Collagen glucosyltransferase activity in human serum

323

Clinica Chimica Acta, 77 (1977) 323-330 @ Elsevier/North-Holland Biomedical Press

CCA 8530

COLLAGEN

GLUCOSYLTRANSFERASE

ACTIVITY

IN HUMAN SERUM

HENRIK ANTTINEN * Department of Medical Biochemistry Oulu, Oulu (Finland)

and Department

of Clinical Chemistry,

University of

(Received December 17th, 1976)

Summary Collagen glucosyltransferase activity was demonstrated in human serum. The assay used for this enzyme was shown to be specific, whereas attempts to measure collagen galactosyltransferase activity were unsuccessful. It is suggested that most of the serum collagen glucosyltransferase activity originates from the extravascular tissue, although it is possible that some is derived from the platelets. The activity of collagen glucosyltransferase in serum varied somewhat with age, the mean value in newborn infants being about 30 per cent higher than the level in adult subjects. In a randomly selected group of 50 hospitalized patients with various diseases, serum collagen glucosyltransferase activity was found to be elevated in 12 cases. The activities of reference enzymes (mainly aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase) were elevated in 10 of these 12 patients and in three additional patients within the group. However, collagen glucosyltransferase activity showed no clear linear correlation with the activities of the reference enzymes within this group of 15 patients with elevated enzyme activities, the correlation being highest in the case of aspartate aminotransferase activity (r = +0.56, P< 0.05). In two patients collagen glucosyltransferase activity was elevated but all reference enzyme activities were normal, this pattern being possibly caused by damage concentrated in the connective tissue. The measurement of collagen glucosyltransferase activity in’ serum may prove useful in studying destructive diseases affecting the collagen-producing tissues.

Correspondence to: Dr. Henrik Anttinen. Department Oulu, Kabanintie 52 A. SF-90220 Oulu 22, Finland.

of Medical Biochemistry,

University of

324

Introduction The intracellular enzymes of collagen biosynthesis have been assayed in numerous physiological, experimental and clinical conditions in a number of human tissues (for a recent review, see ref. 1). In this connection, the measurement of these enzymes in human serum appears to offer a convenient method for studying possible enzyme deficiencies and other pathological states affecting collagen-producing cells. The only enzyme of collagen biosynthesis which has hitherto been identified in serum is prolyl hydroxylase, which has been demonstrated both by activity measurements [2,3] and more recently as an immunoreactive protein by radioimmunoassay [4,5]. In most normal cases, however, serum prolyl hydroxylase activity lies on the borderline of existing detection techniques [ 61. Collagen galactosyltransferase and collagen glucosyltransferase catalyze the post-translational transfer of galactose and glucose from their nucleotide-sugar donors, UDP-galactose and UDP-glucose, to collagen [1,7-g]. The products of these reactions are hydroxylysine-linked mono- and disaccharides, galactosylhydroxylysine and glucosylgalactosylhydroxylysine [1,7-g]. The reactions are specific, and require the presence of Mn*’ or certain other bivalent cations [1,7--9]. Both enzymes are probably located within the cisternae of the endoplasmic reticulum of cells synthesizing collagen (for references, see ref. 10). Collagen glycosyltransferase activities have been demonstrated in human platelets [ll-141, and assayed as a function of age in human skin [15], but no other measurements of these enzymes have been reported in human tissues. The present work set out to investigate the presence of the two collagen glycosyltransferase activities in human serum, the activity of one of these, collagen glucosyltransferase, proving detectable and its assay specific. The serum activity of this enzyme was studied in respect of its origin and its variation with age and under certain pathological conditions in a randomly chosen hospital patient group. Material and methods Material In order to study the normal range of serum collagen glucosyltransferase activity and its age-related variations, samples were obtained from healthy volunteer subjects or patients at the Oulu University Central Hospital without any active disease that could be supposed to affect serum enzyme activity. The blood samples from newborn infants were taken from the umbilical vein. A group of 50 hospitalized patients was chosen randomly in order to study conditions in which serum collagen glucosyltransferase activity may differ from the normal. All serum samples were stored at -20°C until used. Venous blood samples for studying the blood distribution of collagen glucosyltransferase were drawn from healthy volunteers. UDP-[ 14C]galactose and UDP-[ 14C] glucose were purchased from New England Nuclear Corp., Boston, Mass., U.S.A. and the unlabelled compounds from Sigma Chemical Co., St. Louis, MO., U.S.A. The sources of the other materials were the same as specified elsewhere [ 7,8,16].

325

Preparation of various fractions of blood Samples of 100-200 ml of venous blood were drawn. Portions of 10 ml were allowed to clot and were centrifuged to obtain the serum. The remainder of each sample was anti-coagulated with a solution consisting of 85 mM trisodium citrate, 65 mM citric acid, 2% glucose and 6.6 mM EDTA (18 ml of solution per 100 ml blood). All subsequent procedures were carried out at 0-4°C. Various fractions of the anti-coagulated blood were prepared by differential centrifugation, initially twice for 15 min at 350 X g and then for 10 min at 4300 X g. Platelet-rich plasma was obtained as the 350 X g supematant, leucocytes and erythrocytes as the 350 X g pellet, plasma low in platelets as the 4300 X g supematant and platelets as the 4300 X g pellet. The pellets were carefully washed with 154 mM NaCl and 10 mM Tris/HCl, pH 7.5 at 4”C, and homogenized in a Teflon and glass homogenizer with 60 strokes in a solution consisting of 200 mM NaCl, 0.1% Triton X-100, 50 /.IM dithiothreitol and 20 mM Tris/HCl buffer adjusted to pH 7.5 at 4°C [ 81. Assays The activities of collagen galactosyltransferase (EC 2.4.1.?) and collagen glucosyltransferase (CGlcT, EC 2.4.1.?) were measured as described previously [ 7-9,15], except that in most experiments 17 PM UDP-[14C]galactose (55 Ci/mol) (for galactosyltransferase) or 17 PM UDP-[ 14C]glucose (59 Ci/mol) (for glucosyltransferase) was used for the assay. All assays were carried out in duplicate. The purification of the reaction products included the ion-exchange chromatography step of the original assay procedures [ 71, but the last step, paper electrophoresis, was omitted. The specificity of the assays with the serum enzymes was evaluated using an amino acid analyzer [15,17]. A program was used which clearly separates the hydroxylysine glycosides, radioactivity in the fractions then being measured in a liquid-scintillation counter [l&19]. One unit of collagen glucosyltransferase activity was defined as the activity required to synthesize an amount of the 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 collagen is used as a substrate and the concentrations of the other reactants are those indicated previously [ 81. The reference enzymes were aspartate‘aminotransferase (GOT, EC 2.6.1.1), alanine aminotransferase (GPT, EC 2.6.1.2), alkaline phosphatase (AP, EC 3.1.3.1), lactate dehydrogenase (LDH, EC 1.1.1.27) and 2-hydroxybutyrate dehydrogenase (HBDH). The measurements of the reference enzyme activities were performed in the laboratory of the Department of Clinical Chemistry at the University of Oulu as described previously (for GOT, GPT, AP and LDH, see ref. 20; for HBDH, see ref. 21), except that the incubation temperature in the assay of HBDH was 37°C. The protein contents were assayed [22] using bovine serum albumin as standard. The statistical significances were determined by Student’s t test and the correlations by calculating the linear regression.

326 Results

Comments on assays with serum collagen glycosyltransferases

Initial results suggested the presence of both collagen galactosyltransferase and collagen glucosyltransferase activities in the human serum. Further experiments were then performed to examine the reliability of this finding. The specificity of the assays with the serum enzymes was evaluated as described in Methods. As illustrated in Fig. 1, about 80% of the ~dioactivi~ found in the glucosylation reaction was present as the specific product, glucosylg~a~~sylhydroxylysine, whereas the assay of serum g~ac~syl~~sfer~e activity was entirely unspecific. Fig. 2 illustrates the linearity of the assay of serum glucosyltransferase activity. Distribution of collagen glucosyltransferase activity in blood

Detectable amounts of collagen glucosyltransferase activity were found in the serum, the platelet-rikh plasma, the plasma low in platelets and the platelet homogenate (Table I) (for activity in platelets, see also ref. 14). The enzyme activity in the erythrocyte and leucocyte pellet homogena~ lay at the borderline of detection and the specificity of the assay could not be tested. However, the specific activity of the serum enzyme was at least five times that observed in the erythrocytes and leucocytes, and thus this serum enzyme could not originate from the blood cells. Essentially no collagen glucosyltransferase activity was sedimented with the platelets, nor was the clotting of the blood found to account for the serum activity. The specific activity of the platelet enzyme was about one hundred times that of the serum enzyme, but the total activity in the platelet preparation was about a half of that in the serum in an equal blood volume. lwo

1500

1250

1000

1250

750 500 I ::

1000

250

I :

0 1250 1000

750

500

750 500

250

250 0

0

a

80 TIME

120 (min)

160

200

0 SERUM

(~1)

Fig. 1. Specificity of assays OX cwlagen galactosyltransferase (A) and glucosyltransferase (B) activities in human serum. The final products obtained in the assay of the glycosylation reactions were analyzed using an amino a&d analyzer. fractions being collected every 2 min and radioactivity measured in a liquidscintiIlation counter. The dashed lines represent the elution position of gbaeosyigalactosylhydroxylyaine (tfrst dashed line, at 54 mint and ~actoay~ydroxyly~e (second dashed line. at 89 min) standards. Fig. 2. Linearity of the assay of collagen glu~osyltrausferase activity in human serum. Product formation was measured as dpm of f14CJ~ucosylg~actosy~ydroxy~y~e.

321 TABLE I DISTRIBUTION

OF COLLAGEN GLUCOSYLTRANSFERASE

FraCtiOn SellUll

Platelet-rich plasma Platelet-poor plasma Leucocytes and erythrocytes Platelets *

ACTIVITY

IN HUMAN BLOOD

Specific activity (U/g protein) 0.0063 0.0054 0.0050 <0.0010 0.47

* Specificity of the assay about 95% (see ref. 14).

Age-related changes Relatively minor age-related changes were noted in the serum collagen glucosyltransferase activity, as shown in Fig. 3, the mean value f standard deviation in twelve newborn infants being 0.440 + 0.075 U/l, while it was 0.293 + 0.078 U/l in eight children aged 3 months to 16 years, and 0.331 i: 0.067 U/l in seventeen adult subjects aged 24 to 71 years. The mean value in the newborn infants was thus 30% higher than that in the adult subjects, the difference being statistically significant (P < 0.0005). The difference between the mean value in the children and adult subjects was not statistically significant. Preliminary studies on changes in diseases When the data from the randomly chosen group of 50 hospitalized patients were analyzed, 15 patients with elevated serum activities concerning either the reference enzymes or collagen glucosyltransferase were found. The enzyme activities and clinical data in these patients are summarized in Table II. It was evident that in most cases where the values of the reference enzymes indicated cell damage the collagen glucosyltransferase activity in the serum was also above the normal range. There was no clear linear correlation, however, between collagen glucosyltransferase activity and the activity of the reference enzymes within the group of these 15 patients. The correlation coefficient for collagen glucosyltransferase activity with aspartate aminotransferase activity was +0.56 (P < 0.05), that with alanine aminotransferase activity +0.49 (P = N.S.) and

0.6 z : 2 - 0.5 t I

20 40 60 AGE ( years ) Fig. 3. Effect of age on the activity of collagen glucosyltransferase in human serum.

0

328 TABLE II DATA ON PATIENTS WITH ELEVATED FERASE AND REFERENCE ENZYMES

SERUM ACTIVITIES

OF COLLAGEN

GLUCOSYLTRANS-

Abbreviations: CGlcT, collagen glucosyltransferase; GOT, asp&ate aminotransferase; GPT, alanine aminotransferase; AP, alkaline phosphatase; LDH, lactate dehydrogenase; HBDH, 2-hydroxybutyrate dehydrogenase. Patient

Sex

Age (yea=)

CGlcT w/l,

GOT (UP)

GPT (U/l,

AP w/D

182 159

Others w/l,

Diagnosis or other probable clinical cause of elevated enzyme activities

P.R. N.L. L.J.-R.

M

36 40 62

0.256 0.281 0.325

50

M M

51

135 92 58

H.H.

M

43

0.417

56

163

253

M.M. O.S.

M M

71 53

0.520 0.521

77 70

120 31

271 354.

Hepatitis Pulmonary cancer with metastases in mediastinum and bones

Y.K.

M

69

0.606

28

31

231

V.K.

F

77

0.680

151

243

811

A.P. L.S. V.H. AH.

M F M F

76 55 65 55

0.845 0.846 0.900 0.917

78 49 59 167

71 55 139 184

287

A.Pe E.M. K.K.

F F M

23 60 17

0.927 1.016 1.215

163 19 840

177

386

Pulmonary cancer with metastases in pleura and ribs Choledocholi~i~~, obstructive jaundice Peritonitis, laparotomy Pulmonary emboiism Peritonis. laparotomy Thoracotomy, mediastinitis and pleuritis Recurrent jaundice of pregnancy Erythema nodosum

Upper limits of normal values

0.398

*

40**

13 970

Hepatitis Toxic hepatitis Pulmonary cancer with metsstases in mediastinum and ribs LDH 1072, HBDH 573

LDH 542

124 LDH 1243 HBDH 551

HBDH 872

Hepatitis

Myocardiai infarction, resuscitation

40**250**LDH 450** HBDH350**

* Mean f 1 SD. ** Upper limits of the normal values used in the laboratory

of Department of Clinical Chemistry,

University of Oulu.

that #with alkaline phosphatase activity +0.23 (P = N.S.). Interestingly, the serum activity of. collagen glucosyltransferase was markedly elevated in two patients (Y.K. and E.M.), even though the values for the reference enzymes were within the normal range. Patient Y.K. had lung cancer recidivation with metastasis in pleura and destructive me&stases in ribs, and patient E.M. had erythema nodosum with very pronounced skin lesions. The specificity of the highest collagen ~ucosyltr~sfe~e activity in serum (patient K.K.) was tested as described in Methods, and proved to be the same as in normal serum (80%).

Discussion The intracellular enzymes catalyzing post-translational modifications in collagen biosynthesis are very specific [l], so that it has been possible to develop diagnostic techniques for their measurement in order to study diseases affecting cowmen-produc~g tissues El], Most applications are based on the determination of the enzyme activity in tissue biopsies [l].. The activities of

329

the collagen glycosyltransferases in human tissues have been assayed only in platelets [ll-141 and in human skin [15] as a function of age. The present study demonstrates that the activity of collagen glucosyltransferase is detectable in human serum both in normal cases and in some instances of tissue damage. The assay of collagen glucosyltransferase activity was reliable, whereas attempts to assay collagen galactosyltransferase activity in serum were unsuccessful. The activity of collagen glucosyltransferase in serum did not originate in the leucocytes or erythrocytes, nor was it sedimented with the platelets or derived from the platelets in the blood clotting process. The specific activity of collagen glucosyltransferase in serum was about one hundredth of that in the platelets and in skin [ 151, the concentration of immunoreactive prolyl hydroxylase in serum being likewise one hundredth of that in the skin [ 51, but one tenth of that in the platelets [ 5,141. Both enzymes are located in collagen-producing cells in the cistemae of the endoplasmic reticulum (for references, see ref. 10). If both enzymes originated in the platelets, it could be expected that the serum values would be related to the values in the platelets rather than to those in the skin, a tissue with a high collagen synthesis. Furthermore, it seems unlikely that the elevations in serum collagen glucosyltransferase activity noted here in several different pathological conditions would be determined by the platelets. However, the possibility is not excluded that a minor part of the serum enzyme may be derived from the platelets. It has recently been reported that the specific activity of collagen glucosyltransferase in human skin varies considerably with age [ 151, the mean value in foetal skin being about six times the level in the skin of adult subjects, and the mean value in the skin of newborn infants being about three times the adult level [15]. The serum enzyme activity did not show any age-related changes of such magnitude, although the mean value in newborn infants was 30% higher than that found in adults. The difference between the serum and skin enzyme activities in terms of age-dependency would be understandable if the serum values were to reflect cell destruction rather than the collagen-producing activity of connective tissue cells. Preliminary attempts were made to obtain some information on possible changes in serum collagen glucosyltransferase activity in pathological states by analyzing this enzyme activity in a randomly selected group of 50 hospitalized patients. The changes in this enzyme activity were compared to those in several other enzyme activities which are usually determined to indicate damage in various tissues. It should be noted that different enzyme activities may represent damage in different cells. The results indicated that serum collagen glucosyltransferase activity was elevated in almost every case (10 patients) in which the reference enzymes indicated cellular destruction (in 13 patients). However, the lack of a clear linear correlation between collagen glucosyltransferase activity and the activity of the reference enzymes suggests that the mechanism by which the elevations are mediated or the cells from which the enzymes are derived are different. This is further supported by the finding of two patients in whom the reference enzyme activities were normal even though collagen glucosyltransferase activity was markedly elevated. The pattern was possibly caused by damage concentrated in the connective tissue.

330

Hitherto, no useful method has been reported for measurement of destruction of collagen-synthesizing cells. The data of this study suggest that the assay of serum collagen glucosyltransferase activity may prove to be of value in studying the extent and specificity of connective tissue lesions in various states of tissue damage. Such assays may also reveal cases in which it has not been possible to demonstrate the damage with the presently existing diagnostic methods. Acknowledgements The author gratefully acknowledges the valuable suggestions and comments of Professor Kari I. Kivirikko, M.D. and the expert technical assistance of Mrs Raija Harju, Miss Raija Leinonen and Miss Liisa Joki. My thanks are also due to Professor Reijo Vihko, M.D. for permission to obtain the serum samples. This work was supported in part by Grants from the Medical Research Council of the Academy of Finland. References 1 Kivirikko. K.I. and RisteIi, L. (1976) Med. Biol. 54.169-186 2 Keiser. H.R., Vogel, C.L.. Sadiksli. F. and Looney. W.B. (1975) in Collagen Metabolism in the Liver (Popper, H. and Becker, K., eds.), pp. 78-85. Stratton Intercontinental Medical Book Corporation. New York 3 Langness, U.. Grasedyck. K., Borgmann, J. and Lindner. J. (1975) in Coliagen Metabolism in the Liver (Popper, H. and Becker, K., eds.). pp. 73-77. Stratton Intercontinental MedicaI Book Corporation, New York 4 Tuderman, L., Kuutti, E.-R. and Kivirikko, K.I. (1976) Eur. J. Biochem. 60. 399-405 5 Tuderman. L. and Kivirikko, K.I. (1976) Eur. J. CIin. Invest., in press 6 Takeuchi. T. and Prockop. D.J. (1969) Gastroenterology 66.744-750 7 MyRylli. R.. Risteh. L. and Kivirikko. K.I. (1975) Eur. J. Biochem. 62.401410 8 MyUylli. R.. RisteIi, L. and Kihikko. K.I. (1976) Eur. J. Biochem. 61.69-67 9 Risteli, L.. MylIyM. R. and Kivirikko, K.I. (1976) Biochem. J. 166.146-163 10 Anttinen, H. (1976) Biochem. J. 160.29-35 11 Barber, A.J. and Jamieson, G.A. (1971) Biochim. Biophys. Acta 262.633-646 12 Barber, A.J. and Jamieson. G.A. (1971) Biochim. Biophys. Acta 262.646-662 13 Bosmann. H.B. (1971) Biochem. Biophys. Res. Commun. 43.1118-1124 14 Anttinen, H., Tuderman. L., Oikarinen. A. and Kivirikko. K.I. (1977) Blood. in press 15 Anttinen. H.. Oikarinen. A. and Kivirikko. K-1. (1977) CIin. Chii. Acta76,950-101 16 Anttinen. H. and Kivirikko. K.I. (1976) Biochim. Biophys. Acta 429.750-768 17 Risteli. J. and Kivirikko, K.I. (1976) Biochem. J. 168. 361-367 18 Askenasi. R. (1973) Biochim. Biophys. Acta 304. 375-383 19 Oikarinen, A., Anttinen. H. and Kivirikko. K.I. (1976) Biochem. J. 166,645-661 20 The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology (1974) Stand. J. CIin. Lab. Invest. 33, 291-306. 21 Smith, A.F., Brown, S.8. and Taylor, R. (1970) Ciin. Chim. Acta 30. 106-113 22 Lowry, O.H., Rosebrough. N.J.. Farr, A.L. and RandaB. R.J. (1961) J. Biol. Chem. 193. 266-276