가골조직의 alkaline phosphatase 및 lactic dehydrogenase 활성치 변동에 관한 연구
(A) study on the changes of alkaline phosphatase and lactic dehydrogenase activity in fracture callus of rats
It was Robinson(1923) who first set forth the hypothesis that the alkaline phosphatase found in chondroblasts and osteoblasts is a local factor which results in precipitation of calcium salt through the libeation of inorganic phosphate by the hydrolysis of phosphate ester. Alkaline phosphatase activity increased in early
stage of fracture callus was observed (Swenson 1946; Enneking 1948; Pritchard and Ruzicka 1950; Ghatak and Sinha 1960; Semb et al. 1971). They then concluded that the alkaline hosphatase plays an important role in fracture healing. Jackson(1957) demonstrated granules which pair within the osteoblast and approach the cell
membrane together prior to and during the secretion of collagen. These granules are thought to be aminopolysacchride (similar to hyaluronic acid) and alkaline phosphatase which is essential for developing calcifiable matrix. Sulphation first appearts at the cell membrane on association with the prefibrillar collagen
formation, and then the matrix contains the sulphated mucopolysacchride, chondroitin sulphate. The funtion of alkaline phosphatase is still obscure but is in some way related to the mineralization process and it was significantly lower in fracture with delayed union as compared to normally healing fracture(Semb and Gudmundson, 1971). It has been suggested that alkaline phosphatase limits the accumulation of calcification ingibitor, e.g. inorganic pyrophosphate(Jibril, 1967). Russel et al. (1959,1970) have suggested that the inorganic anion, pyrophosphate, present in bone other tissues, is an important element in calcification, and alcification or mineral resorption is initiated by a specific enzyme known as pyrophosphatase which removes inhibiting pyrophosphate by splitting it into two molecules of orthophosphate. Semb and Gudmundson(1971) concluded that alkaline phosphatase is probably of considerable importance in the premineralization phase of the fracture healing, and decreased enzyme activity in delayed union may be an impotant limiting factor for normal fracture healing. The function of alkaline phosphatase is related to production of ground substance
(McLean and Urist, 1955), cellular and in the cytoplasm of cells, substantiating the idea that it has a role in protein synthesis(Jaffe, 1972). The bone cells possess a non-specific alkaline phosphatase activity on the outer surface of the
cellular membrane and menbrane-bound alkaline phosphatase is part of a transport system ofr phosphate and/or calcium ion which is influenced by parathyroid hormone(Hekkelman, 1970). Duthie and Ferguson(1973) reported that in general, alkaline phosphatase is concerned with preosseous cellular metabolism, with subsequent elaboration of bone matrix before the crystallization of calcium and phosphate ions.
Lastic dehydrogenase mediates carbohydrate metabolism both through the anaerobic Embden-Myerhoff pathway, the citric acid cycle, and the direct oxidative pathway.
High activity of lactic dehydrogenase ahs been demonstrated histochemically in osteoblasts as wall as in osteoclasts in fracture(Balogh and Hajek, 1965). They found marked proliferation and increased enzyme antivity in the osteoprogenitor cells in the inner layer of periosteum alone the entire diaphysis 3 days after
fracture. Semb and Gudmunson(1971) observed increased lactic dehydrogenase activity to about the same level independent of normal or delayed fracture healing. These results support the theory of a high remodelling rate also in fracture with delayed
Battistone et al. (1972) studied that the effect of zinc cysteamine-N-acetic acid on the healing of experimentally injured guinea pig bone. They found that the correlation of zinc incorporation and healing in the early stages of repair suggests that zinc availability is a rate-limiting step immediately following injury and that, within certain limit, more rapid mobilization of zinc to the repairing tissue will accelerate healing. On macroscopic, roentgenographic, and histologic study about healing of experimental fracture in rabbits fed pyrite, the
callus formation and bony union occurred earlier than in the control group(Choi and Chung, 1970).
The purpose of this study was to elucidate the activity of alkaline phosphatase and lactic dehydrogenase in callus tissue at a time interval of from one day to 15 weeks after fracture, and to investigate the effect of pyrite and ferrous sulfate in fracture healing and enzyme activity.
Materials and Methods
Healthy male rats weighing from 170 to 290 grams were used. The midportion of the femoral diaphysis was manually broken under ether anesthesia. No attempt was made to immobilize the fracture. The animals were kept in nine large cases under the same temperature and humidity and were fed a standard laboratory diet with water ad libitum except for the group fe pyrite and ferrous surfate.
Ninety five rats were grouped as follow;
Ⅰ) Control group; 5 rats
Ⅱ) Fracture group; 90 rats
1. Non-medicated group; 60 rats fed normal laboratory diet only. The animals were killed 24 hours, 48 hours, 1 week, 3 weeks, and 5 weeks after fracture, 5 to 10 rats time. The left femurs of 30 rats were broken manually in the midportion of
the diaphysis and 8 weeks later the right fumurs were broken with the same technique and 7 weeks later these rats were killed for examination.
2. Medicated group; 30 rats fed pyrite and ferrous sulfate for 5 weeks after fracture and then killed for examination.
1) Pyrite fed group
a. Small dose group; 8 rats
b. Large dose group; 8 rats
2) Ferrous sulfate fed group
a. Small dose group; 7 rats
b. Large dose group; 7 rats
Pyrite was obtained near Seoul and was composed mainly of FeS^^2(98.7%) and its minor constituents were Cu, SiO^^2, MgO, CaO, and Al^^2 O^^3. Before killing the animals, the status of fracture healing was group into stable or unstable union under ether anesthesia by manual pressure over the fracture site and recheched by roentgenogram. On sacrifice for examination the fracture site was cleaned of soft tissue and the callus was removed for enzyme analyses. Specimens were kept on ice and the blood was removed by rinsing in isotonic saline at 4 degree centigrade.
Spectimens were crushed into powder in a mortar surrounded by dry ice. Extraction was performed overnight at 4 degree centigrade in distilled water 10 ml. The extracts were centrifuged for 5 minutes and the supernatants were used for the analyses after dilution with distilled water.
Lactic dehydrogenase was measured spectrophotometrically by the technique of Sigma Bulletin No. 500.
Alkaline phosphatase was measured spectrophotometrically by the technique of Sigma Bulletin No. 104.
Before dissection, blood was obtained from the femoral artery to check hemoglobin and bematocrit. Change of body weight was cheched every week in medicated group.
Results and Summary
Alkaline phosphatase activity in fracture callus was maximum after one week following fracture, then gradually decreased but still showed a higher level after 15 weeks following fracture compared with normal diaphyseal bone. Alkaline phosphatase activity was significantly higher in stable union than in unstable union in all the experimental groups. Lactic dehydrogenease activity in fracture callus was maximum in one day after fracture and preserved a high level until on week following fracture. The total activity of lactic dehygrogenase was higher in all fracture callus than in normal bone irrespective of the stage or duration of the fracture healing, but after 5 weeks following fracture it was at almost the same level as in normal control groups. Lactic dehydrogenase activity was significantly increased in rats fed ferrous sulfate 30 mg/day. Alkaline phosphatase activity was increased in both pyrite fed groups of stable and unstable union after 5 weeks following fracture compared with the control group fed normal diet only. On the other hand, the rate of unstable union was significantly decreased in the pyrite fed group of rats after 5 weeks following fracture.
These results suggest that there is a more anaerobic metabolism in callus during fracture healing than in normal bone, and alsoa high remodelling rate in fractured with unstable union. It is felt that the pyrite is effective in fracture healing and the effect seems to be related in part with alkaline phosphatase activity of callus.