Thiamine influence on collagen during the granulation of skin wounds

Thiamine influence on collagen during the granulation of skin wounds

JOURNALOFSURGICALRESEARCH32,24-31 (1982) Thiamine Influence on Collagen OSCAR M. ALVAREZ, *Department of Nutritional TDepartment of Dermatology, ...

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JOURNALOFSURGICALRESEARCH32,24-31

(1982)

Thiamine Influence on Collagen OSCAR

M. ALVAREZ,

*Department of Nutritional TDepartment of Dermatology,

during the Granulation

PH.D.,*,? AND REX L. GILBREATH,

of Skin Wounds’ PH.D.*

Biochemistry, Rutgers University, New Brunswick, New Jersey 08903, and University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261 Submitted

for publication

January

28, 1981

To determine the effect of thiamine (vitamin B,) on collagen production during wound healing, hydroxyproline content (HP), lysyl oxidase activity (LO), and pepsin-solubilized collagen components were examined in dermis, wound fluid, and artificially induced granulation tissue from surgically wounded rats. Rats were divided into three groups: thiamine-deficient diet (-B,); thiamine-deficient diet supplemented with 3 mg thiamine-HCl (+3B,); and thiamine-deficient diet supplemented with 1 mg thiamine-HCl (+B,) (pair fed). When rats from the -BI group were demonstrated to be deficient in urinary thiamine, all of the experimental animals had one polyvinyl alcohol wound chamber implanted subcutaneously. Differences were observed in: HP concentration between -B, and both +3B, and +B, in granulation tissue and wound fluid, and LO between -B, and +B, in skin and granulation tissue. Both high- and low-molecular-weight collagenous components extracted from granulation tissue were greater for +3B, and +B,, than for -B,. The most dramatic findings were in the higher molecular weight component characterized as Type III collagen. The alterations observed in the collagen contents and maturation of granulation tissue demonstrate an involvement of thiamine in wound repair and scar development.

containing amino acids (cisteine and methionine) in type III. Current information suggests that normal mammalian skin collagen is composed of 80-90% type I collagen. In contrast, dermal granulation tissue has a high proportion of type III compared to the smaller amount normally present in mature skin [ 21. More recently, it has been proposed that type III collagen is produced at the earliest phase of wound healing by primitive mesenchymal cells (undifferentiated fibroblastic cells), which is followed by the synthesis of type I collagen by mature wound fibroblasts [8]. In both developmental and skin repair the fibroblast lays down embryonic-type collagen. This collagen (type III) is gradually replaced by the adult-type collagen (type I). It has been reported that thiamine deficiency has a detrimental effect on collagen metabolism [ 191. These conclusions are, however, based only on differences observed in neutral salt-soluble and -insoluble collagen extracts of tissue taken from cotton-induced granulomas in thiamine-deficient rats compared to normal rats. It has been sug-

INTRODUCTION

Research has provided evidence of differences between mammalian collagens, such as variations in amino acid composition, the degree of hydroxylation of proline and lysine, and the type and stability of intermolecular crosslinks [9]. More recently, several chemically and genetically distinct collagens have been classified and demonstrated in tissues from rats [2], guinea pigs [3], rabbits [ 171, and humans [8]. Type I collagen, which has the chain composition a12, (~2 is the major collagen species in mature skin, bone, and tendon [7]. Skin and granulation tissue also contain various amounts of type III collagen, al (III)3 [ 161. Type III collagen often accounts for half or more of the collagen in fetal skin, but less type III is found in the skin of older individuals. One important difference between type III and type I collagens is the presence of sulfur’ Abstract of this work was presented at the 63rd Annual Meeting, Federation of American Societies for Experimental Biology (Fed. Proc. 83: 3, 1979). 0022-4804/82/010024-08$01.00 Copyright Q 1982 by Academic Pm. Inc. All ri@s

of reproduction

in any farm raervcd.

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ALVAREZ

AND

GILBREATH:

EFFECT

gested that thiamine may be involved in collagen biosynthesis during wound healing. The purpose of this study was twofold: (1) to investigate the effect of thiamine deficiency on collagen content and maturation in both wounded and normal skin; (2) To evaluate the effect of hypervitaminosis Bi on dermal wound healing. MATERIALS

AND METHODS

Animals and Diet Fifty-six male Sprague-Dawley rats ( 150200 g) (Charles River Breeding Laboratories, Boston, Mass.) 1-2 months old were used in two experiments. All of the animals were caged individually in stainless-steel cageswith controlled temperature (19-21 “C) and light (12/ 12 LD). The rats were fed Purina Laboratory Rat Chow for a period of 5 days and then randomly divided into three dietary groups as follows: group 1 rats (-B,) were fed a thiamine-deficient diet (ICN Nutritional Biochemicals, Cleveland, Ohio) and 1 ml of distilled water gavage on alternate days; group 2 rats (+B,) were fed the amount of diet consumed by -B,, but supplemented with 1 mg thiamine hydrochloride dissolved in 1 ml distilled water and gavaged on alternate days; and group 3 rats (+3B,) were treated in the same manner as SBi, with the exception of the dosage of thiamine hydrochloride (3 mg on alternate days). Food intake and body weights were measured daily and on alternate days, respectively. Wound Chamber Construction and Subcutaneous Implantation Wound chambers were constructed from Ivalon sponges made of polyvinyl alcohol and formaldehyde (Ivalon Unipoint Laboratories, N. C.). The spongeswere sliced into l-mm-thick sheetsand cut into 2-cm squares. The squares were then rolled into cylinders, and silicone rubber disks (Perkin-Elmer, Norwalk, Conn.) were sutured onto both

OF THIAMINE

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ends to produce a cylinder 2 cm in length and 0.5 cm in outside diameter. These chambers create a stable dead space. After the chamber is implanted, the dead space fills with wound fluid which can be aspirated at will. This inert wound chamber serves as an inductive matrix for collagenous tissue. To implant the chambers each rat was anesthesized under light ether and a 3 X 3cm square of dorsal skin was shaved with standard animal clippers. The skin was swabbed with 20% Zephiran Chloride (Sigma Chemical Co., St. Louis, MO.) and a l-cm incision was made in the dorsal neck skin. The sterile wound chamber was carefully inserted subcutaneously and aligned paravertebrally and the incision closed with two interrupted sutures. Experiments Two experiments were conducted to investigate the effect of thiamine deficiency and a hyperthiamine regimen on: (1) normal collagen synthesis; and (2) collagen synthesis during wound repair. 1. Collagen accumulation and lysyl oxidase activity. Forty-eight Sprague-Dawley male rats were divided into the three dietary treatment groups as previously described. When a decrease in urinary thiamine level was demonstrated in -B, rats, all rats were implanted with a polyvinyl alcohol wound chamber under aseptic conditions as previously detailed. Ten days after implantation, all the animals were killed with chloroform. A full-thickness l-cm’ strip of unwounded normal skin posterior to the incision and the wound chamber were collected and assayed for lysyl oxidase activity, hydroxyproline concentration, and collagen types I and III. In addition, wound fluid from each chamber was assayed for hydroxyproline concentration. 2. Proliferation of dermal collagen types. Eight male rats were divided into two dietary treatment groups. One group was fed the thiamine-deficient diet and the second group consisted of pair-fed controls (see animals

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and diet). When a urinary thiamine decrease was demonstrated in -B, rats, the wound chambers were implanted subcutaneously in all animals. One rat from each of the treatment groups was sacrificed at 24,48,72, and 96 hr after wounding. The granulation tissue infiltrating the wound chambers was then assayed for collagen types I and III. Urinary

Thiamine

Urinary thiamine levels were determined by the thiochrome reaction as described by Hennessy [ 111. Lysyl Oxidase

The method of Pinnell and Martin [ 181 was used to measure the activity of lysyl oxidase. Tritiated water released during enzymesubstrate incubation was separated by small ion-exchange columns (Dowex 5OW X 8, 100-200 mesh, as described by Melet [ 151). Column effluents of 0.75 ml were then mixed with 19.25 ml of Instagel (Hewlett-Packard Instruments, Palo Alto, Calif.) and counted in a Hewlett-Packard Model 3255 liquid scintillation counter. Percentage counter efficiency for tritium was calculated at 39%. Data were computed and statistically analyzed as disintegrations per minute per milligram protein. The method of Hartree [lo] was used to determine the protein content of the tissue extracts. Hydroxyproline

Determination of hydroxyproline in skin, wound fluid, and wound chamber was accomplished by the Alvarez et al. modification [ l] of the Bergman and Loxley method [4]. Cleavage with Pepsin and Isolation Molecular Species

of

The reactive granulation tissue, including the polyvinyl alcohol wound chamber and

1982

skin, were minced and suspended in 100 ml of 0.5 M acetic acid and pepsin (Sigma; twice recrystalized, 300 units/mg) was added to a concentration of 1 mg/ml [ 51. The mixture was stirred for 24 hr at 5°C and the pepsin digest was then centrifuged at 30,OOOgfor 1 hr at 4°C. The tissue residue was redigested for 8 hr with additional pepsin and centrifuged. The supernatants of three extractions were pooled, the pH was adjusted in order to lyse the pepsin, and each was dialyzed 18 hr against 5 liters of 0.02 M NaHPO,. The resulting collagen precipitate was suspended in 2 M guanadine-HCl containing 0.05 M Tris-HCl, pH 7.5 (2-10 mg/ml) and was denatured by heating at 45°C for 30 min. This solution was centrifuged and the supernatant containing the denatured collagen lyophylized. Samples of dried dermal and wound chamber collagen of 75 mg were dissolved in 2 M guanadineHCl 0.05 M Tris-HCl and chromatographed on 1.8 X 80-cm columns of Bio-Gel A- 1.5m (Bio-Rad Laboratories, Richmond, Calif.). The columns were equilibrated and eluted with 2 M guanadine-HCl, 0.05 M Tris-HCl, at pH 7.5 and 22°C with a flow rate of 10 ml/hr. Fractions of 3 ml were collected and the absorbance at 234 nm was monitored with a Beckman DU spectrophotometer (Beckman Instruments, Palo Alto, Calif.). The area under each peak was measured by the trapezoid method [22]. Characterization

of Collagen

Type ZZZ

The higher-molecular-weight components (y) eluted from the Bio-Gel were dissolved at a concentration of 20 mg/ml in a solution of 5.0 M urea, 0.1 M 2-mercaptoethanol, pH 8.0, according to Chung and Miller [6]. Mercaptoethanol breaks down the disulfide links of type III collagen, thereby destroying the helical structure and yielding 3 single (Y, chains chromatographically similar to those of type I collagen. The solution was kept at 22°C for 4 hr and made 0.2 M in iodacetic acid (sodium salt) containing

ALVAREZ

AND GILBREATH:

EFFECT OF THIAMINE

sufficient [ 1-‘4C]iodacetic acid (New England Nuclear, Boston, Mass.) to give a final concentration of 200 &i/ml. After 20 min, a 2-ml portion of this reduced and labeled collagen fraction was chromatographed on Bio-Gel A- 1.5m columns 1.8 X 80 cm). The columns were eluted with 2 M guanadineHCl, 0.05 A4 Tris-HCl, pH 7.5, under conditions previously described. Three-milliliter portions of the column effluent were mixed with instagel and radioactivity counted in a liquid scintillation counter. RESULTS

Experiment I Dry weights of the skin and wound chamber with associated granulation tissue are presented in Table 1. Wound chambers from thiamine-supplemented and pair-fed control rats weighed more (P < 0.05) than those from thiamine-deficient rats. The lighter wound chambers in thiamine-deficient rats reflected a decrease in the amount of collagen present. On an absolute basis, skin weights of all groups were statistically similar. Hydroxyproline concentrations in wound chamber granuloma, wound fluid, and skin, are shown in Table 2. There was no statistical difference in the concentration of total hydroxyproline of unwounded skin between

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ON WOUND HEALING TABLE 2

HYDROXYPROLINE CONTENT OF GRANULATION TISSUE, WOUND FLUID AND SKIN FROM THIAMINEDEFICIENT AND THIAMINE-SUPPLEMENTED RATS

Hydroxyproline (pg/g) Treatment -B, +B, +3B,

Wound chamber

Wound fluid

49.3 f 10hJ 12.1 f Id 441.0 + 20 39.0 f 3 449.0 + 24 53.3 + 3

Skin 4472 11 456 + 12 464+ 12

Note. Wound chamber, n = 12; wound fluid, n = 4; skin n = 12. ’ Expressed as pg/ml. ’ Mean f SEM. c P < 0.001. d P < 0.05.

treatments. However, granulation tissue and wound fluid from thiamine-deficient rats had significantly lower levels of this imino acid (P < 0.001 and P < 0.05, respectively). Differences observed between thiamine-supplemented groups (+B, and f3B1) were not significant. Activity of lysyi oxidase in wound chamber granulation tissue and skin is shown in Table 3. The granulation tissue and skin from rats fed the thiamine-deficient diet had significantly lower (P < 0.05) lysyl oxidase activity than these tissues from thiamineTABLE 3

TABLE 1 DRY WEIGHTS OF SKIN AND EXPERIMENTAL GRANULATION TISSUE” FROM THIAMINE-DEFICIENT AND THIAMINE-SUPPLEMENTED RATS

LYSYL OXIDASE ACTIVITY IN GRANULATION TISSUE AND SKIN FROM THIAMINE-DEFICIENT AND THIAMINE-SUPPLEMENTED RATS

Lysyl oxidase (dpm/mg protein) Treatment

-B1 +BI +3B,

Skin h/cm’) 296 + 4’ 297 + 4 298 f 4

Experimental granulation tissue (mg/mg wound chamber)

Treatment

Wound chamber

Skin

1.53 f 0.2 9.8 f 0.5 7.33 f 0.5

-B, +B, +3B,

407 + 4”.b 687 f 12 715 f 16

255 f 3’ 324+ 3 323 + 3

’ The dry weight of the wound chamber without the silicone disks before implantation was 30 mg. b Mean + SEM.

Note. Wound chamber, n = 12; skin, n = 12. L?Mean + SEM. b P < 0.05.

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supplemented rats. The activity of this enzyme was also similar in the tissues of both thiamine-supplemented groups (+B, and +3B,). Collagen recovery following pepsinization was approximately 87%. Recovery was based on the hydroxyproline concentrations of the tissue before and after digestion with pepsin. Molecular sieve chromatography of the collagen revealed collagenous material eluting at 30 ml, which was a characteristic of a crosslinked collagen of molecular weight 300,000 (7). Reduction and rechromatography of portions of y fraction revealed that this was type III collagen (data not shown). A smaller amount of material eluted in the 200,000 (/3) molecular-weight range and was followed by a slightly slower moving component of molecular weight 100,000 (a) (type I). The proportions of type I (0~) and III (y) collagens from wound chamber granulation tissue are shown in Fig. 1. The amount of type I collagen in the granulation tissue from thiamine-deficient rats was slightly lower than that in the granulation tissue from pair-fed control or thiamine-supplemented rats. However, a dramatic difference was observed in the proportion of granulation tissue collagen type III between the thiamine-deficient and thiamine-supplemented animals. The ratio of type III collagen to type I collagen was decreased 62% due to thiamine deficiency. In normal unwounded skin, thiamine did not influence the magnitude of either collagen type nor their respective ratios (data not included). Experiment

2

Amounts of collagen types I and III synthesized during the first 96 hr postwounding are shown in Fig. 2. The amount of collagen type III depended on the length of time the implant remained in the wound. The chromatographic analysis of wound chambers from supplemented rats after implantation showed a steady increase in type III collagen

VOL. 32, NO. 1, JANUARY

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R=O.lb

0.6 0.2

1.2 v k? -C 0.6 0.2 R=O

37

a

0

20

40

ELUTION

60 VOLUME

80

100

(ml)

FIG. 1. Bio-gel A-l.5 m chromatograph of 75 mg denatured collagen released from wound chamber granulation tissue by pepsin digestion: Type III (y) and I ((Y) collagens with peak areas and ratios (R); thiaminedeficient (-B,), 1 mg thiamine supplementation (+B,), and 3 mg thiamine supplementation (+3BI).

until 72 hr after which time the level was unchanged (Fig. 2, +B,). In the 24-, 48-, and 72-hr samples, only small amounts of collagen Type I were observed. Between 72 and 96 hr, however, a dramatic increase in the amount of type I collagen was detected (Fig. 2, +B,) in granulation tissue from thiamine-supplemented rats. In the thiamine-deficient granulation tissue, the proportions of collagen type I, at 24, 48, and 72 hr were similar to the amounts observed in the control (+B,) wound chambers. Although there was an increased accumulation of collagen type I between 72 and 96 hr in the granulation tissue of deficient rats, at 96 hr the thiamine-deficient tissue contained 28% less type I collagen than that of the controls.

ALVAREZ

AND

GILBREATH:

EFFECT I

6.0

-BI

-

TYPE 111 COLLA(,EN

C--*

TYPE

2- 6.0 t +B,

I COLlA(;EN

.

1.0

_--24 HOURS

*--e--d I 40

I 72

I 96

I

AFTER WOUNDING

FIG. 2. Amount of type I and III collagen synthesis in wound chamber granulation tissue at 24, 48, 72, and 96 hr postwounding. Thiamine-deficient (-B,), thiamine-supplemented (+B,) rats.

The amounts of type III collagen detected in the thiamine-deficient wound chambers was consistently lower, at all time intervals, when compared to the control (+B,) granulation tissue. DISCUSSION

The similarity in the weights of skin between treatment groups, and the observation that deficient animals were losing weight during the final 10 days of the experiment, suggeststhat muscle and organ tissue, rather than skin, was being catabolized. During the sequence of collagen biosynthesis, a necessary step is the hydroxylation of appropriate proline residues in protocollagen to hydroxyproline. The hydroxyproline index has been demonstrated to be a reliable tool for assessingcollagen metabolism in tissue, urine, and serum [20]. As a result of thiamine deficiency, the hydroxyproline concentrations in the granulation tissue, wound fluid, and skin decreased 88, 69, and 2%, respectively. The large differences observed in the granulation tissue and wound fluid

OF THIAMINE

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hydroxyproline in contrast to the slight variations noted in skin imply that the effect of thiamine deficiency on collagen biosynthesis is more pronounced during wound repair. Lysyl oxidase catalyzes the enzymatic reaction that initiates collagen crosslinking [21]. These crosslinks are the basic step in the formation of the stable structure of mature collagen and provide its inherent property of tensile strength. Decreased activity of this enzyme in the tissues from thiaminedeficient rats suggests a defect in the maturation processof the collagen fibers. During wound healing, depressed levels of lysyl oxidase indicate a frail wound and poor scar development. It is unlikely that thiamine affects lysyl oxidase activity directly. However, thiamine may affect lysyl oxidase activity by influencing collagen synthesis, since the activity of this enzyme is closely related to the synthesis of collagen (Siegel).’ These results clearly demonstrate that in response to an inflammatory stimulus, the collagen synthesized is predominantly type III collagen. During thiamine deficiency only granulation tissue collagen type III is affected. This suggests that vitamin B, influences collagen synthesis during wound healing. The differentiation of mesenchymal cells to mature repair fibroblasts has been ccnsidered a fundamental event in dermal wound healing [ 241. In early phases of healing (2472 hr), undifferentiated fibrocytes with various morphology can be seen both in the wound edges and in granuloma tissue from sponge implants [23]. The period between 24 and 72 hr coincides with the “lag” period of collagen type I synthesis in open granulomas and skin wounds [2]. Type III collagen seems to be the product of these cells found early in the wound. It has been speculated [ 141that a function of collagen type III is the creation of a reticular network necessary for the proper synthesis and maturation of type I collagen * R. C. Siegel, personal communication.

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fibers. During thiamine deficiency, an abnormality in the synthesis of type III collagen is observed (-B,, Figs. 1 and 2). This atypical synthesis of collagen type III may lead to the improper development of collagen type I. Considering our knowledge of the biochemical functions of thiamine, it is probable that during wound healing, the role thiamine plays at the cellular level is related to energy metabolism. It is known that the process of wound healing requires increased mitotic and synthetic activities on the part of the cells (fibroblasts, endothelial, and epithelial) adjacent to the site of injury [ 12, 131. The energy production in chemical form (ATP) has been calculated in normal and repairing skin [ 131. These authors report that repairing cells are maintained by energy (ATP) obtained mainly (70%) from the EmdenMyerhof pathway and partially (30%) through the pathways of mitochondria. Thiamine, in the form of thiamine diphosphate (thiamine pyrophosphate) acts as a coenzyme in the system for the oxidative decarboxylation of pyruvate or of a-ketogluterate in the pyruvate or ketogluterate dehydrogenase enzyme complex systems, respectively. Vitamin B, is also a coenzyme in the reactions of transketolation which occur in the direct oxidative pathway for gluocose metabolism. These relationships strongly suggest that during thiamine deficiency, there is a reduced capacity of cells involved in wound healing to generate the sufficient ATP required to meet their migratory and mitotic needs. ACKNOWLEDGMENTS We wish to acknowledge Dr. William H. Eaglstein for his thoughtful advice during the preparation of this manuscript. This work was supported by the New Jersey Agricultural Research Station (HATCH).

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dine-2-carboxylic acid (LACA) on epidermal and dermal wound repair. Plast. Reconstr. Surg., in press, 1981. 2. Bailey, A. J., Sims, T. J., Le Lous, M., and Bazin, S. Collagen polymorphism in experimental granulation tissue. Biochem. Biophys. Res. Commun. 66: 1160, 1975. 3. Barnes, M. J., and Morton, L. F. Presence of type III collagen in guinea-pig dermal scar. Biochem. J. 157: 263, 1976.

4. Bergman, M., and Loxley, R. Two improved and simplified methods for the spectrophotometric determination of hydroxyproline. Anal. Chem. 35: 1962, 1963. 5. ChandraRajan, J. Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation. Biochem. Biophys. Res. Commun. 83: 180, 1978. 6. Chung, E., and Miller, E. J. Collagen polymorphism: Characterization of molecules with the chain composition a, (III), in human tissues. Science. 183: 1200, 1974.

I. Fietzek, P. O., and Kuhn, K. The primary structure of collagen. Inr. Rev. Conn. Tissue Res. 7: 1, 1976. 8. Gay, S., Viljanto, J., Raekallio, J., and Pentinen, R. Collagen types in early phases of wound healing in children. Acta Chir. Sand. 144: 205, 1978. 9. Grant, M. E., and Prockop, D. J. The biosynthesis of collagen (first of three parts). N. Engl. J. Med. 286 (4): 194, 1972. 10. Hartree, E. F. Determination of protein: A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48: 422, 1972. 11. Hennessy, D. J. Chemical methods for the determination of vitamin B,. Ind. Eng. Chem. 13: 216, 1941. 12. Im, M. J. C., Freshwater, M. F., and Hoopes, J. E. Enzyme activities in granulation tissue: Energy for collagen synthesis. J. Surg. Res. 20: 121, 1975. 13. Im, M. J. C., and Hoopes, J. E. Energy metabolism in healing skin wounds. J. Surg. Res. 10: 459.1970. 14. Lapiere, C. M., Nusgens, B., Pierard, G., and Hermanns, J. F. The involvement of procollagen in spatially oriented fibrogenesis. In P.M.C. Burleigh and A. R. Poole (Ends.), Dynamics of Connective Tissue Macromolecules. North-Amsterdam: North-Holland, 1975. Pp. 33-46. 15. Melet, J. Simplified assay for lysyl oxidase.activity. Anal. Biochem. 77: 141, 1977. 16. Miller, E. J. A review of biochemical studies on the genetically distinct collagens of the skeletal system. Clin. Orthop. Relai. Res. 92: 260, 1973.

REFERENCES 1. Alvarez, 0. M., Mertz, P. M., and Eaglstein, W. H. The effect of the proline analogue l-azeti-

17. Peacock, E. E., and Van Winkle, W. Structure synthesis and interaction of fibrous protein and matrix. In Wound Repair, 2nd ed. Philadelphia: Saunders, 1976. Pp. 81-145.

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EFFECT

18. Pinnell, S. R., and Martin, G. R. The cross-linking of collagen and elastin: Enzymatic conversion of lysine in peptide linkage to cY-aminoadipic-&semialdehyde (allysine) by an extract from bone. Biochemistry 61: 708, 1968. 19. Prasad, C. A., and Bose, S. M. Effect of thiamine deficiency on collagen metabolism. J. Nutr. Sci. Vifaminol. 20: 97, 1974. 20. Prockop, D. J., and Udenfriend, S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal. Biochem. 1: 228, 1960. 21. Siegel, R. C., and Martin, G. R. Biosynthesis of

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collagen cross-links: Increased activity of purified lysyl oxidase reconstituted collagen fibrils. Proc. Natl. Acad. Sci. USA 71: 4826, 1974. 22. Tierney, J. A. Integral calculus. In Calculus and Analytical Geometry, 2nd ed. Boston: Allyn & Bacon, 1972. P. 318. 23. Viljanto, J. Biochemical basis of tensile strength in wound healing. Acta Chir. Stand. Suppl. 333: 1, 1964. 24. Viljanto, J. Cellstic. A device for wound healing studies in man. Description of method. J. Surg. Res. 20: 115, 1976.