Review of biochemistry of the calcified tissues

Review of biochemistry of the calcified tissues

Robert Van Reen, PhD, Bethesda, Md

Results of studies on the hormonal control of calcium metabolism, fluoride and ca lcifie d tissues, and collagen, the main protein of connective tis ­ sues, are discussed.

During the past few years, there has been an ac­ celeration in the amount of research in the dental sciences with a concomitant increase in the num ­ ber of scientific publications. Research in the bio­ chemistry o f the calcified tissues has been active, which precludes a comprehensive review of all aspects of work. M ajor breakthroughs have been made in understanding the hormonal control of calcium metabolism and the chemistry and struc­ ture of collagen. Therefore, attention has been focused on these and several other active areas.

Hormonal control of calcium m etabolism ■ Thyrocalcitonin: It has long been known that parathyroid hormone (PTH) has a definite effect on calcium metabolism, and until recently, the 1340

parathyroid glands have been thought to have the hormonal control o f calcium in blood and other fluids and tissues. The concept was developed that a drop in plasma calcium results in the increased production of P T H which, by mobilizing skeletal calcium, raises the calcium concentration to nor­ mal levels. Conversely, a rise in blood calcium concentration was thought to result in a reduction in the secretion o f PTH . Blood calcium homeo­ stasis via a “ feedback” mechanism was a popular concept, although, as pointed out by Rasmussen1 and other researchers, P T H is slow acting, and large changes in blood calcium concentrations would be anticipated if it were the only mecha­ nism of calcium regulation. Copp and others2 proposed that a hypocalcemic hormone was produced by the parathyroid glands o f dogs in response to a perfusion o f hypercalcemic blood. The term calcitonin was coined for the proposed hormone. The release of a hypocalcemic factor has been confirmed,3 but it was pointed out that it was not feasible to separate the thyroid and parathyroid glands in the dog; thus, it was impossible to designate the source o f cal­ citonin. Copp and Henze4 and Care and Keynes5 reported the results of perfusing the superior pair of parathyroid glands in anesthetized and in con­ scious sheep and indicated that these glands are a source o f calcitonin. Hirsch and others6 and Hirsch and Munson7 demonstrated that a hypocalcemic factor is released by the thyroid gland of the rat and that the factor could be extracted from thy­ roid gland tissue o f several species, including pig thyroid which contains no parathyroid gland tis­ sue. They called the factor thyrocalcitonin (TCT). In the period covered by this review, rapid progress has been made in determining the source of T C T , its chemical characterization, its action on bone metabolism and in regulating calcium blood levels, and its action in several species, in­ cluding man. On the basis o f conflicting data, it has been sug­ gested that hypercalcemia elicits the release of two hormones, one from the parathyroid gland and the other from the thyroid. However, the evi­ dence accumulated in the past few years supports the concept that the thyroid gland is the major source o f the hypocalcemic hormone.8-11 Gittes and Irvin12 have presented evidence that the para­ thyroid glands secrete a humoral factor that re­ leases calcium-lowering T C T from the thyroid. Such a hypothesis would reconcile the conflicting reports suggesting that both the parathyroid and the thyroid glands produce a hypocalcemic factor.

More recent studies by Toverud and others13 showed that the thyroid gland alone is critical in protecting the rat against experimental hypercal­ cemia. Furthermore, Foster14 reported that it was not possible to demonstrate a parathyroidreleasing factor in the Wistar hooded rat. Thyrocalcitonin has been extracted from the thyroid gland o f the rat, pig, cow, monkey, dog, and goat, and has been demonstrated in the hu­ man thyroid gland.15 16 The possible use of ani­ mal preparations in man was studied soon after preparations o f the hormone were available. Fost­ er and others17 administered T C T from the pig thyroid gland to three patients with disseminating malignant disease with hypercalcemia. A n intra­ venous injection o f 1 to 2 2 M R C units (a unit is the quantity o f T C T that will lower the serum cal­ cium in a 150 gm rat 0 .1 m g/100 ml at 50 minutes after an intravenous injection) lowered serum cal­ cium in the patients by 0.6 to 0.8 m Eq per liter for up to 14 hours. Ten units produced a slight fall at six hours in a normal patient. The effect of a subcutaneous administration of a crude extract of porcine T C T in three normal humans was stud­ ied by Bell and others.18 Thyrocalcitonin at 0 .1 5 mg/kg body weight produced transient decreases in serum calcium which were maximal at between a half and two hours. Milhaud and others19 20 also noted the effectiveness o f pig T C T in man and in 3 -month-old twins with idiopathic hypercalcemia. A number of investigators have extracted T C T from thyroid gland tissue, purified the prepara­ tions, and deterjnined some of the characteristics of the hormone. On the basis o f Sephadex separa­ tion, Foster and others21 suggested that the molec­ ular weight o f pig calcitonin is less than 3,000. Tenenhouse and others22 concluded that the hor­ mone is a polypeptide with a mol wt 8,700, a single N-terminal threonine, and a single cysteine residue in an unidentified form. The suggested ab­ sence of cystine in a purified preparation as re­ ported by Gudmundsson and others23 leaves some doubt as to the amino acid components of the hor­ mone. Studies of hog T C T by O ’Riordan and others24 in which density-gradient ultracentifugation was used, indicated a mol wt of 5,000 to 6,000. It was suggested that the report by Foster and others21 o f a mol wt 3,000 may have been due to the splitting o ff o f a small active compo­ nent o f the native substance. A 44,000-fold puri­ fication o f hog thyroid gland tissue was obtained by Potts and others.25 The active fraction formed a single band on analytical polyacrylamide gel electrophoresis, did not contain isoleucine, had a Van Reen: BIOCHEMISTRY OF CALCIFIED TISSUES

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1341

mol wt about 4,50 0 , and did not show any cystine. Although the hypocalcemic factor can be ex­ tracted from the thyroid gland tissue of a number of species, on the basis of immunochemical evi­ dence, there may be structural differences in T C T s obtained from animals o f different taxonomic or­ ders.26 The cellular and subcellular location of T C T has been investigated. Bauer and Teitelbaum27 suggested that the parafollicular cells might be the source of secretory granules that show hypocal­ cemic activity. B y means of fluorescent studies of antibodies, T C T was detected in the cytoplasm of all epithelial cells o f the pig thyroid gland.28 It was not present in the follicular colloid but was stored or synthesized in the same cells that elab­ orate thyroglobulin. Cooper and Tashjian29 con­ cluded from results o f studies of differential cen­ trifugation with rat, porcine, and calf thyroid gland tissue that T C T is found mainly in the microsomal fraction of thyroid cells. Pearse30 has reviewed the literature concerning the C cells of the thyroid gland which are distinct from follicular or acinar cells. He has indicated that these cells respond to high blood calcium lev­ els by increased secretion o f a substance that is postulated to be T C T . More recent studies have indicated that the C cells are derived from ultimobranchial bodies and are not of true thyroid tissue origin.3oA T o support this, Copp30B has re­ ported that ultimobranchial tissues o f chickens and a species o f shark contain high levels of the hypocalcemic factor, whereas the thyroid tissues do not. He suggests that the term calcitonin be re­ tained in place o f thyrocalcitonin to designate the hormone. Several investigators have undertaken investiga­ tions to determine the mechanism of action o f T C T . In spite o f some disagreement, it appears most probable that the hormone inhibits bone resorp­ tion or induces bone deposition, or both. Neither the kidney31 nor the gastrointestinal tract32 are essential for T C T to exert its hypocalcemic effect. Aliapoulios and others,33 working with tissue cul­ ture o f mouse calvaria, showed that T C T greatly diminished PTH-induced resorption o f bone. In the studies of W ase and others,34 T C T increased the incorporation o f 45C a into the bones o f rats, caused more positive calcium balances, decreased the output of endogenous calcium, and increased the biological half-life o f 47Ca. Results of in vivo studies by Foster and others35 showed that T C T acts on bone independently of PTH , causes in­ creased trabecular bone in vertebrae of growing 1342 ■ JADA, Vol. 76, June 1968

animals, and a diminution in the number o f osteo­ blasts in vertebrae. A fraction of bone mineral is more labile than others and subject to resorp­ tion in response to PTH . Hirsch36 has indicated that T C T inhibits the release o f calcium induced by P T H from both stable and labile bone. This suggests that the same areas o f bone are responsive to both P T H and T C T . Considerably more work has been reported in this area. ■ Parathyroid hormone: One of the most in­ teresting facets of work on P T H is that concerning the chemical structure of the hormone. Potts and Aurbach37 and Potts and others38 have proposed the amino acid structure and sequence of P T H de­ duced from studies with exopeptidases, the com­ position o f fragihents from cyanogen bromide cleavage, dilute acid hydrolysis, and digestion with pepsin. They have shown that the P T H poly­ peptide is basic with lysine and arginine clustered in certain areas. They showed that the minimum structure required for biological activity resides in a sequence of 20 amino acids at the carboxyl(COOH-) terminus and amounts to about 2 5 % of the native molecule. A variety o f new or improved bio-assays for P T H have been proposed.39-43 One o f the most sensitive assay systems for P T H has been reported by Aurbach and others.44 In this assay, rat liver mitochondria are allowed to react with succinate isotopically labeled in the 1- and 4 - positions with 14C in a suitable medium. PT H stimulates the release o f carbon-labeled carbon dioxide (14C 0 2) with as little as 7 X 1 0 -9 M P T H causing a significant stimulation o f the oxidation of suc­ cinate. That the target tissues for P T H action include bone and kidney has been established by the re­ sults o f numerous studies, although the hormone also affects many other tissues. Several years ago, it was suggested that P T H increased the intestinal absorption of calcium. Although this has been re­ futed by some investigators, recently Shah and Draper45 reinvestigated the in vivo intestinal ab­ sorption o f calcium by growing rats and showed that after a parathyroidectomy, absorption at rela­ tively low levels of calcium intake was significantly depressed but not at a dietary level of 1 .2 % . It can­ not be determined from the results o f these studies whether the decrease in absorption was due to a direct or an indirect effect of P T H on the intestinal mucosa. P T H has been thought to have a direct effect on bone, but this may not be true in view of re­

cent evidence. Borle and Neuman46 observed that H eLa cell cultures show morphological, develop­ mental, and biochemical changes in response to PTH . Increased dissolution of bone powder by the cells was one definite change. Tenenhouse and others,47 working with Ehrlich ascites cell cul­ tures, found that when P T H was incubated with the cells, it caused the release of some factor into the medium that was effective in dissolving cal­ cium from bone when the supernatant from the cells was incubated with bone powder. Measurable effects were observed with as little as 1 0 -8M PTH . The evidence with H eLa cells and Ehrlich ascites cells indicates that PT H has no direct effect on bone dissolution. In view of these findings, it may be necessary to reevaluate a number o f earlier studies. For instance, Raisz48 studied bone resorp­ tion in tissue culture and several factors influenc­ ing the response to PTH . He reported a consid­ erable variation in resorption when bones were cultivated in media made with different samples o f human and rat serum. It may be that serum con­ tains factors whigj] influence the elaboration of a factor into the growth medium, which in turn causes bone dissolution. Almost a decade ago, Harrison and others49 showed that injections of P T H into vitamin D-deficient young rats did not raise the serum calcium level unless the rats were pretreated with vitamin D. The concept that vitamin D is required for the responsiveness of the target organs to physiologic amounts of P T H has been supported by some in­ vestigators and denied by others. This discrepancy in results reported by reliable investigators has continued into the period covered by this review. Toverud50 concluded that the calcium-mobilizing action o f P T H in the rat is independent o f or re­ quires only minute amounts o f vitamin D. To de­ termine whether the parathyroid glands o f vitamin-D-deficient animals could secrete an effective bone-resorbing hormone, A u and Raisz51 cul­ tured glands from D-deficient hypocalcemic rats in low- or high- calcium medium containing serum from vitamin D-deficient rats. The media from parathyroid gland cultures were then applied to embryonic bone. The results indicated that the secretion of bone-resorbing substance by glands from vitamin D-deficient rats was under the con­ trol o f the calcium concentration in the medium. Thus, it was concluded that the functional activity of rat parathyroid glands does not depend on the presence o f vitamin D. Ney and others52 conclud­ ed that all the actions o f P T H are exerted in the D-deficient dog. In contrast to these reports, in

a series o f elaborate experiments, Arnaud and others53 perfused control and vitamin-D-deficient parathyroidectomized, growing rats with PT H and studied urinary and serum calcium and phos­ phorus. There was a sharp contrast in the response of the control and D-deficient rats to PTH , with the latter group being unable to sustain the phosphaturia observed in the control rats. Harrison54 has reviewed in detail the recent literature con­ cerning this area. During the past three years, numerous reports have appeared on the influence o f P T H or para­ thyroid extract (P T E ) on a variety o f metabolic processes, tissues, subcellular particles, and so on. A recent book by Gaillard and others55 on the parathyroid gland contains 2 3 review articles by various authors.

Vitamin D and calcium metabolism Vitamin D deficiency in man and experimental animals results in abnormal bone and tooth forma­ tion. This can be prevented by vitamin D which maintains the serum (Ca++) X (HPO4 = ) product at a level which is supersaturated with respect to bone mineral. The vitamin increases the serum concentrations o f calcium and phosphate by medi­ ating increased absorption o f these ions from the intestine and by mobilizing miperal from the skel­ eton. Increased intestinal absprption appears to be due to a vitamin D stimulation o f active trans­ port of calcium. Results of more recent studies by Harrison and Harrison56 on the permeability of intestinal mu­ cosa to calcium support the concept o f active trans­ port since the intact intestinal mucosa o f the rat presented a diffusion barrier to calcium which was. lessened by vitamin D treatment o f the ani­ mals from which the intestines were obtained. In these latter studies, it was not possible to influence calcium transport by the addition of vitamin D in vitro to surviving intestinal loops arid the D effect could be obtained only by administration of the vitamin to rats some hours before the intestinal preparations were prepared. This time lag has also been observed by Norman57 58 in studies o f calcium absorption across the intestinal mucosa o f the chick. The rat intestine accumulated 1 1 % o f an oral dose o f 500 IU vitamin D 3-3H within four hours.59 The delayed effect of vitamin D in stimu­ lating calcium absorption indicates a turnover time o f 2 4 to 3 6 hours for the biochemical ma­ chinery associated with the effect. In further Van Reen: BIOCHEMISTRY OF CALCIFIED TISSUES ■ 1343

work ,57 data were presented demonstrating a di­ rect stimulating effect o f vitamin D on ribonucleic acid labeling by 3H-uridine, which indicates that the primary biochemical response o f intestinal mucosa cells to vitamin D is the initiation o f R N A and protein synthesis. In this regard, several investigators have demon­ strated that actinomycin D blocks the physiologi­ cal action o f vitamin D .60-63 Actinomycin D blocks deoxyribonucleic acid-directed R N A syn­ thesis and subsequent protein synthesis. There­ fore, the suggestion that vitamin D induces the syn­ thesis of a specific protein required for calcium transport is strengthened. Another suggestion has been that vitamin D interacts with nuclear mem­ branes and causes a change in calcium permea­ bility. The calcium is then described as the con­ trolling agent for the synthesis o f a calcium trans­ port protein.64 Whether vitamin D directly or cal­ cium elicits a response is not known, but evidence for a vitamin D-induced protein in chick intestinal mucosa has been found. Wasserman and Taylor 65 administered vitamin D 3 to rachitic chicks and in­ duced the formation o f a calcium-binding factor in intestinal mucosal tissue. The binding activity was associated with a protein fraction that could be destroyed by treatment with trypsin. The ap­ pearance o f the induced protein occurred simul­ taneously with increased absorption o f calcium. In further work ,66 the vitamin D-induced calcium binding activity was noted also in the duodenal mucosa o f the rat and squirrel monkey. The cal­ cium-binding protein has been separated from other proteins in the mucosa and purified 30-fold. The protein was detected in all segments of the small intestine and kidney of D 3-treated rachitic chicks but not in the colon, liver, and muscle. Further developments in this area will be eagerly awaited. Several other facets of vitamin D metabolism have been reported on but cannot be reviewed here. Neville and DeLuca 67 and Callow and others68 have investigated the synthesis and tissue distribution o f tritiated vitamin D. Also, the rat serum proteins that were found associated with vitamins D 3-3H and D 4-3H were studied by Rikkers and DeLuca .69

Collagen During the period covered by this review, con­ siderable work has been reported on collagen, the main protein o f connective tissues including den­ 1344 ■ JADA, Vol. 76, June 1968

tin. Only the highlights can be discussed. ■ Chemistry: It has been known for almost 20 years that the mammalian collagens have a char­ acteristically high glycine and proline and low tyrosine content. They alone contain hydroxyproline and hydroxylysine. One of the features of in­ tact collagen or tropocollagen (acid soluble) is the lack of free a-amino acid groups that would normally be at the end o f an amino acid chain, which indicates the blocking of these groups. Hormann and Joseph 70 have presented evidence that the a-amino groups of tropocollagen are blocked by acetyl groups. However, other investi­ gators71 have reported results which indicate that the terminal amino groups of insoluble collagens from hides are blocked by hexoses and therefore are not available to chemical reagents that detect amino groups. More definitive work on the car­ bohydrate component of collagen has been pre­ sented,72 which indicates that the majority of the carbohydrate from soluble collagen o f guinea pig skin is glucosyl-galactose and that it is attached to the 8 -hydroxyl group o f hydroxylysine. Collagens probably also contain some organic phosphorus. Glimcher and others73 administered injections of 32P phosphate intraperitoneally to rabbits and guinea pigs and then isolated the neu­ tral and acid-soluble collagens of skin and healing skin wounds and demonstrated organically-bound phosphorus in all preparations. They suggested that the phosphorus is bound to the carbohydrate found in the collagens. The findings o f phosphorus in collagens might prove of considerable signifi­ cance in understanding the mechanism o f calci­ fication since phosphates bind calcium strongly. Pine and Holland 74 have presented the concept that perhaps isoproteins of collagen exist in dif­ ferent human tissues. The suggestion was made as a result o f findings concerning the heterogeneity in the composition o f collagens. This would cor­ respond to the isozymes of lactic acid dehydro­ genase, and so on, which recently have been shown to exist. In this regard, Bornstein 75 also presented data which indicated that the primary structure of collagen is heterogeneous as a con­ sequence o f incomplete hydroxylation o f proline. ■ Structure: M any advances have been made in our knowledge of the structural features o f col­ lagen. There is considerable evidence that the col­ lagen macromolecule consists of three singlestranded polypeptide chains, each chain dis­ tinguished by differences in amino acid composi­

tion. Earlier, two o f the chains were thought to be similar, «i-type, and only a 2 thought to be dif­ ferent. Recently, however, Piez76 77 and Heidrich and Wyston78 have fractionated the <*i component of codfish skin and calf skin into an « 1 and a 3 component. In addition to the single-stranded components, dimers (^-components), trimers (y-components), and high molecular weight poly­ mers formed by intramolecular and intermolecular cross-linking of «-chains have been reported. The evidence indicates that the three chains are parallel in direction and extend the full length of the molecule in a helical configuration.79 In a series of studies, Bornstein and others80 and Bornstein and Piez81 have been able to determine the nature of the cross-linkages of soluble rat skin collagen. They have concluded that the intra­ molecular interchain cross-link is formed from the side chains of two lysyl residues on adjacent a chains. Only one specific lysyl residue on each « chain appears to be available for this reaction.82 Oxidative deamination o f the lysine moiety to an aldehydic group appears to be an intermediate step in the cross-link formation. Other forms o f cross-linking single chains are possible in lower forms of animals. McBride and Harrington83 have presented evidence for disul­ fide cross-linkages in the neutral salt soluble col­ lagen extracted from the cuticle of Ascaris. Simi­ larly, Blanquet and Lenhoff84 have studied the collagen-like protein present in the capsule and thread of a sea anemone and have reported di­ sulfide bonds. Little or no cystine has been de­ tected in mammalian collagens, but those from a variety of fishes show about 20 half-cystine resi­ dues per 1,000 amino acids. Thus, in some species, cross-linking other than through lysine residues is possible. The studies on cross-linkages in mammalian collagens have determined the possible mecha­ nism of toxicity in lathyrism, a disease primarily affecting connective tissues. Lathyrism in man has been reported in several countries and is re­ lated to the eating of large quantities of sweet peas of certain varieties. A similar disease can be produced in experimental animals by feeding ¡3aminopropionitrile or related nitriles. The inhi­ bition of cross-linking in collagen by lathyrogenic agents was demonstrated a number of years ago, but the exact mechanism was not shown. The work o f Bornstein and Piez81 mentioned previously indi­ cates that a single specific lysyl residue in each «chain of collagen is converted to an aldehyde be­ fore the cross-link is formed. These workers have

shown that this conversion to an aldehyde is blocked in lathyrism. Considerable evidence has also been found that lathyrogens inhibit crosslinking in elastin and that this is related to a de­ crease in aldehydic groups.85-88 Penicillamine, which has been suggested for the treatment of cystinuria and Wilson’s disease, has been shown to cause a condition in rats which is similar to lathyrism after prolonged use.89 90 ■ Synthesis: The synthesis of collagen has now been studied in a number of biological systems: the intact animal, skin, healing wounds, embryonic tissues, bone or cartilage cultures, bone-cell cul­ tures, and in cell-free preparations. In almost all studies, the formation o f collagen was detected by the presence o f hydroxyproline in protein ma­ terial. Firschein91 studied the incorporation of labeled hydroxyproline in various calcified tissues after L - 3H-proline was administered. He reported that the highest rate of collagen synthesis oc­ curred in the proximal end o f the tibia, followed by that of the mandibles, tibial shafts, and the calvaria, in decreasing order. Although some of these differences may be due to relative amounts of cellular material, collagen synthesis in various cell strains of fibroblastic origin varies consider­ ably.92 The results o f numerous studies on protein syn­ thesis have led to the recognition that synthesis oc­ curs on clusters o f ribosomes that are assembled on a messenger R N A coded for each particular protein. In several instances, there has been a re­ lationship between the size o f the protein mole­ cule being synthesized and the size o f the polyribosomal cluster involved. The formation of col­ lagen on polyribosomes from chicken eggs was studied by Manner and others,93 who concluded that collagen is synthesized on extremely large polyribosomes and that hydroxylation of proline occurred before peptide chain assembly was com­ plete. Goldberg and Green94 have studied colla­ gen synthesis in a cultured mouse fibroblast line and have shown that about 7 % of the protein being synthesized was collagen. In later studies,95 it was suggested that the synthesis o f collagen subunits (mol wt about 17,000) might occur on a structure o f five ribosomes. Correspondingly, synthesis of complete a chains was assigned to larger ribosome complexes. One o f the problems of collagen biosynthesis is related to the mechanism by which hydroxypro­ line is incorporated. Proline and not hydroxypro­ line has been shown to be the precursor o f the Van Reen: BIOCHEMISTRY OF CALCIFIED TISSUES ■ 1345

hydroxyproline residues present in collagen. Two general pathways have been proposed to explain the incorporation o f hydroxyproline. One sugges­ tion has been that “ activated” proline, probably in the form o f prolyl-sRNA, is hydroxylated be­ fore it is incorporated into peptides. The second pathway indicated is that proline residues are in­ corporated into microsomal peptide linkages, and subsequently, hydroxylation occurs. A t present, the evidence supports the latter theory.96-102 In recent studies, Lukens103 has studied the size of the substrate required in the cell-free hydroxyla­ tion reaction that converts peptide-bound proline residues to hydroxyproline residues. He has indi­ cated that the substrate is of a size comparable to the a component o f gelatin and does not require attachment to an s R N A molecule. The hydroxyla­ tion o f proline in synthetic polypeptides with purified protocollagen hydroxylase has been ac­ complished.104 Although the synthetic polypep­ tide was not as good a substrate as protocollagen, the observations substantiate the concept of poly­ peptide hydroxylation. Hydroxylysine is another component o f colla­ gen and lysine is an obligatory precursor o f the hydroxylysine. Prockop and others105 have sug­ gested that the hydroxylation of lysine probably occurs in the same polypeptide precursor of colla­ gen, which is a substrate for the hydroxylation of proline. In the presence of metal chelating agents, no collagen hydroxyproline is formed. A similar in­ hibition has been found in regard to hydroxyly­ sine formation, which indicates that the mecha­ nisms involved in the two hydroxylations are es­ sentially the same in regard to metal ion partici­ pation.106 Popenoe and others107 108 have investigated the mechanism of lysine hydroxylation and have shown that during the reaction, no hydrogen atom other than the one replaced by a hydroxyl group becomes exchangeable, thus suggesting that there is a direct addition of an oxygen atom to lysine carbon 5. ■ Collagen and vitam in C: There is consider­ able evidence that ascorbic acid is necessary for the accumulation o f collagen in connective tissues. Studies in vivo have been primarily with guinea pigs since this mammal requires dietary ascorbic acid, whereas most other experimental species are able to synthesize the vitamin. One o f the early suggestions concerning the etiology o f vitamin C-deficiency syndrome was 1346 ■ JADA, Vol. 76, June 1968

that the polymerization of collagen into fibrils was defective. Recently, however, Bentley and Jackson,109 working with skin preparations from ascorbic acid-deficient guinea pigs, have con­ cluded that although deficiency greatly impairs collagen biosynthesis, it does not affect the aggre­ gation o f newly formed protein or increase the catabolism o f the aggregates thus formed. A number of years ago, it was suggested that ascorbic acid may exert an effect on biological hydroxylation reactions by participating in the transfer o f electrons to oxygen. More evidence has been accumulated to support this idea. Barnes and others110 have indicated that in scurvy there is an impairment in the hydroxylation o f lysine to collagen hydroxylysine in the tendon and skin o f guinea pigs. Tissue culture work has also implicated ascor­ bic acid in the synthesis o f collagen. Deletion of ascorbic acid in the medium for cultures o f chick tibias reduced collagen synthesis after two days.111 Findings o f further investigations of this system indicated that ascorbic acid exerts a direct action on collagen synthesis by stimulating the hydroxyla­ tion o f peptide-bound proline.112 This latter sug­ gestion has been made also by Peck and others113 as a result o f studies on tissue culture o f bone cells from rat calvaria.

Fluoride and calcified tissues Research on fluoride continues to be carried out by a number of investigators in dental science. The role of fluoride in preventing dental caries and its possible action in preventing osteoporosis are the two main subjects of intensive interest. The effect of fluoride in reducing the occurrence o f dental caries in man and experimental species is so well documented that only brief mention of this area will be made. The maternal transfer o f fluoride to offspring was studied in rats by Zipkin and Babeaux.114 The effects o f prenatal exposure to fluoridation on dental caries was investigated in man by Horowitz and Heifetz115 and in rats by Babeaux and Zipkin,116 with essentially negative results in both studies. The site of fluoride uptake in enamel studied by Little and others,117 and the influence o f various other materials on fluoride uptake by Goodman118 shed more light on prob­ lems of fluoride metabolism. The chemistry of caries inhibition is discussed by Brudevold and others119 in an excellent review of the current status of our knowledge.

During the past three years, considerable data have appeared in the literature indicating that a lack o f fluoride over a period o f years may be an important etiological factor in osteoporosis. Bern­ stein and others120 compared radiographs of the lateral lumbar area o f the spines of 300 persons who lived in an area where the fluoride content o f the water supply was high with those of 7 1 5 persons who lived in an area where the water fluoride was low. Evidence o f osteoporosis was considerably higher in the low-fluoride area, es­ pecially in women. A few reports have appeared concerning the treatment o f osteoporosis with sodium fluoride. A slight positive calcium balance and recalcification o f the skeleton have been noted. Encouraging but not overly enthusiastic re­ sults o f case studies continue to be reported121-124 which indicate that investigations in this area should be intensified. Saville125 studied the effect o f fluoride in the drinking water on bone fragility and skeletal cal­ cium in the rat. He reported that the calcium con­ tent of the axial skeleton and the forelimb, which is related to body weight, increased in rats given water containing 20 ppm fluoride. Steier and others126 studied the healing o f fractured bones of mature rats over a period of 30 days. A syn­ ergistic healing effect o f vitamin D and 50 ppm fluoride in the drinking water was observed. A recent review of the relationship o f fluoride deficiency and osteoporosis was made by Hegsted.127

T h e opinions in th is paper are those of th e author and do not necessarily reflect th e view s o f th e Navy D epartm ent or th e naval service at large. T h is investigation was supported by research task no. M R 0 0 5 -0 6 -0 0 0 7 from th e bureau of m edicine and surgery, Navy D epartm ent. Doctor Van Reen’s address is Naval M edical Research In­ stitu te, N ational Naval M edical Center, Bethesda, Md.

1. Rasmussen, H. Parathyroid horm one— nature and m echanism of action. Am er J M ed 3 0 :1 1 2 , 1961. 2. Copp, D.H.; Davidson, A.G.F., and Cheney, B.A. Evi­ dence for a new parathyroid horm one which lowers blood calciu m . Proc Can Fed Biol Soc 4 :1 7 , 1961. 3. Kumar, M.A.; Foster, G.V., and M acIn tyre, I. Further evidence fo r calcito nin— a rapid acting horm one which low­ ers plasm a calciu m . Lancet 2 :4 8 0 , 1963. 4. Copp, D .H ., and Henze, K.G. Parathyroid origin of cal­ cito n in — evidence from perfusion of sheep glands. Endocrin­ ology 75 :49, 1964. 5. Care, A.D., and Keynes, W .M . T h e secretion o f c a lc it­ onin by th e parathyroid glands o f th e sheep. J Endocr 31: xxxi, 1965. 6 . Hirsch, P.F.; G authier, G.F., and Munson, P.L. Thyroid

hypocalcem ic p rin cipal and recu rrent laryngeal nerve injury as factors a ffe c tin g th e response to parathyroidectom y in rats. Endocrinology 7 3 :2 4 4 , 1963. 7. Hirsch, P.F., and M unson, P.L. H ypocalcem ic e ffe c t of th yroid extract in rats. Parm acologist 5:2 7 2 , 1963. 8 . Talmage, R.V.; N euenschwander, J., and Kraintz, L. Presence of th yrocalcito nin in rats. Fed P ro c 2 3 :A -6 1 8 ,1964. 9. Talm age, R.V.; N euenschwander, J., and K raintz, L. Evidence for th e existence of thyrocalcito nin in th e rat. En­ docrinology 7 6 :1 0 3 , 1965. 10. M a cIn tyre, I.; Foster, G.V., and Kumar, M.A. Thyroid origin o f calcito nin . In G aillard, P.J.; Talm age, R.V., and Budy, A .M . (eds.). T h e parathyroid glands; ultrastructure, secretion and fu nction . Chicago, U niversity o f Chicago Press, 1965, p 8 9 . 11. Foster, G.V., and others. Thyroid origin o f calcito nin . N atu re 2 0 2 :1 3 0 3 , 1964. 12. Gittes, R .F., and Irvin, G.L. Thyroid and parathyroid roles in hypercalcem ia: evidence fo r a th yrocalcito nin -releasing factor. S cien ce 14 8 :1 7 3 7 , 1965. 13. Toverud, S .U .; Gittes, R.F., and Munson, P.L. Thy­ roid and parathyroid glands in recovery from hypercalcem ia. Fed Proc 2 5 :3 4 7 , 1966. 14. Foster, G.V. Th yrocalcitonin: fa ilu re to dem onstrate a parathyroid releasing factor. N atu re 2 1 1 :1 3 1 9 , 1966. 15. Aliapoulios, M.A.; Voelkel, E.F., and Munson, P.L. Assay of human th yroid glands fo r thyrocalcito nin activity. J C lin Endocr 2 6 :8 9 7 , 1966. 16. W illiam s, G.A., and others. Evidence fo r thyrocal­ citon in in man. Proc Soc Exp Biol M ed 1 2 2 :1 2 7 3 , 1966. 17. Foster, G.V., and others. E ffect o f thyrocalcito nin in man. Lancet 1 :107 Jan 15, 1966. 18. Bell, N .H .; B arrett, R.J., and Patterson, R. E ffects of porcine th yrocalcito nin on serum calcium and phosphorus and m agnesium in th e m onkey and in m an. Proc Soc Exp _ Biol Med 12 3 :1 1 4 , 1966. 19. M ilhau d, G., and others. Existence et a c tiv ité de la thyrocalcito nin e chez l’homm e. C om pt Rend Acad Sci Paris 2 6 1 :4 5 1 3 , 1965. 20. M ilhau d, G., and Job, J.-C. Thyrocalcitonin: e ffe c t on idiopathic hypercalcem ia. S cience 15 4 :7 9 4 , 1966. 21. Foster, G.V., and others. Som e chem ical and physical properties o f ca lcito n in . Biochem J 9 4 :2 5 P 1965. 22. Tenenhouse, A.; Arnaud, C., and Rasmussen, H. The isolation and ch aracterizatio n of thyrocalcitonin. Proc N at Acad Sci USA 5 3 :8 1 8 , 1965. 23. Gudm undsson, T.V.; M acIntyre, I., and Solim an, H.A. T h e isolation o f thyrocalcitonin and a study o f its effects in th e rat. Proc Roy Soc [B io l] 1 6 4:46 0, 1966. 24. O’Riordan, J.L .H ., and others. Th yrocalcitonin: u ltra ­ centrifu gation in gradients o f sucrose. S cience 1 5 4:88 5, 1966. 25. Potts, J.T., Jr., and others. P urificatio n o f porcine thyrocalcitonin. Proc N at Acad Sci USA 5 8 :3 2 8 , 1967. 26. Tashjian, A .H ., Jr., and Munson, P.L. A ntibodies to porcine thyrocalcitonin: effects on th e hypocalcem ic ac­ tiv ity of calf, rat, and m onkey extracts. Endocrinology 77: 520, 1965. 27. Bauer, W.C., and Teitelbau m , S.L. Thyrocalcitonin ac tivity of p a rtic u la te fraction s of th e th yroid gland. Lab Invest 15:323 , 1966. 28. Hargis, G.K.; and others. Thyrocalcitonin: cytological localization by im m unofluorescence. S cien ce 152:73 , 1966. 29. Cooper, G.W., and Tashjian, A.H ., Jr. S u b cellu lar lo­ calization o f thyrocalcito nin . Endocrinology 7 9 :8 1 9 , 1966. 3 0 . Pearse, A.G.E. T h e cytochem istry o f th e th yroid C cells and th e ir relationship to calcito nin . Proc Roy Soc 164: 4 7 8 , 1966. 30A. Pearse, A.G.E., and Carvalheire, A.F. Cytochem ical evidence fo r an ultim obranchial origin o f rodent th yroid C cells. N atu re 2 1 4 :9 2 9 , 1967. 3 0 B . Copp, D.H. Hormonal control of hypercalcem ia.

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Am er J M ed 4 3 :6 4 8 , 1967. 3 1 . Hirsch, P.F.; Voelkel, E.F., and Munson, P.L. Thyro­ calcito nin : hypocalcem ic-hypophosphatem ic p rin cip le of the th yroid gland. S cience 14 6 :4 1 2 , 1964. 32 . Aliapoulios, M.A., and Munson, P.L. Thyrocalcitonin. Surg Forum 16:55, 1965. 33 . Aliapoulios, M.A.; Goldhaber, P., and Munson, P.L. Th yrocalcitonin in hibition o f bone resorption induced by parathyroid h o rm o n ein tiss u ec u ltu re . S cience 1 5 1 :3 3 0 ,1 9 6 6 . 34. Wase, A.W., and others. Som e effects of thyrocal­ cito n in on th e calcium m etabolism o f th e rat. Endocrinology 7 9 :6 8 7 , 1966. 35 . Foster, G.V., and others. E ffe ct o f thyrocalcito nin on bone. Lancet, 1:1 4 2 8 Dec 31, 1966. 36 . Hirsch, P.F. Thyrocalcitonin in hibition of bone re­ sorption induced by parathyroid ex tra ct in thyroparathyroidectom ized rats. Endocrinology 8 0 :5 3 9 , 1967. 3 7 . Potts, J.T., Jr., and Aurbach, G.D. T h e chem istry of parathyroid horm one. In Gaillard, P.J.; Talm age, R.V., and Budy, A. (eds.). Th e parathyroid glands; ultrastructure, se­ cretio n and fu n ctio n . Chicago, U niversity o f Chicago Press, 19 65, p 53. 38. Potts, J.T., Jr., and others. S tructural basis o f biolog­ ical and im m unological activity of parathyroid hormone. Proc N at Acad Sci 5 4 :1 7 4 3 , 1965. 39. Causton, A.; C horlton, B., and Rose, G.A. An im ­ proved assay for parathyroid hormone, observing th e rise of serum calcium in thyroparathyroidectom ized rats. J En­ docr 33:1, 1965. 4 0 . Treacher, R.J. Bioassay o f parathyroid hormone in rats by determ ination o f plasm a calcium , urinary 32P excre­ tion and serum alk a lin e phosphatase. J Endoc 35 :2 2 9 , 1966. 4 1 . Bethune, J.E.; Inoue, H., and Turpin, R.A. A bioassay for parathyroid horm one in m ice. Endocrinology 8 1 :6 7 ,1 9 6 7 . 4 2 . Tashjian, A .H ., Jr. Effects o f parathyroidectom y and cautery of th e thyroid gland on th e plasma calciu m level of rats w ith au totransp lan ted parathyroid glands. Endocrinology 7 8 :1 1 4 4 , 1966. 4 3 . Schlueter, R.J., and Caldwell, A.L., Jr. Thyrocal­ cito n in : param eters o f bio-assay. Endocrinology 81 :854 , 1967. 4 4 . Aurbach, G.D.; Houston, B.A., and Potts, J.T., Jr. Control o f parathyroid horm one o f energy u tilizatio n in m ito­ chondria. In G aillard, P.J.; Talm age, R.V., and Budy, A. (eds.). Th e parathyroid glands; ultrastructure, secretion and fu n ctio n . Chicago, U niversity of Chicago Press, 1965, p 197. 4 5 . Shah, B.G., and Draper, H .H . Depression of calcium absorption in parathyroidectom ized rats. Am er J Physiol 2 1 1 :9 6 3 , 1966. 4 6 . Borle, A.B., and Neum an, W .F. E ffects o f parathyroid horm one on HeLa cell cultures. J Cell Biol 2 4 :3 1 6 , 1965. 4 7 . Tenenhouse, A.; M eier, R., and Rasmussen, H. Para­ th yroid horm one and bone m obilization in vitro. J Biol Chem 2 4 1 :1 3 1 4 , 1966. 4 8 . Raisz, L.G. Bone resorption in tissue cu ltu re. Factors in fluencin g th e response to parathyroid horm one. J Clin Invest 4 4 :1 0 3 , 1965. 4 9 . Harrison, H.C.; Harrison, H.E., and Park, E.A. V ita­ m in D and c itra te m etabolism : e ffe c t of vitam in D in rats fed diets adequate in both calcium and phosphorus. Amer J Physiol 19 2:43 2, 1958. 50. Toverud, S.U . The effect o f parathyroid horm one and vitam in D on serum calcium in rats. Acta Physiol Scand 6 2 :3 9 1 , 1964. 51 . Au, W .Y.W., and Raisz, L.G. E ffect o f vitam in D and d ietary calcium on parathyroid activity. Am er J Physiol 2 0 9 :6 3 7 , 1965. 52 . Ney, R.L., and others. Action o f parathyroid hor­ m one in th e vitam in -D -d eficien t dog. J Clin Invest 4 4 :2 0 0 3 , 1965. 53 . Arnaud, C.; Rasmussen, H., and Anast, C. Further studies on th e in terrelatio nship between parathyroid hor­

1348 ■ JADA, Vol. 76, June 1968

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