activity was found in all the patients with progressive pulmonary fibrosis and in about half of the patients with acute stages of farmer’s lung and infectious pneumonia. In one third of the patients with stage I sarcoidosis the serum enzyme activity was slightly increased, whereas in bronchial asthma and chronic bronchitis the values were mostly within the normal range. In conclusion, elevated serum enzyme activity was demonstrated in connection with those respiratory diseases in which pulmonary fibrosis was already verifiable or relatively often develops later. Measurements of serum galactosylhydroxylysyl glucosyltransferase may, thus, be useful in evaluating actual lung collagen synthesis in human pulmonary diseases.
Introduction
No serum parameter has hitherto been reported which reflects changes in the rate of collagen biosynthesis in the lung tissue. Measurement of such a parameter would * Correspondence to: Dr. Hemik Anttinen, Department of Medicine, University of Turku, Kiinamyllynkatu 4-8, SF-20520 Turku 52, Finland. 0009-8981/85/$03.30
0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
be valuable in connection with those pulmonary diseases which lead to fibrosis in which excessive collagen deposition is a central pathological feature. Galactosylhydroxylysyl glucosyltransferase (GGT, EC 2.4.1.66) is an intracellular enzyme required in one of the unique post-translational modifications in collagen biosynthesis which catalyzes no significant transfer of glucose to glycoproteins other than collagens [l]. This enzyme activity can reliably be determined in human serum (S-GGT) by a relatively simple procedure feasible for large series of samples [2-51, the activity being very stable during the storage of the serum [4]. Previous reports indicate that S-GGT reflects the actual collagen synthesis in certain liver [4,6-91 and neuromuscular [lo] diseases and is even sensitive enough to detect collagen scar formation following acute myocardial infarction [5]. Furthermore, this measurement can be used in the evaluation of treatment in fibrosing liver diseases [7]. However, S-GGT is normal in the great majority of localized skin diseases [ll] and in many malignant diseases not involving liver [6]. At the moment, no information is available on S-GGT in human lung diseases. The present work sets out to study whether detectable amounts of GGT are liberated into the serum from the lung tissue in human pulmonary disorders, and whether elevated S-GGT values can be demonstrated in those pulmonary diseases in which lung collagen synthesis is increased and local or generalized pulmonary fibrosis develops. Patients and methods Patients
Serum samples were obtained from 101 patients (35 males and 66 females, aged TABLE I Patient characteristics and mean activity of serum gaIactosyIhydroxylysy1 ghmosyltransferase (S-GGT) in various groups of pulmonary diseases Diagnostic group
Bronchial asthma chronic bronchitis Sarcoidosis Infectious pneumonia Farmer’s lung Pulmonary fibrosis
No
Sex
Mean age (range) yr
Stage of disease
Mean S-GGT f (U/I)
SD
p (compared with reference mean ” )
15
3M,12F
56 (28-78)
Severe
0.49 f 0.06
< 0.01
8 6
8M 3 M, 3 F
68 (52-77) 41 (29-67)
Exacerbated Stage I
0.51 f0.06 0.5OkO.12
< 0.01 < 0.05
22
12 M, 10 F
56 (15-93)
Acute
0.58kO.18
< 0.001
45
7M,38F
46 (21-69)
Acute
0.63 f 0.17
< 0.001
5
2M,3F
53 (35-64)
Progressive
0.71 f 0.06
< 0.001
a S-GGT reference mean f
SD
is 0.43 f 0.07 U/l and the upper normal Iimit 0.57 U/I (71.
5
15-93 yr, mean age 51 yr). Fifty-six of the patients were admitted to hospital for diverse pulmonary disorders and 45 with an acute stage of farmer’s lung but without any detectable pulmonary fibrosis. The subjects fulfilled the criterion of not suffering from any other disease known or assumed to affect tissue collagen synthesis or S-GGT activity [1,3-7,9-111, but were otherwise consecutive and unselected. The patient characteristics are shown in Table I. Progressive pulmonary fibrosis was documented by follow-up of the chest X-ray findings and in all but one of the patients, also by lung biopsies. This group consisted of five patients, two having fibrosing alveolitis and the other also pleural fibrosis. Both were treated with corticosteroids. In one patient the fibrosis resulted from farmer’s lung and in two from recurrent infectious pneumonias. These three patients were receiving no anti-inflammatory or antifibrotic treatment. Methoh The blood samples were allowed to clot for 1.5 h at room temperature before the separation of the sera. S-GGT activity was assayed in duplicate with 40-~1 samples of the serum by determining the amount of radioactive glucosylgalactosylhydroxylysine formed from uridine diphosphate-[‘4C]glucose in a gelatinized collagen substrate [2,3,12]. The gelatin substrate was prepared from insoluble calf skin collagen as described in detail previously [2]. One unit of GGT activity was defined as the activity required to synthesize an amount of radioactive glucosylgalactosylhydroxylysine in dpm corresponding to 1 pmol in 1 h at 37°C under conditions in which a saturating concentration of denatured purified citrate-soluble rat skin collagen is used as a substrate [12]. The reference S-GGT value for 77 healthy control subjects (aged 14-61 yr) determined in our laboratory is 0.43 + 0.07 U/l (mean f SD) [7], and values exceeding 0.57 U/l (mean + 2 SD) were considered abnormal. No changes are found in this serum enzyme activity with age or with sex in subjects aged > 12 yr [3]. Samples of three control sera were assayed together with the sera of the patients in each enzyme series. The statistical significance of the differences between the means was calculated using the two-tailed Student’s t test. Results
Individual S-GGT values in various diagnostic groups are shown in Fig. 1. Elevated values were recorded most frequently in progressive pulmonary fibrosis (all 5 patients) and in the acute stages of farmer’s lung (23 out of 45, 51%) and infectious pneumonia (10 out of 22, 45%). One third (2 out of 6) of the patients with stage I sarcoidosis had slightly increased S-GGT. In bronchial asthma and chronic bronchitis S-GGT was mostly within the normal range. The highest mean S-GGT was found in progressive pulmonary fibrosis, but the mean S-GGT also exceeded the upper normal limit in the acute stages of farmer’s lung and infectious pneumonia (Table I). In stage I sarcoidosis, bronchial asthma and chronic bronchitis the mean S-GGT, although being slightly higher than the reference mean, was within the normal range (Table I).
6
1.1
c
I
0.9 -
5
v
0 _____
v)
0.5
0-e .*& 0
0
0 I____
l
G0
L
0 00 0.3
---_,
__--
_-_
-.
---_
OL
Fig. 1. Individual values of S-GGT activity in various groups of pulmonary diseases. The horizontal solid and dashed lines indicate the mean and limits of the mean f 2 SD for the reference material.
Very high individual S-GGT values exceeding the reference mean +4 SD were found in 60% (3 out of 5) of patients with progressive pulmonary fibrosis, 29% (13 out of 45) of those with the acute stage of farmer’s lung and 23% (5 out of 22) of those with the acute stage of infectious pneumonia, but they were lower than this value in all the patients in the other groups. Discussion Human S-GGT measurements have previously been used for evaluating fibrotic processes and treatment in certain liver diseases [4,6,7,9] and diseases affecting muscular tissue [5,10]. The present results clearly show that detectable amounts of GGT are also liberated into the serum from the affected tissue in human pulmonary diseases.
7
Recent animal studies with experimentally induced lung collagen accumulation demonstrate increased lung GGT activities which correlate in a statistically significant manner with increased lung collagen content, and that S-GGT parallels these changes (H. Anttinen, U. Puistola, A. Oikarinen, L. Ryhanen, unpubl. obs.). Thus, it seems likely that S-GGT reflects the actual changes in tissue collagen synthesis occurring in human pulmonary diseases. The elevated S-GGT values were highest and most frequently encountered in progressive pulmonary fibrosis and the acute stages of farmer’s lung and infectious pneumonia. Concerning the present results it is significant, that the rate of collagen synthesis usually increases in inflammation of the lung [13,14] and in the early inflammatory stages preceding fibrosis, even though no evident collagen accumulation can yet be observed [13,14]. In the sarcoidosis group the chest X-ray findings and pulmonary function tests of the patient with the highest S-GGT value revealed progressive impairment in 6 mth, whereas in the other sarcoidosis patients no progression of the disease was found during this follow-up period. According to our clinical experience, roentgenologically verifiable pulmonary fibrosis seldom develops in farmer’s lung, but pulmonary function tests far more often remain permanently impaired, suggesting the development of irreversible pulmonary fibrosis. In the pathogenesis of farmer’s lung both immune complex-mediated (Type III) and cell-mediated (Type IV) immune reactions seem to be involved [15], which both tend to lead to fibrotic tissue changes. The high frequency of elevated S-GGT values found here in the acute stage of farmer’s lung led us to start a follow-up study on this disease. The results, although still preliminary, suggest that the S-GGT values at the acute stage can be used to predict irreversible pulmonary impairment due to fibrosis (H. Anttinen, EO. Terho, R. Myllyl$ E-R. Savolainen, unpubl. obs.). Monitoring of a serum indicator of changes in lung collagen synthesis would be a convenient way of gaining useful information for evaluating the fibrotic process and the need for, or efficiency of, therapeutic intervention. S-GGT may prove to be such an indicator, even in early stages of fibrosing pulmonary diseases. Acknowledgements This study was supported by grants from the Sigrid Juselius Foundation, Finland, and the Medical Research Council of the Academy of Finland. We thank Professor Kari I. Kivirikko for his valuable suggestions and comments, and Mrs. Ritva Pihkakoski and Miss ArJa Oja for their expert technical assistance. References 1 Kivirikko KI, Myllyla R. Collagen glycosyltrausferases. Int Rev Connect Tissue Res 1979; 8: 23-72. 2 Myllylil R, Risteli L, Kivirikko KI. Assay of collagen-galactosyltransferase and collagen-glucosyltransfemse activities and preliminary characterization of enzymic reactions with transferases from chick-embryo cartilage. Eur J Biochem 1975; 52: 401-410. 3 Anttinen H. Collagen glucosyltransferase activity in human serum. Clin Chim Acta 1977; 77: 323-330.
8 4 Kuutti-Savolainen E-R, Anttinen H, Miettinen TA, Kivirikko KI. Collagen biosynthesis enzymes in serum and hepatic tissue in liver disease. II. Galactosylbydroxylysyl glucosyltransferase. Eur J Clin Invest 1979; 9: 97-101. 5 Anttinen H, Jarvensivu PM, Savolainen E-R. Serum galactosylbydroxylysyl glucosyltransferase in acute myocardial infarction and during subsequent collagen scar formation. Eur J Clin Invest 1981; 11: 375-379. 6 Bolarin DM, Savolainen E-R, Kivirikko KI. Enzymes of collagen synthesis and type III procollagen amino-propeptide in serum from Nigerians with hepatocellular carcinoma and other malignant diseases. Int J Cancer 1982; 29: 401-405. 7 Savolainen E-R Miettinen TA, Pikkarainen P, Salaspuro MP, Kivirikko KI. Enzymes of collagen synthesis and type III procollagen aminopropeptide in the evaluation of D-penicillamine and medroxyprogesterone treatments of primary bihary cirrhosis. Gut 1983; 24: 136-142. 8 Anttinen H, Ryhanen L, Puistola U, Arrant0 A, Gikarinen A. Decrease in liver collagen accumulation in carbon tetrachloride-injured and normal growing rats upon administration of zinc. Gastroenterology 1984; 86: 532-539. 9 Boiarin DM, Savolainen E-R, Kivirikko KI. Three serum markers of collagen biosynthesis in Nigerians with cirrhosis and various infectious diseases. Em J Clin Invest 1984; 14: 90-95. 10 Myllyla R, Myllyla W, Tolonen U, Kivirikko KI. Changes in collagen metabolism in diseased muscle. I. Biochemical studies. Arch Neurol 1982; 39: 752-755. 11 Kuutti-Savolainen E-R. Enzymes of collagen biosynthesis in skin and serum in dermatological diseases. II. Serum enzymes. Clin Chim Acta 1979; 93: 53-58. 12 Myllyla R, Risteli L, Kivirikko KI. Collagen ghtcosyltransferase. Partial purification and characterization of the enzyme from whole chick embryos and chick-embryo cartilage. Eur J Biochem 1976; 61: 59-67. 13 Karlinsky JB, Goldstein RH. Editorial. Fibrotic lung disease - a perspective. J Lab Clin Med 1980; 96: 939-942. 14 Laurent GJ, McAnulty RJ. Protein metabolism during bleomycin-induced pulmonary fibrosis in rabbits. In vivo evidence for collagen accumulation because of increased synthesis and decreased degradation of the newly synthesized collagen. Am Rev Respir Dis 1983; 128: 82-88. 15 Terho EO. Extrinsic allergic alveolitis - the state of the art. Eur J Respir Dis 1982; 63 (suppl 124): 10-26.