TUDIE 0 EXPERIME TALLY PRODUCED FRACTURE I DOGS PART I-CHA GES I VITAMI C METABOLISM By

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1 TUDIE 0 EXPERIME TALLY PRODUCED FRACTURE I DOGS PART I-CHA GES I VITAMI C METABOLISM By S. GHATAK,* C. W. BROACH* AND B. N. SINHA** Central Drug Research Institute and Medical College, Lucknow. (Received September 5, 1957) Since the initial reports of Sayers et al" a number of investigators have confirmed the fall in adrenal ascorbic acid as a result of stress in the intact animal. In our earlier communication,2 it was shown that the experimental fracture of tibia and fibula in guinea pigs-a condition of stress, causes a decrease in the total ascorbic acid content of venous blood, urine and of most of the organs and tissues, adrenals being the most affected. In view of the importance of ascorbic acid in osteoblastosis, it was considered desirable to investigate whether the decrease of ascorbic acid in various tissues was due to the increased demand for this a.cid at the site of fracture. Changes in the concentration of this vitamin and its oxidised and reduced forms, in the circulation as also at the site of fracture have been studied and the results obtained are reported in the present communication. MATERIALS AND METHODS Male Mongrel Dogs weighing between were used as experimental animals throughout these studies, in preference to guinea pigs because adequate quantities of blood and the exudate from haematoma at the site of fracture could be collected easily from the former. Middle third of the shaft of the femur was chosen for the site of fracture as it is known to produce the maximum stress and can be splinted properly. The selected dogs were fed with bread, boiled milk and meat for two weeks before operation, so that a constant level of vitamin C may be attained in the body. Water was given ad-lib except during the experimental procedure when the intake was regulated. Pre-operative preparation of the anidlal (A) On the morning of the operation the animal was given soap water enema and its water intake was restricted. (B) The thigh was shaved and cleaned with a swab of ether and painted with 2 per cent mercurochrome in alcohol and covered with sterile dressings. (C) Blood was withdrawn from the vein for control estimations just before the operation. *Central Drug Research Institute, Lucknow. *Department of Orthopaedics, Medical College, Lucknow.

258 S. GHATAK, C. W. BROACH AND B. N. SINHA All possible aseptic precautions were taken for conducting the operation. The dog was anaesthetised with ether. No premedication was given.

2 258 S. GHATAK, C. W. BROACH AND B. N. SINHA All possible aseptic precautions were taken for conducting the operation. The dog was anaesthetised with ether. No premedication was given. Blood samples after anaesthesia and before operation, were collected for measuring the changes, if any, in the ascorbic acid content. Operative technique An incision about 2"-3" long was made on the outer aspect of the middle ofthe thigh. The skin and the subcutaneous tissues were retracted and the bone exposed. An incision was made on the periosteum and this was lifted up from the bone. Osteotomy was carried out 011 the exposed and cleaned portion of the femur. The periosteum and the muscles were replaced, bleeding points ligatured and skin and subcutaneous tissues sutured with sterile linen thread. The site of operation was painted with 2 per cent mercurochrome and sterile dressing applied. A splint, improvised for the animal as represented in sketch No. I, was tied on the leg. Post-operative care After the animal recovered from anaesthesia, it was given isotonic saline drinks and was placed on a specially made bed. Hard, broad leather neck collar, losely fitted round the neck was used to avoid licking of the operation site and double chain was used to prevent the dog from tearing the bandage (sketch No.2). The animal was sacrificed on the fifth day after the operation. During tbis period, it lost about 250 gm. in weight.

CHANGES IN VITAMlN C. METABOLlSM 259 Samples of blood and the exudate were collected at regular intervals with sterilized all-glass syringes. The serum was separated by centrifugation.

3 CHANGES IN VITAMlN C. METABOLlSM 259 Samples of blood and the exudate were collected at regular intervals with sterilized all-glass syringes. The serum was separated by centrifugation. It may be mentioned that closed facture of the femur could not be produced. Osteoclasis was employed in the beginning but it produced a large contusion of the muscles of the thigh, and this was likely to vitiate the results. The open method, under aseptic precautions was, therefore, employed so as to produce the minimum amount of trauma during the operation. The wound was subsequently closed. Collection of urine as it was passed was not possible. Therefore, four hourly catheterisation using a rubber catheter was resorted to for first 3-4 collections. Further collection thereafter was not possible because of abrasion and inflamation of urethra. For these reasons the analysis of urine has been left out in the present series of experiments. METHOD FOR ESTIMATlON The dehydroascorbic acid (DHA) in the protein free filtrate of serum and the exudate was determined by the method of Lowry, Lopez, and Bessey,3 while the total ascorbic acid () in the protein free filtrate of serum and the exudate was measured by the method of Bessey, Lowry and Brock 4 using 0'03 per cent CuS0 4, 5H 2 0 as the oxidising agent. The spectrophotometric measurements were carried out in a Klett-Summerson colorimeter at 540 mj-l.

4 260 S. GHATAK, C. W. BROACH AND B. N. SINHA Values for reduced ascorbic acid were calculated by substracting the "alues for DHA from the total. The data obtained were statistically analysed. RESULTS Variation in normal blood samples Five dogs were kept under standardised dietary conditions, bled on alternate days for five days and the total and DHA contents determined in the serum after precipitation of the proteins by 10 per cent trichloracetic acid. Data were statistically analysed. Analysis of variance gives the F 4' 16 ratio (days to error mean S. S.) = 1.508, P > 0.20, not significant for total ascorbic acid; F" 16 = 0.575, P> 0.20, not significant for DHA and F" 16 = 0.951, P> 0.20, not significant for reduced. There was, therefore, no significant day-to-day variation in the total, DHA and the reduced contents ofthe normal blood samples over a period of ten days during which the animals were on the standard diet. Effect of ether anaesthesia on vitamin C content Blood was withdrawn soon after the animal had been anaesthetised and total and DHA contents were determined. Statistical analysis of the data obtained in nine dogs given in Table I would indicate that the differences from the normal are not significant at 5 per cent level. TABLE 1 Statistical analysis of the data on the effect of ether anaesthesia on the vitamin C content of blood. mean difference of 9 dogs S. E. of mean difference t P Difference in total >0.40<0.50 >0.20<0.30 (after anaesthesia-normal) DHA red >0.10<0.20 Effect of fracture of the femur on the vitamin C content. Blood-Blood samples were withdrawn t, 24, 48, 72 and 96 hours after fracture and the values obtained in 9 dogs are recorded in Table II. It would be seen that in certain cases, ascorbic acid value rises above the normal soon after the fracture while in others this rise takes place as late as 24 hours after the fracture. This is followed by a progressive decrease in total, DHA and reduced values. Maximum decrease was observed 72 hours after fracture, the time at which usually the callus formation sets in. Statistical analysis (Cf. Table III) of the data shows that the differences in the total, DHA and reduced before and three days after fracture are significant at 5 per cent level. The same is true with regard to the differences in the respective values after anaesthesia and three days after the fracture.

5 S. No. of animals and weight CHANGES IN VITAMIN C METABOLISM 261 TABLE II Effect of fracture on the Vitamin C content of blood rfloocc. Interval. when the Interval after fracture.. WIthout blood EstimatIOn Anaesth. was dra- t hr 24 hr 48 hr 72 hr 96 hr wn after anaesth. DHA DBA DBA DBA ]50 R llo ISO ]COO CO ] TABLE III Statistical analysis of the data on the effect of fracture on the vitamin C content of blood. Difference in (after 3 days of Difference in (after 3 days of _ fracture-normal) fracture-anaesthesia) Oxidised Reduced Oxidised Reduced Mean differ ence of 8 dogs S. E. of mean difference ± * * * * * * p <0.t02 <0.02 <0.02 <0.01 <0.01 <005 * The differences in means are significant at 5 per cent level of significance.

6 262 S. GHATAK, C. W. BROACH AND B. N. SINHA Exudate-The fluid collected at the site of injury was withdrawn t, 24, 48, 72 and 96 hours after the facture by piercing aseptically, a blood donor needle attached to a syringe. The volume of the exudate at the half hour interval was 3-5 cc mixed up with bone marrow, but it increased with time; in certain cases as much as 20 cc could be collected 72 hours after fracture. It was centrifuged and estimations made in the clear supernatant. Results obtained are presented in Table IV. It would be seen from the data that the total in the exudate soon after the fracture has a tendency to rise much above the corresponding value in the blood. DHA values in dogs no. 4,5, 7, 8 and 9 rose much above those in blood and kept at a high level through out the experiment. Reduced level similarly, rises during the half hour interval after the fracture) but shows a fall later on. Satistical analysis of the data (Cf. Table V) would indicate that the fall in the values of total and reduced after 3 days of fracture, as compared with the figures just after fracture, is significant. DHA remains practically unchanged. TABLE IV Effect of fracture on the Vitamin C content of Exudate rfl 00 cc. No. of animals and weight Estimation Interval after fracture t hr. (initial) 24 hr 48 hr 72 hr 96 hr DHA DBA DBA DBA ]50 75 ]05 '1 ] DBA ] DBA ] DBA

7 CHANGES IN VITAMIN C. METABOLISM 263 TABLE V Statistical analysis of the data on the effect of fracture on the vitamin C content of the exudate. mt'an difference of 7 dogs S. E. of mean difference t p *Significant at 5 per cent level. at 5 percent level. Difference in (after three days just after fracture) red * * <0.02 >0.10<0.20 <0.01 difference just fails to be significant DISCUSSION The observations described above would indicate that the total ascorbic acid, its oxidised and reduced forms are all affected due to the fracture of the middle third of the shaft of the femur. The increase in the reduced content of blood noted! hour after the fracture in some of the experimental dogs would indicate that there is a Row of from the tis ues into the circulation to meet the increased demand at the site of fracture. In certain cases this ri e could not be detected because of the possibility of this occurence either before or after taking the half-hour sample. This ini tial ri e is followed by a progressive fall up to 72 hours, when callus formation is known to set in. The DHA level shows a similar trend. In the exudate, however, the total and reduced rise much above the corresponding values in blood soon after fracture but decrease 3 days later, values of the exudate rise even higher than the corresponding values in Llood and keep at that level throughout the period of the experiment. Considering the fact that there is an increase in the volume of the exudate with the lapse of time, the total quantity of present at the site of fracture would be much higher than reported here. From the higher values of, it would appear that on reaching the -ite of injury ascorbic acid is oxidised before utilisation. The above observations support the view that vitamin C plays an important part in the healing process that sets in after fracture. The increased demand for this vitamin is met by its release from various tissues. CONCLUSIONS I. There was no significant day to day variation in the total, and reduced content of normal blood samples over a period of 10 days of dogs kept under animal house diet. 2. Ether anaesthesia had no effect on the reduced, DHA and total content of blood.

8 264 S. GHATAK, C. W. BROACH AND B. N. SINHA 3., reduced and oxidised forms of ascorbic acid of blood decreased progressively after femur fracture though in certain cases the t hour after fracture value for reduced was higher than the normal level. 4. The level of reduced of the exudate oon after fracture was much higher than the normal level; thereafter there was a gradual decrease. The DHA levels were more or less con tant throughout the period of experiment. ACKNOWLEDGEMENTS The authors are deeply indebted to Dr. B. Mukerji, Director, Central Drug Research Institute for his kind permission to carryon the experiments in the laboratories of the Institute, thus making the present collaboration possible. Dr. D. L. Shrivastava, Incharge, Division of Biochemistry, took an active interest in the investigation throughout for which the authors are grateful to him. Thanks are also due to Mr. N. Sen of the Statistics Department for all the statistical analysis and to Mr. Ajit Malaviya for the technical help. REFERENCES I. Sayers, G., Sayers, M.A., Liang, T. Y. and Long, C.. H. Endocrinology 1945,37, Ghatak, S., Dhar, D. C., Agarwala, S. C. and Shrivastava, D. L. Jour Sci. Ind. Res., 195-1, 13B, Lowry, 0., Lopez,]. A. and Bessey, O. Jour. Biol. Chern., 160, 1945, Bessey, 0., Lowry, O. and Brock, M.]. Jour. Biol. Chern., 168, 1947,196.

STUDIES ON EXPERIMENTALLY PRODUCED FRACTURE IN DOGS: PART II-CHANGES IN TOTAL, NON-PROTEIN AND PROTEIN NITROGEN CONTENT.

STUDIES ON EXPERIMENTALLY PRODUCED FRACTURE IN DOGS: PART II-CHANGES IN TOTAL, NON-PROTEIN AND PROTEIN NITROGEN CONTENT. By S. GHATAK AND B. N. SINHA (With the technical ahistance ofu. C. Pande) (Central