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Heterogeneity of the antigens related to the aminoterminal propeptide of type III procollagen in human serum
Clinica Chimica Acta, 124 (1982) 39-44 Elsevier Biomedical Press
39
CCA 2233
Heterogeneity of the antigens related to the aminoterminal propeptide of type III procollagen in human serum Onni Niemel;i a, Leila Risteli a, Eero A. Sotaniemi b and Juha Risteli a** a Depariments
of Clinical Chemistry and Medical Biochemistty, and b Clinical Research Unii, Department Internal Medicine, University of Ouly Oulu (Finland) (Received
November
of
23rd, 1981; revision April 21st, 1982)
Inhibition curves that are considerably less steep than the reference peptide curve are a constant finding when human serum samples are studied with the radioimmunoassay for the aminoterminal propeptide Co1 l-3 of type III procollagen. This is due to the presence in the serum of three main peptide forms which differ in their antigenic properties and can be separated by gel filtration. Their molecular sizes are, respectively, larger than, equal to and smaller than the peptide Co1 1-3. The proportions of these forms were different in a number of serum samples tested. An elevated value in the Co1 l-3 radioimmunoassay need not reflect increased deposition of type III collagen in the liver, but could also be due to increased degradation of a newly-synthesized type III procollagen or degradation of a tissue form still containing the aminoterminal propeptide. This should be considered when interpreting elevated serum values.
Introduction Collagen is synthesized as a larger molecule, procollagen, which contains additional peptide extensions at both the aminoterminal and the carboxyterminal end [ 11. These peptides are cleaved off by specific proteases during the conversion of procollagen to collagen [2]. The level of the aminoterminal propeptide of type III procollagen in serum can be measured by radioimmunoassay, and it has been suggested that this reflects connective tissue formation activity in the liver [3,4]. The carboxyterminal propeptide of type I procollagen can also be detected in serum [S]. * Address for correspondence: SF-90220 Oulu 22, Finland.
OOOS-8981/82/0000-0000/$02.75
Dr. Juha Risteli, Department
0 1982 Elsevier Biomedical
of Clinical
Press
Chemistry,
University
of Oulu,
40
When fibrosis develops, the collagen first deposited in the tissue contains increased amounts of type III collagen [l], whereas type I collagen predominates in advanced liver cirrhosis and the ratio of type I:type III increases [6]. Type III collagen has a more rapid turnover than type I collagen [6], and it has been suggested that most of the collagen degradation in the liver occurs with newlysynthesized or only loosely organized collagen [6]. Type III collagen is also found in liver in a form which still contains the aminoterminal propeptide [7,8]. The radioimmunoassay for the aminoterminal propeptide of type III procollagen, now available commercially, is based on the use of the bovine peptide, which shows a significant cross-reaction with human samples [3]. The slope of the inhibition curve for human serum is less steep than that for the bovine reference peptide, however. Although this was not recognized in previous studies [3,4]. it has been constantly observed recently, and the manufacturer of the commercial radioimmunoassay kit now recommends the use of a 50% intercept method for the evaluation of the data from human samples [9]. In the present study the reason for the antigenic difference between the bovine peptide and the cross-reacting material in human serum was carefully investigated using several serum samples representing different pathologies of the liver. Materials and methods Antigens and immunization Type III procollagen was isolated from foetal calf skin, and its aminoterminal propeptide, Co1 1-3, was obtained by collagenase digestion and purified as described previously [lo]. A rabbit antiserum was prepared by a subcutaneous injection of 300 pg. of the Co1 l-3 peptide in Freund’s complete adjuvant. Two similar booster injections were given at three-week intervals. Serum samples and gel filtration procedure Forty-two human serum samples were obtained both from healthy volunteers and from patients with various liver diseases. Samples of 2 ml were chromatographed on a column (1.5 X 110 cm) of Sephacryl S-300 equilibrated in phosphate buffered saline, pH 7.2, containing 0.04% Tween 20. Fractions of 3.3 ml were collected. Each fraction was directly analyzed for the presence of Co1 l-3 related material with Co1 l-3 radioimmunoassay. The column was calibrated with bovine serum albumin, the labelled peptide Co1 l-3 and tritiated water. Radioimmunoassay The peptide Co1 l-3 was labelled with “‘1 by the chlorarnine T method [ 111. In the standard inhibition assay (sequential saturation type) 0.1 ml of an antiserum dilution capable of binding 50% of the labelled antigen was incubated for 16 h at 4’C together with 0.2 ml of non-labelled antigen solution or unknown sample. After addition of 1 ng labelled antigen (35000 dpm in 0.1 ml), incubation was continued for 6 h. The free and bound antigens were then separated by precipitation (16 h at 4°C) with 0.01 ml of goat antiserum to rabbit immunoglobulin G. All dilutions were
41
made in phosphate buffered non-specific binding,
saline, pH 7.2, containing
0.04% Tween
20 to prevent
Computer analysis of radioimmunoassay data The slopes of the inhibition curves obtained with the reference peptide Co1 1-3, serum samples and different gel filtration pools were calculated with an Apple II computer using a program described in [12]. The method uses logit and log transformations to obtain a linear dose response curve, followed by iterated weighted least squares regression analysis. Statistical methods A one-way analysis of variance the slope differences between the filtration pools. The significance tested using a modified t test and method (see [ 131).
was used to analyze the statistical significance of reference peptide Co1 l-3 and the different gel of the difference from the peptide Co1 l-3 was calculating the critical t value by the Bonferroni
Results The rabbit antiserum against the aminoterminal propeptide of bovine type III procollagen bound the protein in radioimmunoassay, and the 1: 200 dilution, giving 50% binding of the labelled peptide, was selected for the inhibition tests. Less than 1% of the label was precipitated in the presence of non-immune rabbit serum. A 50% displacement of the label in the Co1 1-3 radioimmuno-inhibition assay was achieved with a concentration of about 1 pg/l of the non-labelled peptide (Fig. 1). The inhibition curves obtained with human serum samples were less steep than that of the reference peptide. The mean slope for 42 different sera was 0.82 * 0.14 (range I/DILUTION 256 100.
64
OF SERUM 16
4
--p-1_ s3
1
52 SOSl ? z
z E
40
20
g
60 :/v 0 0.31 INHIBITOR
1.25
5
b&l/l)
Fig. 1. Radioimmuno-inhibition assay using ‘251-labelled Co1 1-3 and a specific antiserum. The inhibitors and the serum samples Sl (control), S2 (chronic of the reaction were the peptide Co1 l-3 (0 -0) active hepatitis) and S3 (acute alcoholic hepatitis).
42 TABLE
I
SLOPES OF THE lNHlBlTlON MUNOASSAY
CURVES
FOR Co1 1-3. pool I. II AND
111 IN (‘01 l-3 RADIOIM_~_
Sample
n
--co1 Pool Pool Pool
6 6 4* 6
l-3 I II Ill
* In the gel filtration
Slope (mean * SD)
Statistical (compared
~______ 1.68*0.10 1.071-0.10 1.67-0.16 0.55 5: 0.07
p
runs for two sera the amount
of antigen
significance with Co1 1-3)
in pool II was too low to be analyzed.
0.6661.07) which is highly significantly (p < 0.001) different from the slope of the Co1 l-3 (1.68kO.10, in Tablel). The inhibition curves obtained for three representative serum samples Sl (control), S2 (chronic active hepatitis) and S3 (acute alcoholic hepatitis) are shown in Fig. 1. The dilution of the con&o1 serum sample Sl giving 50% inhibition in the assay corresponded to 7.5 pg of Co1 l-3/1 of serum. The values for the sera S2 and
INHIBITOR 12.5
50
WI) 201
1oor
30
40 FRACTION
50
60
NUMBER
0.31 INHIBITOR
1.25
5
(&I/I)
Fig. 2. Gel filtration of the serum samples Sl (control), S2 (chronic active hepatitis) and S3 (acute alcoholic hepatitis) on a Sephacryl S-300 column. The elution position of the peptide Co1 l-3 is indicated by the arrow. The relative concentration of the inhibitor in the different fractions is calculated from the observed inhibition using Co1 l-3 as a standard. In reality the main peaks have different inhibition slopes, and thus the quantification is arbitrary. The bars indicate the pools used for the inhibition studies in Fig. 3. Fig. 3. Radioimmuno-inhibition assay for Co1 1-3. The inhibitors 0) and the pools I, II and III from Fig. 2.
(.--
of the reaction
were the peptide
Co1 l-3
43
S3, calculated in the same way, were 2.3 and 10 times higher than this, respectively. A number of the sera tested, representing the whole range of peptide concentrations (5-100 fig/l), were subjected to gel filtration. The antigenicity related to Co1 l-3 resolved into at least four peaks, their proportions varying from one sample to another. In this respect, three basic patterns were observed (illustrated by the sera Sl, S2 and S3 in Fig. 2). Some antigenicity eluted in the void volume of the Sephacryl S-300 column, this proportion being highest in S2. The next peak eluted well before the reference peptide Co1 1-3, and significant amounts of this material were present in all the samples. The third peak corresponded in size to the authentic Co1 l-3. This was the main component in S3, but only visible as a shoulder in the other samples. Finally, all the samples studied contained a similar amount of material clearly smaller than Co1 l-3. For comparison of the inhibition capacities of the three main peaks, three pools were made, as indicated in Fig. 2, from the gel filtration fractions: pool I larger than Co1 l-3; pool II similar in size to Co1 1-3; and pool III smaller than Co1 1-3. These pools were tested in the Co1 l-3 radioimmuno-inhibition assay (Fig. 3). Pool 11 gave an inhibition curve identical to that of the reference peptide, whereas the two other pools revealed less efficient inhibition. The slopes of similar pools from six gel filtration runs are given in TableI. Pools I and III differed highly significantly (p < 0.001) from the reference peptide Co1 l-3 and pool II. Discussion The antigens related to the aminoterminal propeptide of type III procollagen were found to be heterogenous in human serum. Three main species having respectively higher, equal or lower molecular masses by gel filtration than the reference peptide Co1 l-3 were detected. As only one of these components gave an inhibition curve similar in shape to that for the reference peptide (TableI), the overall inhibition curves of the sera were considerably less steep. This heterogeneity has been observed earlier by Rohde et al in biological fluids [3], but the reported inhibition curves with serum samples were identical or very close to that for Co1 l-3. Their assay was nevertheless able to detect the smallest form, which also gave a less steep inhibition, in urine [3]. The heterogeneity and difference in slope are not confined to the assay when performed using our reagents, however, as the commercial radioimmunoassay kit (Behringwerke AG: Lot 17/81) gave similar results. It has been suggested that the increased amounts of free propeptide in the serum are derived from de novo synthesis of type III procollagen in the liver [3,4]. However, type III procollagen is also deposited extracellularly without cleavage of the aminoterminal propeptide [7,8], and so the degradation of the tissue form may contribute to the antigens in the serum. The form larger than the peptide Co1 l-3 (pool I) in particular cannot be derived from normal cleavage by the aminoterminal procollagen peptidase. Rohde et al, who also found three distinct peaks in gel filtration, assumed that this peak might be an aggregated form of the peptide Co1 l-3 [3]. This is not likely, however, since pool I had a slope which differed significantly from that of pool II (Fig. 3, TableI). Furthermore, there was no stoichiometric
44
relationship between pools I and 11. In fact, there was more material in pool 1 than in pool II in most sera. The smallest serum antigen, pool III, was present in constant amounts in all samples and it was the main form in the normal sera. It is probably related to the Co1 1 domain of the Co1 l-3 peptide [3]. Ackermann et al found that the Co1 l-3 level in serum correlated with a parameter of connective tissue degradation in chronic active liver diseases [4]. In addition to the synthesis and deposition of type III collagen, degradation of either newly-synthesized type III procollagen or a tissue form still containing the propeptide could affect the Co1 l-3 level in serum. The contributions of these mechanisms could vary in different diseases of the liver. As long as the origin of the different antigen forms in serum is not known, these alternatives should be taken into consideration when interpreting the results. Acknowledgements The expert technical assistance of Miss Aila Utoslahti is acknowledged. We thank Professor Kari I. Kivirikko for helpful discussions. This study was supported in part by a grant from the Medical Research Council of the Academy of Finland. References 1 Prockop DJ, Kivirikko KI, Tuderman L, Guzman NA. The biosynthesis of collagen and its disorders. N Engl J Med 1979; 301: 13-23, 77-85. 2 Fessler JH, Fessler LI. Biosynthesis of procollagen. Ann Rev Biochem 1978; 47: 129-162. 3 Rohde H, Vargas L, Hahn E, Kalbfleisch H, Bruguera M, Timpl R. Radioimmunoassay for type III procollagen peptide and its application to human liver disease. Em J Clin Invest 1979; 9: 451-459. 4 Ackermann W, Pott G, Voss B, Mtiller K-M, Gerlach U. Serum concentration of procollagen-IIIpeptide in comparison with the serum activity of N-acetyl+-glucosaminidase for diagnosis of the activity of liver fibrosis in patients with chronic active liver diseases. Clin Chim Acta 1981; 112: 365-369. 5 Taubmann MB, Goldberg B, Sherr CJ. Radioimmunoassay for human procollagen. Science 1974; 186: 1115-1117. 6 Rojkind M, Kershenobich D. Hepatic fibrosis. Clin Gastroenterol 1981; 10: 737-754. 7 Gay S, Fietzek PP, Remberger K, Eder M, Kuhn K. Liver cirrhosis: immunofluorescence and biochemical studies demonstrate two types of collagen. Klin Wochenschr 1975; 53: 205-208. 8 Wick G, Brunner H, Penner E, Timpl R. The diagnostic application of specific antiprocollagen sera. II. Analysis of liver biopsies. Int Arch Allergy Appl Immunol 1978; 56: 316-324. 9 Instruction booklet: RIA-Procollagen-III-peptide. Behringwerke AG, 198 1. IO Nowack H, Olsen BR, Timpl R. Characterization of the aminoterminal segment in type III procollagen. Eur J Biochem 1976; 70: 205-216. 11 Timpl R, Risteli L. Radioimmunoassays in studies of connective tissue proteins. In: Furthmayr H, ed. Immunochemistry of the extracellular matrix. Boca Raton: CRC Press, 1982; in press. 12 Rodbard D, Lewald JE. Computer analysis of radioligand assay and radioimmunoassay data. Acta Endocrinol (Kbh) 1970; 147: 79-103. 13 Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res 1980: 47: 1-9.
39
CCA 2233
Heterogeneity of the antigens related to the aminoterminal propeptide of type III procollagen in human serum Onni Niemel;i a, Leila Risteli a, Eero A. Sotaniemi b and Juha Risteli a** a Depariments
of Clinical Chemistry and Medical Biochemistty, and b Clinical Research Unii, Department Internal Medicine, University of Ouly Oulu (Finland) (Received
November
of
23rd, 1981; revision April 21st, 1982)
Inhibition curves that are considerably less steep than the reference peptide curve are a constant finding when human serum samples are studied with the radioimmunoassay for the aminoterminal propeptide Co1 l-3 of type III procollagen. This is due to the presence in the serum of three main peptide forms which differ in their antigenic properties and can be separated by gel filtration. Their molecular sizes are, respectively, larger than, equal to and smaller than the peptide Co1 1-3. The proportions of these forms were different in a number of serum samples tested. An elevated value in the Co1 l-3 radioimmunoassay need not reflect increased deposition of type III collagen in the liver, but could also be due to increased degradation of a newly-synthesized type III procollagen or degradation of a tissue form still containing the aminoterminal propeptide. This should be considered when interpreting elevated serum values.
Introduction Collagen is synthesized as a larger molecule, procollagen, which contains additional peptide extensions at both the aminoterminal and the carboxyterminal end [ 11. These peptides are cleaved off by specific proteases during the conversion of procollagen to collagen [2]. The level of the aminoterminal propeptide of type III procollagen in serum can be measured by radioimmunoassay, and it has been suggested that this reflects connective tissue formation activity in the liver [3,4]. The carboxyterminal propeptide of type I procollagen can also be detected in serum [S]. * Address for correspondence: SF-90220 Oulu 22, Finland.
OOOS-8981/82/0000-0000/$02.75
Dr. Juha Risteli, Department
0 1982 Elsevier Biomedical
of Clinical
Press
Chemistry,
University
of Oulu,
40
When fibrosis develops, the collagen first deposited in the tissue contains increased amounts of type III collagen [l], whereas type I collagen predominates in advanced liver cirrhosis and the ratio of type I:type III increases [6]. Type III collagen has a more rapid turnover than type I collagen [6], and it has been suggested that most of the collagen degradation in the liver occurs with newlysynthesized or only loosely organized collagen [6]. Type III collagen is also found in liver in a form which still contains the aminoterminal propeptide [7,8]. The radioimmunoassay for the aminoterminal propeptide of type III procollagen, now available commercially, is based on the use of the bovine peptide, which shows a significant cross-reaction with human samples [3]. The slope of the inhibition curve for human serum is less steep than that for the bovine reference peptide, however. Although this was not recognized in previous studies [3,4]. it has been constantly observed recently, and the manufacturer of the commercial radioimmunoassay kit now recommends the use of a 50% intercept method for the evaluation of the data from human samples [9]. In the present study the reason for the antigenic difference between the bovine peptide and the cross-reacting material in human serum was carefully investigated using several serum samples representing different pathologies of the liver. Materials and methods Antigens and immunization Type III procollagen was isolated from foetal calf skin, and its aminoterminal propeptide, Co1 1-3, was obtained by collagenase digestion and purified as described previously [lo]. A rabbit antiserum was prepared by a subcutaneous injection of 300 pg. of the Co1 l-3 peptide in Freund’s complete adjuvant. Two similar booster injections were given at three-week intervals. Serum samples and gel filtration procedure Forty-two human serum samples were obtained both from healthy volunteers and from patients with various liver diseases. Samples of 2 ml were chromatographed on a column (1.5 X 110 cm) of Sephacryl S-300 equilibrated in phosphate buffered saline, pH 7.2, containing 0.04% Tween 20. Fractions of 3.3 ml were collected. Each fraction was directly analyzed for the presence of Co1 l-3 related material with Co1 l-3 radioimmunoassay. The column was calibrated with bovine serum albumin, the labelled peptide Co1 l-3 and tritiated water. Radioimmunoassay The peptide Co1 l-3 was labelled with “‘1 by the chlorarnine T method [ 111. In the standard inhibition assay (sequential saturation type) 0.1 ml of an antiserum dilution capable of binding 50% of the labelled antigen was incubated for 16 h at 4’C together with 0.2 ml of non-labelled antigen solution or unknown sample. After addition of 1 ng labelled antigen (35000 dpm in 0.1 ml), incubation was continued for 6 h. The free and bound antigens were then separated by precipitation (16 h at 4°C) with 0.01 ml of goat antiserum to rabbit immunoglobulin G. All dilutions were
41
made in phosphate buffered non-specific binding,
saline, pH 7.2, containing
0.04% Tween
20 to prevent
Computer analysis of radioimmunoassay data The slopes of the inhibition curves obtained with the reference peptide Co1 1-3, serum samples and different gel filtration pools were calculated with an Apple II computer using a program described in [12]. The method uses logit and log transformations to obtain a linear dose response curve, followed by iterated weighted least squares regression analysis. Statistical methods A one-way analysis of variance the slope differences between the filtration pools. The significance tested using a modified t test and method (see [ 131).
was used to analyze the statistical significance of reference peptide Co1 l-3 and the different gel of the difference from the peptide Co1 l-3 was calculating the critical t value by the Bonferroni
Results The rabbit antiserum against the aminoterminal propeptide of bovine type III procollagen bound the protein in radioimmunoassay, and the 1: 200 dilution, giving 50% binding of the labelled peptide, was selected for the inhibition tests. Less than 1% of the label was precipitated in the presence of non-immune rabbit serum. A 50% displacement of the label in the Co1 1-3 radioimmuno-inhibition assay was achieved with a concentration of about 1 pg/l of the non-labelled peptide (Fig. 1). The inhibition curves obtained with human serum samples were less steep than that of the reference peptide. The mean slope for 42 different sera was 0.82 * 0.14 (range I/DILUTION 256 100.
64
OF SERUM 16
4
--p-1_ s3
1
52 SOSl ? z
z E
40
20
g
60 :/v 0 0.31 INHIBITOR
1.25
5
b&l/l)
Fig. 1. Radioimmuno-inhibition assay using ‘251-labelled Co1 1-3 and a specific antiserum. The inhibitors and the serum samples Sl (control), S2 (chronic of the reaction were the peptide Co1 l-3 (0 -0) active hepatitis) and S3 (acute alcoholic hepatitis).
42 TABLE
I
SLOPES OF THE lNHlBlTlON MUNOASSAY
CURVES
FOR Co1 1-3. pool I. II AND
111 IN (‘01 l-3 RADIOIM_~_
Sample
n
--co1 Pool Pool Pool
6 6 4* 6
l-3 I II Ill
* In the gel filtration
Slope (mean * SD)
Statistical (compared
~______ 1.68*0.10 1.071-0.10 1.67-0.16 0.55 5: 0.07
p
runs for two sera the amount
of antigen
significance with Co1 1-3)
in pool II was too low to be analyzed.
0.6661.07) which is highly significantly (p < 0.001) different from the slope of the Co1 l-3 (1.68kO.10, in Tablel). The inhibition curves obtained for three representative serum samples Sl (control), S2 (chronic active hepatitis) and S3 (acute alcoholic hepatitis) are shown in Fig. 1. The dilution of the con&o1 serum sample Sl giving 50% inhibition in the assay corresponded to 7.5 pg of Co1 l-3/1 of serum. The values for the sera S2 and
INHIBITOR 12.5
50
WI) 201
1oor
30
40 FRACTION
50
60
NUMBER
0.31 INHIBITOR
1.25
5
(&I/I)
Fig. 2. Gel filtration of the serum samples Sl (control), S2 (chronic active hepatitis) and S3 (acute alcoholic hepatitis) on a Sephacryl S-300 column. The elution position of the peptide Co1 l-3 is indicated by the arrow. The relative concentration of the inhibitor in the different fractions is calculated from the observed inhibition using Co1 l-3 as a standard. In reality the main peaks have different inhibition slopes, and thus the quantification is arbitrary. The bars indicate the pools used for the inhibition studies in Fig. 3. Fig. 3. Radioimmuno-inhibition assay for Co1 1-3. The inhibitors 0) and the pools I, II and III from Fig. 2.
(.--
of the reaction
were the peptide
Co1 l-3
43
S3, calculated in the same way, were 2.3 and 10 times higher than this, respectively. A number of the sera tested, representing the whole range of peptide concentrations (5-100 fig/l), were subjected to gel filtration. The antigenicity related to Co1 l-3 resolved into at least four peaks, their proportions varying from one sample to another. In this respect, three basic patterns were observed (illustrated by the sera Sl, S2 and S3 in Fig. 2). Some antigenicity eluted in the void volume of the Sephacryl S-300 column, this proportion being highest in S2. The next peak eluted well before the reference peptide Co1 1-3, and significant amounts of this material were present in all the samples. The third peak corresponded in size to the authentic Co1 l-3. This was the main component in S3, but only visible as a shoulder in the other samples. Finally, all the samples studied contained a similar amount of material clearly smaller than Co1 l-3. For comparison of the inhibition capacities of the three main peaks, three pools were made, as indicated in Fig. 2, from the gel filtration fractions: pool I larger than Co1 l-3; pool II similar in size to Co1 1-3; and pool III smaller than Co1 1-3. These pools were tested in the Co1 l-3 radioimmuno-inhibition assay (Fig. 3). Pool 11 gave an inhibition curve identical to that of the reference peptide, whereas the two other pools revealed less efficient inhibition. The slopes of similar pools from six gel filtration runs are given in TableI. Pools I and III differed highly significantly (p < 0.001) from the reference peptide Co1 l-3 and pool II. Discussion The antigens related to the aminoterminal propeptide of type III procollagen were found to be heterogenous in human serum. Three main species having respectively higher, equal or lower molecular masses by gel filtration than the reference peptide Co1 l-3 were detected. As only one of these components gave an inhibition curve similar in shape to that for the reference peptide (TableI), the overall inhibition curves of the sera were considerably less steep. This heterogeneity has been observed earlier by Rohde et al in biological fluids [3], but the reported inhibition curves with serum samples were identical or very close to that for Co1 l-3. Their assay was nevertheless able to detect the smallest form, which also gave a less steep inhibition, in urine [3]. The heterogeneity and difference in slope are not confined to the assay when performed using our reagents, however, as the commercial radioimmunoassay kit (Behringwerke AG: Lot 17/81) gave similar results. It has been suggested that the increased amounts of free propeptide in the serum are derived from de novo synthesis of type III procollagen in the liver [3,4]. However, type III procollagen is also deposited extracellularly without cleavage of the aminoterminal propeptide [7,8], and so the degradation of the tissue form may contribute to the antigens in the serum. The form larger than the peptide Co1 l-3 (pool I) in particular cannot be derived from normal cleavage by the aminoterminal procollagen peptidase. Rohde et al, who also found three distinct peaks in gel filtration, assumed that this peak might be an aggregated form of the peptide Co1 l-3 [3]. This is not likely, however, since pool I had a slope which differed significantly from that of pool II (Fig. 3, TableI). Furthermore, there was no stoichiometric
44
relationship between pools I and 11. In fact, there was more material in pool 1 than in pool II in most sera. The smallest serum antigen, pool III, was present in constant amounts in all samples and it was the main form in the normal sera. It is probably related to the Co1 1 domain of the Co1 l-3 peptide [3]. Ackermann et al found that the Co1 l-3 level in serum correlated with a parameter of connective tissue degradation in chronic active liver diseases [4]. In addition to the synthesis and deposition of type III collagen, degradation of either newly-synthesized type III procollagen or a tissue form still containing the propeptide could affect the Co1 l-3 level in serum. The contributions of these mechanisms could vary in different diseases of the liver. As long as the origin of the different antigen forms in serum is not known, these alternatives should be taken into consideration when interpreting the results. Acknowledgements The expert technical assistance of Miss Aila Utoslahti is acknowledged. We thank Professor Kari I. Kivirikko for helpful discussions. This study was supported in part by a grant from the Medical Research Council of the Academy of Finland. References 1 Prockop DJ, Kivirikko KI, Tuderman L, Guzman NA. The biosynthesis of collagen and its disorders. N Engl J Med 1979; 301: 13-23, 77-85. 2 Fessler JH, Fessler LI. Biosynthesis of procollagen. Ann Rev Biochem 1978; 47: 129-162. 3 Rohde H, Vargas L, Hahn E, Kalbfleisch H, Bruguera M, Timpl R. Radioimmunoassay for type III procollagen peptide and its application to human liver disease. Em J Clin Invest 1979; 9: 451-459. 4 Ackermann W, Pott G, Voss B, Mtiller K-M, Gerlach U. Serum concentration of procollagen-IIIpeptide in comparison with the serum activity of N-acetyl+-glucosaminidase for diagnosis of the activity of liver fibrosis in patients with chronic active liver diseases. Clin Chim Acta 1981; 112: 365-369. 5 Taubmann MB, Goldberg B, Sherr CJ. Radioimmunoassay for human procollagen. Science 1974; 186: 1115-1117. 6 Rojkind M, Kershenobich D. Hepatic fibrosis. Clin Gastroenterol 1981; 10: 737-754. 7 Gay S, Fietzek PP, Remberger K, Eder M, Kuhn K. Liver cirrhosis: immunofluorescence and biochemical studies demonstrate two types of collagen. Klin Wochenschr 1975; 53: 205-208. 8 Wick G, Brunner H, Penner E, Timpl R. The diagnostic application of specific antiprocollagen sera. II. Analysis of liver biopsies. Int Arch Allergy Appl Immunol 1978; 56: 316-324. 9 Instruction booklet: RIA-Procollagen-III-peptide. Behringwerke AG, 198 1. IO Nowack H, Olsen BR, Timpl R. Characterization of the aminoterminal segment in type III procollagen. Eur J Biochem 1976; 70: 205-216. 11 Timpl R, Risteli L. Radioimmunoassays in studies of connective tissue proteins. In: Furthmayr H, ed. Immunochemistry of the extracellular matrix. Boca Raton: CRC Press, 1982; in press. 12 Rodbard D, Lewald JE. Computer analysis of radioligand assay and radioimmunoassay data. Acta Endocrinol (Kbh) 1970; 147: 79-103. 13 Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res 1980: 47: 1-9.