Henrik Anttinen

Decrease in Liver Collagen Accumulation in Carbon Tetrachloride-Injured and Normal Growing Rats Upon Administration of Zinc

Several attempts have been made to develop antifibrotic drugs for human use, but their success has been limited. The present data suggest that peroral zinc treatment has a direct and selective inhibitory effect on carbon tetrachloride-induced collagen accumulation in rat liver. Zinc did not normalize the carbon tetrachloride-induced increases in either liver relative weight, liver total protein content, fat accumulation, or the standard liver function tests, but it did efficiently inhibit liver collagen accumulation. It also reduced skin and liver collagen content and urinary hydroxyproline excretion in normal growing animals, indicating that the inhibition is not limited to the fibroproliferative inflammation associated with carbon tetrachloride injury. Neither inhibition of polysomal protein synthesis nor increased degradation of mature collagen fibers was found to play any major role in the effect of zinc. Instead, a plausible mechanism is inhibition of proline hydroxylation.

Assay of protocollagen lysyl hydroxylase activity in the skin of human subjects and changes in the activity with age

Abstract A method is described for the assay of protocollagen lysyl hydroxylase activity in human skin specimens of 100 to 300 mg wet weight. With the procedure reported here, about 90% of the enzyme activity could be extracted from the skin, and results for six skin samples analyzed separately from the same human subject agreed within a variation coefficient of 13%. The lysyl hydroxylase in human skin resembled that from chick embryos in requiring ascorbate, α-ketoglutarate, ferrous iron and atmospheric oxygen. In human skin the activity was highest in foetuses, and higher in infants than in adult subjects. The mean value in young foetuses was about 40 times, and the mean value in two infants of 2 and 4 months of age about 8 times that found in adult subjects.

Collagen biosynthesis enzymes in serum and hepatic tissue in liver disease. II. Galactosylhydroxylysyl glucosyltransferase.

Abstract. Serum galactosylhydroxylysyl glucosyltransferase activity exceeded the 95% confidence limit of the controls in eleven out of thirteen patients with primary biliary cirrhosis, four out of seven with portal cirrhosis, all four with acute hepatitis and all seven with cancer having liver metastases, whereas it was below this limit in most patients with fatty liver, extrahepatic cholestasis and cholangitis, and in seven out of fourteen cases with cancer without liver metastases or with other malignant diseases. Liver galactosylhydroxylysyl glucosyltransferase activity was elevated in the same diseases, and there was a high correlation between these two measures (P < 0.005). Serum galactosylhydroxylysyl glucosyltransferase activity also correlated with serum immunoreactive prolyl hydroxylase protein (P < 0.001), and liver galactosylhydroxylysyl glucosyltransferase activity with liver prolyl hydroxylase activity (P < 0.001), but not with liver immunoreactive prolyl hydroxylase protein. Serum galactosylhydroxylysyl glucosyltransferase activity did not correlate with liver hydroxyproline, and liver galactosylhydroxylysyl glucosyltransferase activity only weakly. Serum galactosylhydroxylysyl glucosyltransferase activity correlated with serum alkaline phosphatase in primary biliary cirrhosis (P < 0.001) but not in any other disease, and with serumaspartate aminotransferase (P < 0.05) and total bilirubin (P < 0.05) in the pooled group of all patients with non‐malignant liver disease. The results suggest that assays of serum galactosylhydroxylysyl glucosyltransferase activity and liver galactosylhydroxylysyl glucosyltransferase activity in liver disease may give useful information on the actual hepatic collagen synthesis.

Collagen glucosyltransferase activity in human serum

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.

Age-related changes in human skin collagen galactosyltransferase and collagen glucosyltransferase activities

Collagen galactosyltransferase and collagen glucosyltransferase activities were assayed in human skin specimens of about 100 mg wet weight. The assay of the glucosyltransferase activity was found to be highly specific. The assay of the galactosyltransferase activity was somewhat less specific, but there was no difference in specificity between the foetal and adult human skin samples. The activities of the two collagen glycosyltransferases in human skin extract were found to vary with age, being highest in foetal skin, and higher in the skin of young children that in that of adults. The galactosyltransferase and glucosyltransferase activities in foetal skin were respectively about 4 times and 6 times those in adult skin. The magnitudes of the changes with age in the two collagen glycosyltransferase activities were smaller than those occurring in the activities of the two other intracellular enzymes of collagen biosynthesis namely prolyl and lysyl hydroxylase. This difference suggests that the four intracellular enzyme activities of collagen biosynthesis are not regulated in an identical manner.

Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos.

Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain.

Affinity chromatography of collagen glucosyltransferase on a UDP-glucose derivative coupled to agarose.

UDP-glucuronic acid from the carboxyl group was coupled to agarose via a six-carbon atom spacer, and columns prepared from this material were used in an affinity chromatography of collagen glucosyltransferase. The enzyme was found to have a high affinity for such columns in the presence of Mn2+ in the buffer, whereas a considerably lower affinity was noted in the absence of such ions. The enzyme could be eluted from the column with either EDTA, UDP-glucose, or small peptides prepared from collagen, the peptides being the most effective eluting agent. After elution the enzyme was separated from the peptides by gel filtration. With this procedure a collagen glucosyltransferase putification of about 3000-fold was obtained from extract of chick embryos by relatively simple steps. Collagen galactosyltransferase was found to have no affinity for the column, suggesting that the binding was not only due to the UDP moiety, but that the uronic acid derivate of glucose also contributed to its specificity.

Differences between proline and lysine hydroxylations in their inhibition by zinc or by ascorbate deficiency during collagen synthesis in various cell types.

The addition of Zn2+ inhibited lysine hydroxylation markedly less effectively than it did proline hydroxylation in chick embryo tendon cells, 3T6 fibroblasts and lysyl hydroxylase-deficient Ehlers-Danlos Syndrome Type VI fibroblasts. With low Zn2+ concentrations, a similar difference was also seen in chick embryo cartilage cells, whereas with high concentrations both hydroxylations were affected to the same extent in this cell type. Ascorbate deficiency likewise had a much less effect on lysine than proline hydroxylation when studied with 3T6 fibroblasts. As these two effectors involve quite different mechanisms, it is suggested that relative insensitivity to inhibition may be a property of lysine hydroxylation seen in many cell types with a number of agents. Studies on the mechanism of the difference in the inhibition indicates that the phenomenon is probably not due to differences in the kinetic constants of Zn2+ and ascorbate for the two enzymes. Neither is it probably to any major extent due to delayed procollagen triple helix formation nor a difference in the location of the two hydroxylases within the cisternae of the rough endoplasmic reticulum. The difference similarly cannot be explained solely by an excess of lysyl hydroxylase in the cell. It may thus be due either to some other intracellular property or to the combined effect of several factors.

Effects of divalent cations on collagen biosynthesis in isolated chick embryo tendon cells

Isolated chick embryo tendon cells were used in [14C]proline and [14C]lysine labelling experiments to investigate the effect of divalent cations on collagen biosynthesis with a special reference to prolyl hydroxylation and lysyl modifications. The following metals were studied by adding them to the incubation medium of the cells: Ca2+, Cd2+, Co2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+. Zn2+ caused a potent reductin in collagen prolyl hydroxylation with a concomitant increased cellular retention of collagenase-digestible material. These effects were detectable even at physiological concentrations. At the same concentrations of Zn2+, lysyl hydroxylation was considerably less inhibited than prolyl hydroxylation, and the extent of hydroxylysyl glycosylation was even increased. Co2+ was also an efficient inhibitor of collagen prolyl hydroxylation, but at concentrations ten times higher than those of Zn2+. In the presence of other metal ions, no or only up to 10% inhibition of prolyl hydroxylation was noted even at those concentrations at which [14C]proline incorporation into the protein was decreased. However, an increased cellular retention of collagen was detected in the presence of some metal ions. No reduction in lysyl hydroxylation was found in the presence of Ca2+ or Mg2+.

Serum galactosylhydroxylysyl glucosyltransferase in acute myocardial infarction and during subsequent collagen scar formation.

Abstract. Changes in serum galactosylhydroxylysyl glucosyltransferase, an enzyme catalysing one of the intracellular post‐translational modifications in collagen biosynthesis, were studied in twenty‐four patients with acute myocardial infarction. The enzyme activity was monitored for 18 days from the onset of infarction, and at least a two‐peaked pattern was observed. The first peak corresponded to the stage of acute myocardial injury, there being a highly significant correlation between the maximal values for serum glucosyltransferase and a‐hydroxybutyrate dehydro‐genase. An average decrease in serum glucosyltransferase activity of 41% was noted during the following 24 h.