Abnormalities in fracture healing induced by vitamin B6-deficiency in rats

Bone, 7, 489-495 (1986) Printed in the USA. All rights reserved

Copyright

8756-3282186 $3.00 + .OO 0 1986 Pergamon Journals Ltd.

Abnormalities in Fracture Healing Induced by Vitamin BE-Deficiency in Rats R.A. DODDS,’

A. CATTERALL,2

1 Division of Cellular Biology, 2 Department of Orthopaedic

L. BITENSKY,’

and J. CHAYEN’

Kennedy institute of Rheumatology, Bute Gardens, Surgery, Chafing Cross Hospital, London, U.K.

Address for corresDondence and reknts: Gardens, London, ‘W6 7DW, U.K. ’

Dr. J. Chaven. D/vision of Cellular Bfology, Kennedy ,

institute of Rheumatology,

Bute

Recently (Dodds et al, 1986) we have shown that the GGPD activity of rat metaphyseal osteoblasts may be regulated by putrescine, the immediate product of ornithine decarboxylase (ODC) activity. It is well known that ODC activity depends on pyridoxal-phosphate (a congener of vitamin B6). Moreover two groups have independently shown that when young Wistar rats are fed for at least 21 days on a particular carbohydrate-rich, pyridoxine-depleted diet, that the content of putrescine in various tissues was reduced by 50% (Eloranta et al., 1976; Pegg, 1977). Consequently it seemed pertinent to test GGPD activity, in response to fracture, in rats fed on this diet and to investigate whether the speed and quality of fracture-healing was affected.

Abstract Vitamin K, is the intermediate carrier of reducing equivalents in mineralization. In fracture-healing in the rat metatarsal the primary source of these reducing equivalents appears to be NADPH, generated from glucose 6-phosphate dehydrogenase (GGPD) activity. Because recent evidence indicated that stimulation of GGPD activity can be induced by putrescine, derived from pyridoxal phosphate-dependent ornithine decarboxylase activity, the effect of pyridoxine (vitamin 8,) deficiency has been studied in this system. Vitamin Be-deficiency caused marked diminution in the GGPD activity in the periosteal region of bone-formation and in the developing callus, with significant delay in the maturation of the callus and union. It also caused changes in the bone suggestive of imbalance in the coupling between osteoblasts and osteoclasts. These results suggest that the vitamin Be-status may be important in fracturehealing.

Materials and Methods Animals

Key Words: Fracture 6-phosphate

London.

Healing-Rat Metatarsal-Glucose Dehydrogenase-Putrescine-Vitamin Be.

Introduction Calcification may depend on the conversion of peptidebound glutamate residues (Glu) to y-carboxyglutamate (Gla). This is achieved by the vitamin K-cycle (Hauschka et al., 1978) in which vitamin K, acts only as a carrier of reducing equivalents from NAD(P)H generated in boneforming cells. In fracture-healing in rat metatarsals (Dodds et al., 1984) it was shown that the major source of these reducing equivalents is periosteal glucose 6-phosphate dehydrogenase (GGPD) activity. In this study, as well as in that of Dunham et al. (1977) it was shown that an early response to fracture was activation of periosteal GGPD activity both in relation to the growth of the callus and, at a specific site along the shaft of the bone, in the periosteum one day before the emergence of new bone at this specific site. Thus it seemed that fracture-healing and the normal maintenance of bone might depend both on adequate circulating levels of vitamin K, (Dodds et al., 1984; Hart et al., 1985) and on sufficient GGPD activity in bone-forming cells. 489

Male albino Wistar rats (40-50 g) were fed for 24 days on a standard, carbohydrate-rich pyridoxine-deficient diet (B.P. Nutrition, Special Diets Services, Whitfield, Essex), or on the same diet to which pyridoxine hydrochloride (Evans Medical Ltd., Greenfield, Middlesex: 6 mg per kg) had been added. Each rat was given up to 25 g daily, and unlimited distilled water. Under general anesthesia, induced by intraperitoneal pentobarbitone-sodium, closed fractures were produced by digital pressure in metatarsals of one hind-limb. At this time, the mean (2 SD) body weight was 164 2 21 g in the controls (n = 23) and 138 ? 13 g (n = 23) in the Be-deficient rats. The rats were allowed to revive; maintained on their respective diet; and subsequently killed at prescribed times up to six weeks later.

Experimental

procedures

The fractured bones with the associated plantar muscle were dissected free and immersed briefly in 5% (w/v) polyvinyl alcohol (PVA: G04/140: Wacker Chemicals Ltd., Walton, Surrey). Bones were then chilled to - 70°C in hexane (BDH, ‘low in aromatic hydrocarbons’ grade, boiling range 67-70X), as described previously (Dunham et al., 1977). They were stored in dry tubes (- 70°C) for up to seven days. The bones were sectioned at 10 km in a Brights bone cryostat equipped with a tungsten-tipped steel knife (Autoradiographic Products, Cheshire) with the haft further cooled with solid carbon dioxide.

Histological

assessment

For histological examination, sections were cut in the antero-posterior plane; those that passed through the midline of the endos-

490

R.A. Dodds et al.: Fracture healing in vitamin Be-deficient

Table I. Areas (mm2 x 10: mean ? SD) of components Be-deficient

or B,-supplemented

rats

of the callus 12 days and 22 days after fracture in rats fed either the

diet.

Time after fracture

Supplemented diet (15 bones; 9 rats) Be-deficient diet (16 bones; 10 rats) Supplemented diet (5 bones; 3 rats) B,-deficient diet (5 bones; 3 rats)

a0.01

(days)

Total callus

New bone

Soft callus

12

22.8 * 5 3

9.2 ? 2.4

13.6 2 32

12

15.6 ? 4.6a

6.2 2 1 .5a

22

19.2 k

22

18.4 2 2 3

i a

9.5 2 4.4”

10.8 2 0.4

a.4 I

i 3

17

10.0 k

ia

8.6 ”

> p > 0.001

teal space were selected for detailed examination and measurement. They were stained with toluidine blue, pH 6.2. The image of each section was enlarged by use of a photographic enlarger: the bone length and width of the shaft were measured directly on the enlarged image and the area of the tissue components, as detailed in Table I, was measured by planimetry. A similar procedure was shown by Aro et al. (1985), to give a more accurate assessment of the development of hard callus than microradiography. The bone length and the width of the shaft in the 12.day fractures were measured manually on the enlarged image and the results were evaluated by the Student t-test. The relationship between the areas of the soft callus and the areas of new bone formation, measured by planimetry of the enlarged images, were evaluated by regression analysis.

Fig. 1. 5 day fracture in a rat fed the supplemented differentiating soft callus which is remarkably deficient

Cytochemical

reactions

Acid phosphatase activrty was demonstrated by the standard naphthol AS-BI post-coupling method (Bitensky and Chayen. 1977). Alkaline phosphatase was assayed by the simultaneous a-naphthyl acid phosphate (Sigma) method, with Fast blue RR as the coupler (Dodds et al., 1964). Glyceraldehyde 3-phosphate dehydrogenase (1.2.1.12), lactate dehydrogenase (1 .l 1.27), hydroxyacyl dehydrogenase (1 .l 1.36) and glucose 6-phosphate dehydrogenase (1 .l .1.49) were assayed by the standard neotetrazolium methods (Dunham et al., 1983) with phenazine methosulphate as intermediate hydrogen-acceptor, and with PVA (30% w/v) as colloid-stabilizer (Chayen et al., 1973). The amount of reaction-product, of alkaline phosphatase activity and of the activrties of the oxidative enzymes, was measured in individual,

diet (Fig. la) and the Be-deficient diet (Fig. 1 b). In the former there IS a large in the latter Sections stained with toluldrne blue Magnification x 15

R.A. Dodds et al.: Fracture healing in vitamin B,-deficient

rats

Fig. 2. 12 day fracture in a rat fed the supplemented diet [Fig. 2a (left)] and the Be-deficient diet [Fig. 2b (right)]. in the former there is extensive external callus with mature cartilage and with woven bone, external to the periosteum. In contrast, in the latter the callus is smaller, consisting mainly of granulation tissue with relatively scanty cartilage. Sections stained with toluidine blue; magnification x 15.

histologically defined cells, by means of a Vickers M85A scanning and integrating microdensitometer (with a mask that encompassed a single cell; a x40 objective; a scanning spot of 0.4 pm diameter in the plane of the section; at 585 nm for the neotetrazo-

lium formazan and 580 nm for the alkaline phosphatase reaction). For comparing the various activities of the different cetl-populations in the 12-day fractures as detailed in the relevant table, ten measurements of the specific cell-type were made in each of two duplicate sections, making a total of 20 measurements. Results were evaluated by the Student t-test.

Results Histology Course of fracture-healing in the controls. Since these were younger rats than in our previous studies (Shedden et al., 1976; Dunham et al., 1977) and were on an unusual diet (supplemented with vitamin B,), it was first necessary to establish the normal course of fracture-healing. In these Wistar rats, fracture resulted in a variable degree of overlap between the two broken ends. By day 5 (Fig. la) this overlap region as well as the area surrounding the broken ends was filled with a considerable soft callus which had already differentiated with extensive areas of cartilage. There was new bone-formation on the periosteum beginning at about 1.4 mm from the fracture-site. By day 12 the overlap region had almost fully resorbed and

was surrounded by extensive external callus composed largely of mature cartilage with a small internal callus of granulation tissue. The woven bone external to the periosteum was capped by areas of calcifying cartilage (Fig. 2s). By 3 to 4 weeks virtually complete bony union had occurred (Fig. 3a). Fracture-healing in the &-deficient rats. Up to day 5: Although a good fracture haematoma occurred there was very little replacement by granulation tissue even by day 5 (Fig. lb). At first sight there seemed to be some periosteal proliferation but this was found to be invasion of the preexisting shaft (as measured by decreased width of shaft, as discussed below) by peculiar osteolytic granulation tissue. Day 12: At day 12 the callus was still small (Table I) although the overlap region of the broken ends of the shaft had been largely resorbed (Fig. 2b). In contrast to the findings in the controls, there was very little mature cartilage and extensive granulation tissue. There was a significant decrease in area, measured in these sections, of both the soft callus and of the newly formed bone (Table I). The overall lengths of the bones were the same as in the controls (14.5 * 0.7 mm, mean 2 SD, n = 15 bones from 9 rats in the B,-deficient vs. 75.1 & 0.2 mm, n = 15 bones from 9 rats in the controls). Although there was a clear correlation between the size of the soft callus and the amount of new bone formed in the control animals (Fig. 4), this

R.A. Dodds et al.: Fracture healing in vitamin BE-deficient rats

Fig. 3. Three weeks after fracture in a rat fed the supplemented diet [Fig. 3a (left)] and in one fed the B,-deficient diet [Fig 3b (right))) In the former there is virtually complete bony union whereas the latter has a large soft callus which resembles that seen in the 12-day control (on the supplemented diet. Fig. 2a). Stained with tolurdine blue; magnification x 15

correlation was not present in the Be-deficient rats, indicating that in the latter there was a defect of new bone-formation irrespective of the amount of callus produced. At 3 to 4 weeks: After 3 to 4 weeks there was still soft callus present with areas of cartilage and calcifying cartilage capping the periosteal woven bone (Fig. 3b). The size of the callus was now very similar to that found in the controls: this was compounded of increased new bone but less diminution of the soft callus (Table I). Full union was achieved by six weeks although remodelling was still incomplete. Bony peculiarities in Be-deficient fracture-healing. Epiphysea/ abnormalities were more marked in the fractured bone but, to a lesser degree, were also found in the unfractured metatarsal bones of rats fed on the Be-deficient diet. They were not found in bones of rats fed the supplemented diet. In agreement with the findings of Rodda (1975), they consisted of sparse, irregular, misshapen and effete metaphyseal trabeculae; the whole epiphysis was narrowed and irregular; the epiphyseal cell formations of the growth plate were smaller and irregular, as were the chondrocytes themselves. Abnormalities of the shaft: The shaft had become narrower (0.24 * 0.04 mm, mean ? SD, 68 measurements in 17 metatarsals from 10 rats, vs. 0.38 % 0.06 mm, 72 measurements in 15 control metatarsals from 9 rats: p < 0.001). There was extensive longitudinal invasion below

the line of the periosteum and endosteum with semr-crrcular deeper pockets (Fig. 5). The vascular spaces were also increased in number and extent; frequently obvious osteoid was present. Some deeper pockets contained multinucleate cells (Fig. 6) that showed strong acid phosphatase activity. By day 8 all these processes, together. resulted in the otherwise intact shaft being broken up into islands of bone resembling trabecular bone (Fig. 7). This was most marked at the margin of the fracture and immediately below the metaphysis. Metabolic

Activities

Detailed studies were made at day 5 and at day 12. At day 5 periosteal GGPD activity, measured in individual periosteal cells at defined distances from the fracture, was elevated (Fig. 8). In the control rats there was elevated activity within the first 6 mm from the fracture (designated as peak l), followed by a region of lesser activity, and then another region of very elevated activity (designated peak 2) at about 1.5 mm from the fracture-site. In the B,-deficient rats although peak 1 was well represented, there was marked depression of peak 2. Since the precise location of peak 2 could vary from 0.8 to 1.6 mm from the fracture site, the mean activities, in all the bones, measured at each location would not be representative. Consequently the results were recorded as follows: (i) the maximal activity found within peak 1; (ii) the maximum activity in peak 2, at whatever site this was found in that particular bone (Table

R.A. Dodds et al.: Fracture healing in vitamin Be-deficient

01

0

1

5

10 aread&tcalus

rats

I

15 (rml2x10)

2b

Fig. 4. Correlation between the area of new bone (mm* x 10) and the total area (mm2 x 10) of the soft callus in 12-day fractures in rats fed the supplemented diet (filled circles) and those fed the BE-deficient diet (open circles). Although there is good correlation for the former (r = 0.82; p < 0.001) there is no correlation for the latter, indicating depressed formation of bone irrespective of the size of callus that formed in the latter.

II). These results showed that there was a highly significant depression of the second peak of GGPD activity. Alkaline phosphatase activity was remarkably similar in both the control and the Be-deficient bones, with decreased activity close to the fracture-site rising to normal levels about 1.4 mm from the fracture. Similar results have been reported in normal rats (Shedden et al., 1976). By day 12 it was possible to study enzymatic activities in the various cell types (Fig. 2) in the callus: cells of the cellular granulation tissue; cells of the loose granulation tissue; mature chondrocytes; calcifying chondrocytes; and osteoblasts. The results are detailed in Table III. B,-deficiency had no effect on the activities of alkaline phospha-

Fig. 5. Bony peculiarities in Be-deficient specimens: abnormality of the shaft in a 3-week fracture. The shaft is becoming narrowed by extensive longitudinal invasion of both periosteal (a) and endosteal (b) surfaces with semi-circular deeper pockets of invasion Sections stained with toluidine blue. Magnification x 140.

Fig. 6. Bony peculiarities in Be-deficient specimens: 12-day fracture. Many spaces are partly lined by osteoid and some contain multinucleate osteoclasts. Section stained with toluidine blue, magnification x 120.

tase, glyceraldehyde 3-phosphate dehydrogenase (GAPD) or hydroxyacyl dehydrogenase (HOAD) in any of the cell types of the callus. Lactate dehydrogenase (LDH) activity was depressed in mature chondrocytes and, to a lesser degree, in the calcifying chondrocytes and in the cellular granulation tissue. The predominant effect was on the GGPD activity which was depressed in all the celltypes of the B,-deficient bones. This depression was highly significant in the mature chondrocytes and osteoblasts.

Discussion Rodda (1975) showed that although the weight of the rats fed the vitamin Be-deficient diet was less than that of the

Fig. 7. Bony peculiarities in Be-deficient specimens: day-8 fracture. The shaft close to the callus is broken up into islands of bone making the shaft resemble trabecular bone Section starned with toluidine blue; magnification x 60

494

R.A. Dodds et al.: Fracture healing in vitamin Be-deficient

Distance from fracture (mm) Fig. 8. Glucose

6-phosphate dehydrogenase activity (MIE x 100) in individual periosteal cells at defined distances (mm) from the fracture-site in 5-day fractures. Filled circles: in a rat fed the supplemented dret; open circles: in a rat fed the Be-deficient diet. The markedly depressed activity of the second peak (around 1.4- 1.7 mm in the control) in the latter is apparent.

paired-fed control rats, the tibia1 bone length was the same up to 50 days on the diet. Despite this he found marked abnormalities of the metaphyseal plate even after as short a time as three weeks on the diet. We have found changes in the epiphyseal and metaphyseal regions of the metatarsals without change in bone length in the rats fed on the Be-deficient diet for 24 days before fracture and for 12 days after fracture. Our previous findings (Dunham et al., 1977) showed that a major metabolic response to fracture in rat metatarsals is an increase in periosteal GGPD activity which is most marked in the cells that grow out, close to the fracture site, to form the developing callus and then, a few days later, specifically at the site at which, one day later, the first new bone is formed on the shaft (about 1.2 mm from the fracture-site). Later evidence (Dodds et al., 1984) indicated that the reducing equivalents for the vitamin K cycle in mineralization may be derived from ~ADPH derived from GGPD activity. It therefore seemed possible that fracture-healing could be regulated by such activity. If this were correct, it would be of value to determine the metabolic control of bony GGPD activity. Recently it has been suggested (Howat et al., 1985) that putrescine, the imme-

diate product of ornithine decarboxylase activity, could play some role in the control of GGPD activity. This concept was appealing, since ornithine decarboxylase activity is readily stimulatable in many cells (e.g. Bachrach, 1984) and might well be activated by the trauma of fracture. Hence we investigated the process of fracture healing in the metatarsals of rats that had been maintained on a vitamin B, deficient diet that has been shown (Eloranta et al., 1976; Pegg, 1977) to deplete rats of putrescine. Vitamin B, is an essential co-factor for ornithine decarboxylase The lack of vitamin B6 in the diet for only four weeks caused marked changes in the histology of the bone and also in the state of the growth-plate even in unfractured rat metatarsals. The effects were grossly exacerbated as a consequence of the fracture, with the shaft and, more particularly the region close to the growth-plate, almost resembling osteoporotic bone (Fig. 7) with cavities, lined with osteoclasts (Fig. 6). Although the deficiency of vitamin B, did reduce the overall size of the developing callus, the most striking effect was the inadequate production of new bone. Whereas in the controls, the amount of new bone formed correlated with the size of the callus, this correlation was not found in the Be-deficient fractures (Fig. 4) which took much longer to unite and which, at all the earlier times investigated, was more undifferentiated than the controls. Moreover, in B,-deficient fracture-healing, a peculiar periosteal and endosteal invasion of the shaft was recorded, diminishing the actual size of the shaft. Although Rodda (1975) had reported similar abnormalities of the growth-plates of the long bones of weanling rats maintained on a pyridoxine-deficient diet for up to 170 days the effects recorded here have presumably been exacerbated by the trauma of fracture. The activities of GAPD, HOAD and alkaline phosphatase were unaffected by the diet (Table Ill); GGPD activity was consistently depressed in all cell types studied (Tables II and III); to some degree LDH was also depressed in cellular granulation tissue and calcifying and mature chondrocytes (Table Ill). This depressed LDH activity may be related directly to the depressed GGPD activity since it has been shown (Dunham et al., 1983) that LDH activity appears to be responsive to flux of glycolytic activity which can be regulated by the extent of GGPD activity. The marked decrease in GGPD activity in the cells of the Be-deficient rats was in accord with the possibility that a system that depended on pyridoxal phosphate could play a regulatory role in the activity of this enzyme. Thus it seems possible that, in considering how to enhance fracture healing, the possible role of pyridoxal phosphate,

Table II. Glucose 6-phosphate dehydrogenase activity (mean integrated in periosteal cells five days after fracture.

Control Mean 2 SD Range Be-deficient Mean t Range

SD

a not significant. bp < 0001

rats

extinction

x 100) per cell for 10 min reaction time measured

Maximum activity at first peak

Maximum activity at second peak

Location of second peak (mm from fracture site)

24.3 -t 10.9= 6 - 54

39 7 * 9 2b 19 - 63

17.8 ” 5~6~ 7 - 37

17.1 2 9.3b 8 - 48

Number of Bones

Rats

1.36 ? 0.2 0.8 - 1 6

12

6

1.38 s 0.43 0.8 - 2 2

13

7

RA

Dodds et al.: Fracture healing in vitamin &-deficient

Table III. Activities of various enzymes (Mean Integrated

495

rats

Extinction

x iOO/cell) for 10 min reaction: mean -+ SD in the various cell

types of the 12.day callus.

Enzyme activrty Alkaline phosphatased GAPD

: T C

LDH HOAD

: T

GGPD

Note. C = control =p < 005.

C T C

Loose granulation tissue

Cellular granulation tissue

4.1 4.9 3.1 4.5 15.6 15.1 1.9 1.9 10.6 8.9

16.9 ? 178240 10.7 2 13.1 + 34.5 * 25.4 ? 35220 32?18 33.0 2 23.9 2

2 2.3 IT 2.6 ” 0.4 2 1.4 i 3.3 1?11.8 ? 0.8 2 08 2 1.8 2 1.9=

rate; T = Be-deficient

Number of Mature chondrocytes

2.8 4 7 34 9.1 2 8a

9 1 4 7b

27.2 27.4 6.4 6.0 52.9 35.1 3.9 3.7 23.7 14.6

2 2 2 2 2 ” 2 ” 2 2

5.0 44 3.3 2.5 2 5 3.2c 15 19 4.5 3.4c

Calcifying chondrocytes 11.9 2 13.4 * 2 2 2 2.1 2 15.9 2 128 ? 17 2 14 2 62 ? 54207

4.5 4.4 0.3 02 1.1 1.3b 0.5 0.3 1.3

Osteoblasts 301 32.2 4.0 4.6 251 23.3 5.2 5.2 18.3 12.6

?Z 65 t 70 t 0 5 2 18 221 2 1.1 ? 1.4 ? 1.8 2 4.5 2 4.6b

Bones

Rats

15 17 6 6 8 9 8 9 15 17

7 8 4 3 4 5 4 5 7 8

rats

b 0.01 > p > 0.001. =p < 0001 d 3 minute reaction

both as a possible regulator of GGPD activity, for providing reducing equivalents for the vitamin K cycle, and as the essential cofactor for the carboxylase of this cycle (Kappel and Olson, 1984), might merit consideration.

Acknowledgement: We are grateful to the Percy Bilton Charity for supporting this work, and to the Arthritis and Rheumatism Council for Research for general support

References Aro H Eerola E and Aho A J Determination of callus quantrty in 4-weekold fractures of the rat trbia J Orthop Res 3 101~108, 1985 Bachrach U Physrologrcal aspects of ornithine decarboxylase. Cell B/othem and funct. 2 6- 10, 1984 Bltensky L and Chayen J Histochemical methods for the study of lysosomes In Lysosomes, A Laboratory Handbook (J T Dangle ed ) 2nd edn North Holland, Amsterdam, 1977, pp 209-243 Chayen J , Bitensky L and Butcher, R G. Practical Histochemrstry Wrley, New York and London, 1973 Dodds R A, Catterall A, Brtensky L and Chayen J Effects on fracture healrng of an antagonrst of the vitamtn K cycle Calof. 71s~. Int. 36 23% 238, 1984 Dodds R A Dunham J., Bitensky L and Chayen J : Putrescrne may be a natural strmulator of glucose G-phosphate dehydrogenase FESS Letters 201 :105- 108, 1986 Dunham J , Shedden R G ,Catterall A, Brtensky L and Chayen J Pentose-shunt oxidation TISS Res 23 77-81,

rn the perrosteal cells in healrng fractures 1977

Calc

Dunham J Dodds R A, Nahrr A.M., Frost G T.B Catterall A, Bitensky L and Chayen J Aerobic glycolysls of bone and cartilage the possrble rnvolvement of fatty acid oxidation Cell Biochem and funct 1 168172, 1983 Eloranta T 0 Kafander E 0 and Raina A M Effect of pyridoxine deflciency on the metabolism of S-adenosylhomocysteine, S-adenosylmethionine and polyamrnes rn rat lrver Blochem. J 160.287-294, 1976 Hart J P Shearer M J Klenerman L , Catterall A, Reeve J Sambrook P.N Dodds R A Bttensky L and Chayen J Electrochemical detection of depressed circulating levels of vrtamrn K, In osteoporosis J Clan. Endocrinol. Metab. 60:126881269. 1985 Hauschka P V , Lian J B and Gallop P M Vrtamrn K and mrneralrzatlon Trends !n Biochem/ca/ Sciences 3 75- 78, 1978 Howat D W Chayen E N Bitensky L and Chayen J Polyamrnes rn strmulus. response coupling C//n. So 66(suppl. 11):21p, 1985 Kappel W K and Olson R E Covalent modification of the solubillzed rat liver vrtamrn K-dependent carboxylase with pyndoxal-5’.phosphate Arch Eiochem. fliophys 235.521-528, 1984 Pegg A E Role of pyridoxal phosphate rn mammalian polyamine brosyntheses lack of requirement for mammalran S-adenosylmethronrne decarboxylase activity &ochem J. 166 81-88. 1977 Rodda R A Bone growth changes rn pyrrdoxine-defrcrent rats J Path 117 131-138, 1975 Shedden R Dunham J , Bitensky L Catterall A and Chayen, J Changes in alkalrne phosphatase activrty in pertosteal cells in healing fractures Calcif TE. Res 22 19-25. 1976

Recefved March 1 I, 1986 Reused June 11, 1986 Accepted July 21, 1986