THE EFFECTS OF MODERATE DOSES OF VIOSTEROL AND OF PARATHYROID EXTRACT UPON RATS*

BY AGNES FAY MORGAN, LOUISE KIMMEL, RACHEL THOMAS, AND ZDENKA SAMISCH

(From the Laboratory of Household Science, University of California, Berkeley)

(Received for publication, April 4, 1934)

Much work has been done in recent years upon the toxic effects of overdoses of viosterol and of parathyroid extract in the effort to discern the mode of action of these potent substances. Shelling and Asher (18), Hess, Benjamin, and Gross (6), Jones and Robson (9), Harris and Innes (5), and others have tried the effects of vary- ing the calcium and phosphorus contents of the experimental diets and have found that the pathological manifestations of hypervita- minosis are more obvious and severe usually on diets which pro- vide generous amounts of these elements, although even calcium- and phosphorus-free diets may allow the symptoms to appear if a large enough excess of viosterol is used (6). The clinical problem, however, is most concerned with the limits of toxicity of this sub- stance as well as that of parathyroid extract as they ooour in diets of normal calcium and phosphorus content and ratio. It seemed worth while therefore to make a detailed study of the bones, soft tissues, and serum of rats fed such a diet and given varying and moderately excessive doses of viosterol and parathyroid extract both together and separately.

The diet used had the composition shown in Table I. This diet allows normal growth either with or without viosterol in therapeu- tic amounts. The rats which were used in the present study were placed on the diet at 28 days of age and during-the following 6 to 8 weeks gained 18 gm. a week when given viosterol, 3 D or 10 D, and

* A brief report of this study was presented before the meeting of the American Society of Biological Chemists at Cincinnati, April 12, 1933 (J. Biol. Chem., 109, lxxi (1933); Proc. Am. Sot. Biol. Chem., 8, lxxi (1933)).

531

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532 Viosterol and Parathyroid Extract

15 gm. a week without such additions. The calcium and phos- phorus content and ratio, 0.62 and 0.40 per cent and 1.55 respec- tively, are close to the range usually suggested as optimal for rats. The Ca: P ratio is also similar to that of cow’s milk, a food making up much of the diet of infants treated with viosterol. Six groups of animals were used altogether, four for comparison of effects of varying doses of viosterol’ and two for observation of similar re-

TABLE I Composition of Diet

Wheat gluten ................... Alcohol-extracted egg albumin . . Agar ............................ crisco .......................... Corn-starch. .................... Salt mixture * ...................

. . . . . . . . .

. . .

.......... .......... .......... .......... .......... ..........

per cent ............... 10 ............... 10 ............... 2 ............... 15 ............... 59 ............... 4

Calcium. . . . . . . . . . . . . . . . . . Phosphorus _ . . . . . Ca: P ratio, 1.55

................................ 0.62

................................ 0.40

Supplement (these were fed as sources of vitamins B, G, and A) Yeast Vitamine-Harris.. . . . . . 50 Carotene in ethyl laurate. . . . . . . . . . . . . . . . 0.006

* The composition of the salt mixture is as follows :

K~HPOI. .............. 18.5 KI ................... 0.005 Ca(H2P0& ........... 51.2 NaF ................. 0.062 MgSO,. ............... 4.5 MnS04 ............... 0.019 NaCl ................. 20.4 KAl(SO& ........... 0.006 Fe citrate ............ 2.0 Ca lactate. .......... 89.7

.sults of the injection of parathyroid extract.2 The procedure consisted of feeding the diet to the rats beginning at the age of 28 days for 5 to 8 weeks, usually 6 weeks. Calcium and phosphorus balance studies were made on three of the groups during most of

1 We are indebted to Mead Johnson and Company through Dr. C. E. Bills for the viosterol 10,000 X which was used in these experiments.

2 We are indebted to E. R. Squibb and Sons for the parathyroid extract used in this part of the study.

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Morgan, Kimmel, Thomas, and Samisch 533

this time. At the end of the feeding period the animals were killed and blood was removed without anticoagulant from the beating heart. Calcium and inorganic phosphorus were deter- mined in the serum. Kidneys and usually hearts as well as femurs and tibias were dissected out, dried, and analyzed for ash, calcium, and phosphorus. The bones were extracted thoroughly with alcohol and ether before being incinerated and were divided accord- ing to the suggestion of Hess, Berliner, and Weinstock (7) into portions consisting of metaphyses, or the spongy epiphyseal por- tion, and shafts, or diaphyses. These parts were ashed separately. However, only the epiphyseal portion of the tibias was examined. The line test technique was applied in most cases to one of the tibias and a record made of the amount of spongiosa seen under the binocular microscope.

Calcium and Phosphorus Retentions-As may be seen in Table II, the retention of calcium on this diet is favorably affected by viosterol fed at the level of 10 D. Throughout our study we have used the Steenbock vitamin D unit to designate the amount of irradiated ergosterol given. The commercial 250 D preparation used at first was tested and found to contain the full potency in- dicated. The first three groups of rats used were divided into two parts, one-half receiving no addition of viosterol and one-half a corn oil dilution of the viosterol equivalent to 10 times the thera- peutic dose of cod liver oil.

Eight balance studies of 1 or 2 weeks duration each were made upon these six groups, during two of which 80 units of parathyroid extract were injected in four doses into each of the rats. In all but one of the balance studies, which were uncomplicated by para- thyroid treatment, the calcium retentions of the viosterol-fed rats were considerably greater, ranging from 26 to 39 per cent more than were those of the group given no viosterol. The phos- phorus retentions during these periods were usually but not in- variably better in the viosterol-fed group. This more specific effect upon calcium retention is reflected in the larger Ca: P ratio of retentions of the viosterol-fed animals.

During Periods 2 and 4 for Group II-b, in which 80 units of parathyroid extract were administered, the excretion of both cal- cium and phosphorus was increased in both viosterol-fed animals and those not fed viosterol. In Period 2, moreover, the intakes

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534 Viosterol and Parathyroid Extract

of these elements were only slightly depressed below those seen in Periods 1 and 3 for the same animals without parathyroid treat- ment. During Period 2 the retention of calcium by the viosterol- fed rats was‘reduced by about 53 per cent from that of the com- parable Periods 1 and 3 while the calcium retention of the rats without viosterol was reduced by 35 per cent. The oorrespond-

TABLE II

Calcium and Phosphorus Retentions of Rats with and without Viosterol and Parathyroid Extract (My. per Rat per Week)

Groo”p 2 rats

__ --

I 6 8

II-a 6 6 6 8 6 7

II-b 5 7 3 8 3 8 7 8

zays

49

28

42

49

28

42

49

63

eriod 1 I NO.

bra tion

&Is

14

14

7

14

14

7

14

7

-___ Total P&l%-

Vias- ted E%

ex- tract

-. I Calcium P1108ph0rus

-

ntekc , ( IIt-

Put

- T- Re-

I ;tVl- 11 1 ;ion

-

ltakt

units 10 D None

293 116 177 190 244 156 88 163

10 D 229 102 127 145 None 229 118 111 145 10 D 227 130 97 142 None 228 158 70 144 10 D 250 149 101 157 None 248 127 121 157

10 D 244 105 139 158 None 243 142 101 158 10 D 80 213 148 65 140 None 80 220 149 71 144 10 D 239 102 137 155 None 240 123 117 156 10 D 80 120 89 31 83 None 80 167 121 46 111

-

- jut- Put

- R.3 ;*ll- 1 ;ion

78 112 94 69

65 80 58 87 68 74 82 62 87 70 76 81

60 98 76 82 95 45 96 48 76 79 77 79 71 12 78 33

ing reductions in phosphorus retention during Period 2 from the averages for Periods 1 and 3 were 49 and 40 per cent. The influ- ence of the viosterol, if any exists, on the losses occasioned by parathyroid treatment is to increase such losses.

The changes in the retentions due to parathyroid treatment in Period 4 of Group II-b are less easily assessed because of the greater lowering of food intake due to the effect of the parathyroid extract.

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Morgan, Kimmel, Thomas, and Samisch 535

It is obvious, however, that the retentions of both calcium and phosphorus are smaller in proportion to intakes in this period than in the non-parathyroid Periods 1 and 3. Thus in Periods 1 and 3 of Group II-b the percentage of calcium intake retained is 41 to 57 and of phosphorus is 51 to 62. In Period 2 it is 30 to 33 per cent of both calcium and phosphorus intakes and in Period 4, 14 to 29 per cent.

Thus the primary effect of t,he viosterol appears to be upon cal- cium retention, perhaps through a decrease in the ease of calcium excretion into the intestine but without so consistent an effect upon phosphorus output. The parathyroid extract on the other hand appears to act both upon phosphorus and calcium outputs, increas- ing them in both cases. The calcium output is increased more significantly by the parathyroid treatment in the case of the rats receiving viosterol than in those without it. This raises some question as to the validity of the hypothesis t)hat the hormone a&s only by stripping calcium phosphate from the bones. If circulating calcium is less easily excreted in the presence of vita- min D as is often predicated, a less noticeable increase in output should be seen following the administration of the extract to the rats receiving viosterol than to those which did not receive it. But this is apparently not the case.

The study of Pugsley (16) upon calcium and phosphorus output as affected by parathyroid extract was not parallel with ours, since he used adult rats fed a low calcium diet, 0.266 per cent phosphorus, 0.025 per cent calcium, of very low Ca:P ratio, and obtained in consequence the typical immunity or gradual failure of response to the hormone which we have found (15) associated only with bhe low calcium diet. He noted an increase at first in phosphorus and calcium excretion in the parathyroid-treated ani- mals, followed by a decrease in most cases, but made no attempt at calculating gross retentions.

Serum Calcium and Phosphorus-The anticipated larger increase in serum calcium due to parathyroid extract, injection in the pres- ence of vitamin D as compared with the condition in its absence, is actually seen in the serum of our rats which also received vios- terol. Table III shows that the serum phosphate is little changed by this treatment in any of the groups on this diet, but serum cal- cium is more easily raised by parathyroid extract in the groups

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536 Viosterol and Parathyroid Extract

receiving 10 D than in those without it. Thus 60 units of the extract raised the serum calcium of the rats fed viosteroll0 D from 12.0 to 15.5 mg. per 100 cc., but had no effect on the serum cal- cium of the rats without viosterol. However, 160 units raised both serum calcium and phosphorus of the latter group to the high levels of 28.7 and 14.3 mg. per 100 cc. respectively. The same amount of the hormone administered to rats given viosterol 10 D was fatal in nearly all cases and the blood was so dehydrated and viscous as to prove impossible to analyze. Thus the effects of

TABLE III

Serum Calcium and Phosphorus of Rats 63 to 67 Days Old As Affected by Viosterol and Parathyroid Extract

No. of groups

8 24 4 17 1 6 4 11 5 19 7 22 3 6 2 8 2 8 5 18 3 9 3 8

Viosterol

None “ “ ‘I

3D 10 D 10 D 10 D

500D 1COOD 15OOD 2000D

‘1 MS1 parathy- mid extract

units

160 60

160 60

-

I

Total Ca Inorganio P

ql. per 100 cc. 10. per 100 cc. .Ymurn *emb?n 12.0 11.0 12.2 8.9 28.7 14.3 11.6 11.0 13.3 9.4 12.0 10.1

15.5 11.1 13.6 9.0 15.7 12.8 14.5 12.0 17.2 12.2

-

the vitamin and the hormone appear to be similar and cumulative. The less notable changes in serum inorganic P may be due to the greater ease with which this substance is excreted by way of the kidneys. This was not verified in our study, however, because no separation of output in urine and feces was attempted in the bal- ance experiments.

The larger doses of viosterol were given as dilutions of the vios- terol 10,000 X supplied by Dr. C. E. Bills. This was taken to be equivalent to 30,000 D in Steenbock units. Serum analyses were made on rats given viosterol500 D, 1000 D, 1500 D, and 2000 D. As shown in Table III a significant rise in calcium is produced at

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Morgan, Kimmel, Thomas, and Samisch 537

1000 D, 1500 D, and ZOO0 D levels. This rise is of the order of that produced by 60 units of parathyroid extract plus viosterol 10 D.

Calcijication of Kidneys and Hearts-The ash, calcium, and phos- phorus contents of dried kidneys in all cases and dry blood-free hearts in some cases were determined. Table IV shows the strik- ing increase in calcium and phosphorus content of these organs produced by even apparently innocuous doses of parathyroid ex-

TABLE IV

Ash, Calcium, and Phosphorus Content (Measured in Per Cent of Dry Weight) of Kidney and Heart of Rats As Affected by Moderate Amounts o-f

Viosterol and Parathyroid Extract

No. of groupf

6 35 2 19 3 12 1 8 2 18 1 9 1 8 1 2 1 3 2 6 5 13 1 7 3 9

r0. 01 rats

dUY8 units

68 None 64 3D 70 10 D 64 500 D 64 1000 D 64 1500 D 64 2000 D

4000 D 70 10 D 160 60 10 D SO-lo( 70 10 D 60 70 None 160 70 “ 60

-

_-

)

-

Kidney Heart

Ash Ca P Ca:P Ash Ca P Ca:P -- ---_

4.3 0.08 0.87 0.09 4.4 0.050.590.08 4.5 0.08 0.94 0.08 4.2 0.05 0.82 0.06 4.5 0.060.490.12 4.4 0.10 1.02 0.09 5.3 0.13 1.13 0.11 4.9 0.19 1.10 0.18 6.0 0.32 1.25 0.25

7.64 5.29 1.44 4.53 2.60 1.36 1.100.601.83 5.16 2.81 1.83 0.680.800.85

10.1 3.09 2.14 1.44 5.4 0.450.920.49 5.58 2.74 2.00 1.150.67 1.71

9.1 1.91 1.87 1.02 5.0 0.080.590.13

tract. In only one group, that of the non-viosterol-fed rats given 60 units, was a normal content found in the hearts and in no cases in the kidneys. On the other hand moderate excess of viosterol alone produces considerably less mineralization of the kidneys, although from 1000 D up abnormal amounts of both calcium and phosphorus were present. In the case of two rats which by mis- take were given viosterol4000 D for 1 week and which died of this overdosage, very high ash content of the kidneys was found. The six rats given viosterol 10 D and 80 or 100 units of parathyroid extract also died of overdosage and these are seen to have also

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538 Viosterol and Parathyroid Extract

exceedingly high calcium and phosphorus content of kidneys. Apparently surviving animals are able to demineralize these organs to some degree, and death from overdosage either of the extract or the viosterol may be due to renal failure from this calcification. Attention might well be paid to this possibility in the clinical use of both parathyroid extract and viosterol.

The far greater deposition of these elements in the kidneys of the parathyroid-treated animals as compared with those receiving moderate excesses of viosterol is puzzling in view of the fact that the serum values for calcium and phosphorus were even greater in the latter in some cases than in the former. The partition of the serum calcium between diffusible and non-diffusible fractions may be different in the two groups, but no clear demonstration of this is as yet available.

This effect of the parathyroid extract on the kidneys was ob- served by Hueper (8) and has been suggested as the probable cause of death from overdosage. Light, Miller, and Frey (12) offer data on the kidney and heart ash of their animals poisoned by excessive doses of viosterol which are difficult to interpret and apparently erroneous since the calcium and phosphorus content and ratio of the ashes are quite unlike any others reported in the literature. Smith and Elvove (19) found calcium contents in the kidneys of rabbits given 2.5 and 10 mg. of irradiated ergosterol daily, which are of much the same order as those seen in our parathyroid- treated rats. Brown and Shohl (3) noted metastatic calcification in rats beginning at the 5000 D level and Shelling and Asher (18) at 1250 D in animals on a stock diet quite like ours in calcium and phosphorus content. Actual analyses are offered only by Light, Miller, and Frey and by Smith and Elvove.

Composition of Bones-The separate analyses oi the metaphyses and diaphyses of the femurs revealed certain differences not dis- cernible by ashing of the whole bone. The spongiosa-containing portion of the tibias was also examined and this was found to be practically the same in ash, calcium, and phosphorus content as the corresponding part of t-he femurs. As seen in Table V little difference exists in the ash per cent of extracted tibias, metaphyses, and shafts of rats given this normal diet and no viosterol or doses at the levels of 3 D and 10 D. The average ash values are 49.1 for the tibias, 49.9 for the metaphyses, and 63.5 for the shafts.

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Morgan, Kimmel, Thomas, and Samisch 539

There is no significant difference from these averages exhibited by any of the groups on these three regimes. The group given viosterol500 D appears to have a definite hypercalcification, how- ever, the metaphyses being 52.7 per cent ash, although the shafts are about like the others, that is 64.1 per cent ash. The groups given viosterol 1000 D, 1500 D, and 2000 D show significant and progressive lowering of the ash of both metaphyses and shafts in about the same amounts.

The animals given 60 to 160 units of parathyroid extract with no viosterol exhibited no effect of the treatment in the bones, but

TABLE V

Ash Content of Fractions of Bones of Rats, 6.J to 84 Days Old, As Affected by Viosterol and Parathyroid Extract

No. of &FJUPS

5 2 2 3 4 1 2 1 1

Total Ash in extracted bones No. of rats Viosterol pa:~$y-

extract Tibia M$;f&;ia “fE2’

Whole femur

____~ units per cent per cent per cent per cent

42 47.9 49.8 i 0.363.4 f 0.2 57.1 18 6@-160 47.1 48.2 f 0.564.0 f 0.3 56.8 19 3D 51.2 49.5 f 0.363.1 f 0.4 20 10 D 48.3 50.5 f 1.264.1 f 0.4 57.4 23 10 D 60-160 45.1 46.9 f 0.563.5 f 0.3 56.7

8 500 D 50.5 52.7 f 0.464.1 f 0.2 18 1000 D 47.2 46.6 i 0.260.0 f 0.2

9 1500 D 48.1 45.9 f 0.661.9 i 0.3 8 2000 D 44.1 44.5 f 0.457.8 f 0.3

those which received the same amounts of hormone along with viosteroll0 D had a distinct lowering of the metaphysis ash of the same order as that seen in the rats given viosterol 1000 D. How- ever, no lowering of ash content of the shafts was seen. It is possible that larger doses or a longer treatment with the parathy- roid extract might produce a demineraliaation of the shafts. It is interesting, however, to note the rapid depletion of the trabecular ash produced without thinning of the cortex by the extract as com- pared with similar losses due to 1000 D viosterol dosage, for ex- ample, which was accompanied by definite demineralization of the shafts.

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540 Viosterol and Parathyroid Extract

Calcium and phosphorus determinations were made upon the ash of most of the bone fractions examined. Except for certain irregularities in the proportions of these elements in the ash of the tibias, almost constant figures were found. Ash of metaphyses and shafts of the femurs in all cases contained 36.8 to 39.8 per cent calcium and 16.3 to 18.6 per cent phosphorus, the means being 38.0 and 17.6. These are close to the theoretical figures, 38.9 and 17.2, calculated from the dahllite formula for bone, CaC03. 2Ca3- (PO& Greater variability is seen in the corresponding propor- tions in the tibias. Whatever the nature of the ash loss in the bones of animals given excess viosterol and parathyroid extract, it does not involve any notable change in the proportions of cal- cium and phosphorus. This confirms a number of previous ob- servations, among which the x-ray studies of Taylor and Sheard (20) and Roseberry, Hastings, and Morse (17) may be mentioned as indicating a definite crystalline structure of bone. Dislodge- ment of both carbonate and phosphate simultaneously must occur in the dissolution of bone brought about by both the excess vios- terol and parathyroid extract.

Appearance of Cut Bones-It has been noted previously (13) that this normal diet without vitamin D produces bones of normal strength but unusually rich in well calcified spongiosa. Ash analyses do not indicate that these bones are hypercalcified, al- though the metaphyses often appear richer in ash and in calcium than do those of similar animals fed optimal amounts of vita.min D. The orderly and rather scanty trabecuke shown by rats given viosterol are depleted in the bone of the parathyroid-treated animals as are the more profuse deposits seen in rats without vita- min D when similarly treated.

In Fig. 1 are shown illustrative cut tibias treated with silver nitrate in order to darken the calcified areas. These are from three rats fed the normal diet, one without vitamin D, one with viostero13 D, and one with viosterol 1000 D. The heavy trabec- ulae characteristic of no vitamin D are clearly evident, the fine reguIar calcium line of the bone of animals receiving normal doses of vitamin D, and an irregular and thinning deposit in the bone of those receiving viosterol 1000 D. Harris and Innes (5) have described as due to excess viosterol a depletion of the hypercalci- tied spongiosa beginning at the epiphyseal growth cartilage and

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Morgan, Kimmel, Thomas, and Samisch 541

moving toward the marrow cavity. We have seen but little in- termediary spongiosa calcification even in the group receiving viosterol500 D and cannot interpret the appearance of the bones of animals receiving viosterol 1000 D and viosterol 2000 D as representing the progressive depletion of such deposits. The break in the center of the growth cartilage in these bones is char- acteristic and has been described also by Giittsche and Kellner (4). The second’line of trabecuhe apparently receding from the epiphyseal growth area may represent merely the partly completed

FIG. 1. The appearance of cut tibias of rats 65 days old, fed a normal diet, (a) from a rat which received no vitamin D, (b) from a rat which re- ceived viostero13 D, and (c) from a rat which received viosterol 1000 D.

process of resorption of bony material both from the epiphyseal border and from the cortex of the shaft. Our ash figures seem to indicate that both parts of the bone are simultaneously and equally decalcified by excess vitamin D. The apparently clean removal of trabeculze at the growth cartilage which we have seen in these and previous studies (13) of parathyroid-treated bones may mean that moderate doses of the hormone lirst cause resorption of the trabecular deposit and perhaps only in excessive amounts attack the cortex. This is indicated by our figures for ash (Table V).

Hess, Berliner, and Weinstock (7) found for eleven rats on nor-

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542 Viosterol and Parathyroid Extract

ma1 diet values of 63.8 per cent ash in the shafts and 51.2 in the metaphyses. These figures are very close to those shown by our normals. Brown and Shohl (3) saw a rise in total femur ash with viosterol 100 D and 1000 D but a fall from 5000 D on. Light, Miller, and Frey (12) noted a fall in femur ash beginning at 40 D. Jones and Robson (10) have recently described a similar fall in femur ash on 30,000 D. Bischoff (2) using dogs, Bauer, Aub, and Albright (1) using rats, and Taylor, Weld, Branion, and Kay (21) using fowls, found denser bones resulting from parathyroid treat- ment. But Lambie, Kermack, and Harvey (11) in rats, and Bauer, Aub, and Albright (1) in rabbits have observed thinning of trabeculse from the same treatment. It is possible that both these conditions may result even in the same animal if depletion of trabeculze be followed at first by deposition in the shaft of the calcium phosphate thus removed.

In any case, all of the effects noted by us as resulting from the parathyroid treatment are more obvious in those animals which also received the moderate dose of viosterol, 10 D, than in those which received no viosterol. Thus the calcium and phosphorus retentions were more reduced otithe same intake when parathyroid was given the viosterol-fed group, the serum calcium was more elevated in the cases which could be compared, the kidneys and hearts contained more calcium phosphate, the bone ash was more reduced in the viosterol-fed than in the non-viosterol-fed rats. Quite comparable effects have been observed in our laboratory previously on rats and on dogs (14).

If a distinction between the mode of action of these two agencies exists, the parathyroid hormone and vitamin D, it can only be discovered by seeking for clear differences in their effects. So far such differences have not been satisfactorily demonstrated. Such differences as we have here noted may be only matters of degree rather than of kind. The greater calcification of kidneys and hearts produced by the parathyroid extract with a given rise in serum calcium and phosphate as compared with that produced by excess viosterol, may possibly be achieved by the latter in larger concentrations. Indeed, the calcification of kidneys of our two rats which died of 4000 D viosterol appears to illustrate this fact.

The more specific depletion of trabeculs rather than of shafts

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Morgan, Kimmel, Thomas, and Samisch 543

which our parathyroid-treated rats show may possibly be dupli- cated by excess viosterol under other circumstances than those of our experiments.

SUMMARY

1. The retention of calcium and phosphorus was usually better in rats fed a diet of normal calcium and phosphorus content and ratio with viosterol 10 D than without it. When 80 units of para- thyroid extract were given each animal during 1 week the calcium and phosphorus retentions were lowered in both cases but more noticeably in the viosterol-fed group.

2. The serum calcium and inorganic phosphate were more in- creased by parathyroid extract in the rats which received viosterol 10 D than in those which did not and were raised in proportion to the amount of the extract given. Viosterol 1000 D and 1500 D had an effect on serum similar to that of viosterol 10 D with 60 units of parathyroid extract.

3. There was a progressive increase in ash, calcium, and phos- phorus content of kidneys with increasing doses of viosterol up to 2000 D and a similar but far greater increase in the parathyroid- treated animals. Kidney ash in the latter was raised 2 to 3 times the normal values.

4. The metaphysis or head of the femurs and tibias and also the shafts showed progressive loss of ash with increasing amounts of viosterol, 500 D to 2000 D, but the parathyroid treatment pro- duced little ash depletion, particularly in the shafts.

5. The cut tibias showed heavy trabecular deposit in the rats given no vitamin D, a fine clear line of calcium deposition at the epiphyseal margin in those given normal doses of vitamin D, an irregular thin deposit with break in cartilage and secondary deposit in the marrow in those given moderately excessive doses of vios- terol. The calcium and phosphorus content of all bone ashes remained within the normal range.

Thus viosterol and parathyroid extract have similar and additive effects upon serum, kidneys, and bones of animals fed the normal diet but the calcification of kidneys is more marked in the para- thyroid-treated and decalcification of bones more advanced in the viosterol-treated animals of similar serum composition.

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Viosterol and Parathyroid Extract

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Thomas and Zdenka SamischAgnes Fay Morgan, Louise Kimmel, Rachel

PARATHYROID EXTRACT UPON RATSOF VIOSTEROL AND OF

THE EFFECTS OF MODERATE DOSES

1934, 106:531-544.J. Biol. Chem. 

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