On the availability of phosphorus in bonemeal, bonemeal supplemented with red oxide of iron, and bone ash to white rats

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OndetstezJOort Journal of Feterinary Science and Animal lnd?I.Stry, Volume 23, :"hnnlJe1·.s_ 1 and 2, March, 1948.

1Printed in the Union of South Africa by the Government Printer, Pretoria.

In

Red On the Availability of Phosphorus

Bonemeal Supplemented with Iron, and Boneash to White Rats.

Bonemeal, Oxide of

By P. 0 . . f. LOU,V, Sedion of Biochemistry and Kutrition, Onderstepoort.

Acconliug- to <·onespondeace which commenced in the issue of the Fanuer':-; 'Veekly for 18th April, 19J4, good results "·ere obtained by feeding a lick containing 125 lb. salt, 25 lb. red oxide and 1 lb. copper sulphate to lactating cows in Southern Rhode::;ia.

. Although iron is frequently incorporated in phosphatic lic:rs, either a,;

uon sulphate or as iron oxide, the suggested lick would not he recommeiHled in the Fnion where phosphates ;ne in such short supply in the natural herbage. Furthermme, it iH doubtful whether continued good results can be expected with a lick ('ontaining 0·6 per cent. eopper sulphate instead of the generally recommen<led one containing only 0·] per cent. of this con- stituent.

The work 1·efen ed to has popularised the u::;e of 1·ed oxide in phosphate licks. After the treatment of animals fo1· internal parasites, it is not infre- quently found that they may benefit h,v the addition of a small amoullt of iron in the li<·k. Ho"·eve1·, the solubilit.v of reel oxide is relatively lo"·,

<:on:-;equentl,v OoYemment Kotice No. 1E'J75 of August, 1943, specifies that an? phosphate-salt-iron oxide lick offered for -~ale should contain a minimum of 1:1 per ('ent. of iron oxide. 'l'he que~:;tiou arise:; as to what extent the incorporation of this form of iron in phosphatic lir:ks influenf'es the retention of phosphorm.

From the literature it i,; evident that iron has a g-eneral detrimental effect on phosphorus ancl <·al<:ium metabolism. Rhem and 'Vinters (194()) AhmYed that ferri(' ('hloricle has a <lefinite a<hero;e effed on phosphorm;

anrl calcium utiliz:1tion. Waltner (1927) showed that the addition of 2 per cent. of reduced iron to :McCollum's stock diet produc:ecl rieketA in rats within four weeks. Analyt:;i;; of blood sennn d rachiti(' rats showPd a ]o\1·

phosphorus but a normal c·alcium c-ontent.

It '"a.~ .ac<:onli ng-ly planned to conduct an experiment to t.e~-Jt the availability of pho,;phorus in bonemeal against honemeal supplemented with 1·ed oxide, and at the same time the aYailability of phosphorus in honeash to white ratR.

261

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AVAILABILITY 0}' PHOSPHORUS IN BONK~iEAT,.

As regards the availability of phosphorus in several phosphatic supplements, Malan and du Toit (1932) tested these supplements and placed them in the following- order of availability of P. : -

(1) Sodium Phosphate.

(2) Precipitated Calcium Phosphate.

(3) Bonemeal and degel.atinised bone flour.

'l'heir conclusions were based on the weight increase of the experimental animals.

Otto (19;18) tested the availability of P in several commercial phosphates.

The phosphates were ferl at .a level well below that of the P requirement of the class of animal concerned. Throughout a Ca: P ratio of 2: 1 was maintained. Availability was determined by means of the retention of P.

From the experiments rli-soclium phosphate appeared to be slightly more available at the given level of

P

intake than either eli-calcium phosphate or bonemeal. '!.'here was no sig-nificant difference between the retentions of eli-calcium phosphate and honemeal. SummariRing his findings he eonsiclers that " taking all the results obtained into consideration it is doubtful whether animals when fed phosphates well below their requirements will retain more P when it is given as eli-sodium phosphate than the relatively insoluble cli-

<"alei um phosphate or bonemeal ".

PltELIMINARY BxPEHDIE:r>T.

A 111: mals.

Ten quartofi of albino rats of the same age, (27-35 dayfi), sex and weight (44-77 gm.) were ehosen. Here it was impracticable to select quartos from litter mates, and as these animals were highly in-bred, quartos were selectecl

with restrietions only to age, sex and weight.

At the beginning of the experiment one rat out of every quarto wat> :,;acri- ficed. The entrails were removecl and the digestive tract freed of its content:,;.

'l'he left femur was dissected out and .analysed for its ash percentage on a dry fat-free basis. The entire rat was then dried at 100° C. and subse- quently ashecl at a low temperature and the calcium and phosphorus content (leterminecl after addition of the a shed femur.

The remaining three rats .out of every quarto were assigned to the three different diets and the paired-feeding method was employed. 'fhe food intake of the trio was restricted by the amount consumed hy the member eating the least. In the exploratory experiment, tap water was given ad hln:twn.

The animab 'Yere kept in cages fitted with screened floors thus preventing- access to excreta.

The constituents of the three diets in which honemeal (Diet A,) honemeal plus reel oxide (Diet B), and bone ash (Diet C) was incorporated, are given in '!.'able 1.

'I'he bonemeal and boneash were both of 70 mesh fineness. The boneash that was used was prepared by ashing bonemeal in .an electric furnace, keeping it at a dull red heat for one hour and grinding to 70 mesh fineness.

262

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.Egg WI! ire (heated) ............... . Celatine ............ . B11tterfat.. . . . . ......... . Bacto Agar ............... . Harris Yeast ..... .

Co<l Liver Oil. ..... .

1-illcrose ............. . I'extrinized , 'tnrch ..

Snlt :I.Iixture ............... . Bone i\1enl ................... . RC'cl Oxide .................... . Bone ..\.~h ........ .

Pcrccn tngC' Ca ......•........ Pcr0cn tagc P ... .

Ca.fP..... . ......... .

'1'.\BLF.

Diet A.

gr./Kg.

150 ::)0

so

20 15 20 40 Ml6·:)

30 18·.)

0·4673

o-:?:no

1 • rn 1.

Diet.· H.

gr./Kg.

li>O

~0 80 20

].)

20 40 1)92 30 18· :>

-i·i)

0·4670 0·2368 1 ·07

P. G. J. LOC\L

Diet c.

gr./K§!'.

1:10 30

so

20

].) 20 .HJ

(l()~ .• )

~0

JO· ;)

()·.(.)iii' 0·23:!1 1 ·06

The "·eight of iro11 oxide arldetl wa;; equal to the \Yeig-ht of plw.~phonu' p~nto:si(le in the ~upplement, "·hich is in the same proportion as that incOJ"poratE'cl in phosphate-iron oxide licks.

Supplemeuti11g- of the diets \\'CIS made at the expense of the dextrinizecl starc-h. The salt mixtUJ·e of Day and McCollum (19:~9) '.Yith the omissi-on of

calci UJJl c-arbonate, was employed.

The h<tsal dids ("Ontained 0 · 00 per c-ent. P aiHl 0 · O(i per ("t>ll t. Cn and

•vere aecorcling-l~- supplementer] to contain 0-2:~ pe1· cent. P <llHl 0 ·4G per ("ent.

Ca, g·iYing- fl Ca/P ratio of 2 to l. The n~.t~ throve on these diets <IS was manifested b:· tlwir g·eneral appearances and gain in weight.

The exrerimental feeding \\'US c-ontinued for 42 clays \\·hen the rat:=; Welt' killed, tbe per('eniag-e ash on the left femun; and the calcium and phosphonw content of the entire rat <letenninerl ns before.

The calcium was determined b,· the ,·olumehi(" method of McCrud<len (1911), while the pho~phoru:-; wa,;· deterlllined l.y the method of Fi::;ke and Subharow (1!)2:)) using photo-electric technique.

lcesults.

He~ulb of the preliminary experin1eni are g-iYen in Table 2.

'J'be phosphorus ("Ontent of the experin1eutal !"dis :~t the beginning of the ex perimeu t wa ~ estimated from the an alysi,; obtai ned from the con trob.

From the data the aYerage ash percentages of the femurs of the three groups are 54·90, 55·15, and 0-l-·9:i, ''hi("h manifest a reflsonahle good hone fonna- tion. 'l'he aYerflge percentage retentions of phosphorus £01· tl1e three groups a1·e 53·():~, 01·7G and 55·70.

Applying the analysis of variaH(·e technique to the iu<li,·idual ash pel·- cenbg-es and percentages of phosphorus retentions, no significant difference:;

'"ere found, "·hic-h was to be expected from the hig-hly variable inllividual data.

9928-13

263

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1\;) C':> ~

'l'AilLE 2. RA'l' vVEIOH'l' (gm.)

I

I l' 1 Total P . p p d Pcrcen-A vcrage Ash

I

Average

l c

^•ooc

d P in in rat m 't·at gfame t~tge percen-perccn-Ash onsumc a ron\ · . ( m.) Food a.t end be<>innin food P tage t~tge pe"•cn- .1 g (gm.) (gm.) ~( ) g ( ) g>tlllecl. g~t•nerl. of femur tagc. gm. gm. · _c_ __ ___c, ___ ..,:.._ ___ : ___ _c_ ____ .!.__ __ ~----'-------- 307 ·7276 ·8104 ·434H

·3 7 5 -~

51·61 57·06

I

231·-5 ·5486 -5~99 ·2948 ·2351 42·85 54·18 I 255·8 ·6062 .,,701 ·3050 -2651 4:3·73 5:i·3l I 253. 3 . 6003 . 6601 '3;153 . 3048 60. 77 54. 94 2;)7·5 ·6103 ·6R02 ·3601 ·3201 52·45 M·84 237 '6 . 5631 . G401i '2800 . 3605 64' 02 :)5. 49

I

251 '5949 . 6405 ':3052 . 3353 56.36 ' ;)2. 79 256 ·6067 ·6252 ·3150 ·3102 51·12 53·46 318 . 75?.7 1. . 9205 . 4050 . 4255 .~6. 4:) i\(j. 04 , _ ~~--_ · 7465 -~04 ~ 435.5 _· ~~~-~~~ 53· 03 _ ~4 · 92 M · 90 _ 308 . 7293 . 7602 . 4349 . 3253 44. 60 5!). 80 238 · 5636 · 5802 · 2948 I · 2854 -50 · 63 ii4 · 29 258 . 6109 . 5902 '3050 '2852 46' 68 fi2. 86 263 . 6228 . 6903 . 31\53 . 33ii0 i'\3' 78 -~6' 24 255 · i\ · 6050 · 6802 · 360 I · 320 I li:? · 90 ;'\5 · 09 ' 232·8 ·551:3 ·5701 ·2800 ·2901 .)2·62 fil-9:)

I

²⁶⁰

'61.57 . 7200 . 3052 '4148 (i7. 37 fi2 -:'i3 250-1 . •>922 . mo 1 · 3150 . 2551 4?. · 07 fi4 ·50

I

318 ·7530 -910'' -4950 -4155 .55·17 .)7·61i

1

^-^-~~:^__¹^__

·7187 ·8004 . ·43fili -,~3()49 __ 2~7_ ~~-~~~-~~~- 310

I

·7195 -8904 -4349 ·455-'i 63-:lo .)6·96 238 .. v;24 . 6003 · 2948 · 301\i\ •'\5 · :->.0 .'\•> · 3n ''4 · 79 1 258 · 6988 · 6300 · 30ii0 · 3250 M · 27 :18 · 20 I 263 · r, 104 1 . rmr;:> · a.'i•'\3 · 340:! ''" · 73 "'' · 01 262 ·()081 I ·7602 1 -:~601 ·4001 65·79 ;)2·48 243 · :>640 · 6003 · 2800 · 320:i i\6 · 7P .'i:l·lO 240 · ~ · ii784 · i'ii\:):-l · 30.~~ · ~~>O I 43 · 23 i\0 · 94 2.~() '1>942 '6090 . 3150 I . 2949 49.62 I :)3. 79 318 . 7381 . 9302 -4950 I . 4352 :38.96

m ·

97 :nr, ·7311 -8305 ·43115 1 ·:l950 54·02 .J5·70 56·10 1 54·!J:~

---·----!----1---- n. 1 : J 73 12+ 1

?1

nd ~ l~ . ~ 8J 47 lOR 61 I I d 53 107 5! 14 Q fi7 102 .J,) I 7

J-

.J.t'i 96 ,-; l :20 <jl 4() 122 73 :23 2 48 94 4f:i 2() Q 77 149 7~ :29 2 70

I

l2S M' -----~---------~----- c. 3d 1 73 140 67 6J ~ ]~ ~ • 9 d

I

4 7 106 ,)() l2 J ;)3 103 .)0 v; <jl m 122 6ii 1.8 d 415 108 6:i 2l Q 49 !06 57 24 9 48 104 56 27 2 77 160 83 30

s

70 145 ?i\

I

~'tat l'\c. lliet. and

I

Sex. ···-·---,- A.

I

l d 4J ?J

I

lOJ 13 Q lfi J 19 Q ZZ<jl :l:) Q :?8 Q

Initinl.

I

Final. .1 .. C~tin. 73 I 130 57 49 I !)0 41 +7 I 92 .j.;) 5:~ 107 54 i'i7 I 112 -~5 4:) 100 55 +9 lOS 59 48 lUl 6.'i 77 152 75 70 14() 7()

··- --- -- - - ---

... :;

H r

...

t:o .... r H >-' ~ ;:;; ;? u: >-:: ~ 0 ~ u: H '7, .... 0 '7, l'i ~ M :>· r

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The lo\Y phosphoruR retention may in•li,·ate that this element was fe1l in the diet at a level higher than that required for normal growth uncl phosphorus retention. Aceording to Utley and Macleod (1935) a diet l'Ol1-

taining- O·lG per cent P anfl a Ca/P ratio of !3 to 1 would supply either tlw minimal m· slightly under the minimal :unount of phosphorus for normal gTOIYth :mel phosphorus retention.

SJ·:CoX!l ExrEIIDlEXT.

The lo,,- phtl~phorus retention;; oh;erYecl in the preliminary experin1ent 11·hich muy indicate that the P in the diet~ of the prelimiu;~ry experiJlleni 11·as in t'X<·es.-; of thP minimul requirenwuts of the animals, might C'loutl any lliff<"J'encp,.; in the .U:-lsim ila.tion of J:> hon1 the three diets. Aceordingly an i<lenhc<ll experiment \Yith seYen tlll<ntm; of albino rats wa::; eontludecl <Iii

before, ex('ept that the phosphorus in the 1liets was supplie<l at a leYel of ()·Hi pPr eent P. The Ca (J:> 1·atio of the,.;e 1liets wa::; 1·87 in eYeJ',\' ('Use.

J)i.~hlle<l IYater was g-ive11 ud liiJI.fUIII. The 1·ntions were made up a~ hefore. :-lupplemeutiug 1rac; made acconling to 'J'ahlf' :1,

Diet.

A B

c

13A·'[C .RATION. I

1 - - - - -- - - 1

l ";~~~~)t I ^(g~.) I ^(g~.,

^I

98!'!

· 1 8 1 . .

^')!l3!'i ^·4054

9"(i·.')8 ·5!Jl!l ·-Hl3~

()93

·67 1

^·.59()2^I^··lfl5S

Bone- meal (gm.)

10 · 1"~ I

10·82

TuTAL(Kc.

- - - -- -- -

nollC- ,1

FeO

I

C·l

~~~;'.)

^(gm.)^I^(g;,^.)

( )

^p ¹ ^Ca

I

^P ^I

I ( ) ( )

I

^Ca/^P

gm. I gm . . gm.

1

~

^.³⁸⁹⁰

1 1 .

⁰⁹⁹³¹^2.^!JR2f)

I t .

^{!)94 7}¹ ^l^·87

:2-(i

I '

^"90

^I

¹

^·0!>03 ¹

^{2. !lSO!l}

^!

I . 592.') I ¹·87

6-:3~-~ ~-383711·0999 2. !J7!)91l . 5fl67 i ^1.⁸⁷

'l'lw re<l oxide in diet B

'"a"

n<lfle<l in the ;:;ame :11uount .a~ the P ₂0., in the phoshate ~upplement. l<'ee<ling was ('ontinuerl for 42 days. The animals fed well anrl their general appearances were good. Except that :ieYend l'ctt~ kuibhled at the ,;ol<lel' of the metal SCI'eem; nothing nhnonnal

,,·as u bsel'Ye<l.

H?sults of thP experiment are c:ummm·i,.;ecl in 'l'able 4.

The aYeruge ash pe1 <·eJJtagec; on a <lr:y fat-free basis of the femurs of ihe three groups are !)l·G9, 52-:H an<l 51·05. The imlividual nsh perrPntag-e;-;

<·xhihit 110 ,.;ig-nifieant differenee on statisiical analy::;is.

,b 11·as expecte1l the }Jen·en tage phosphorus retentiDns Kere consi rlera hly higher than in the preliminar:y experiment. The average percentage phos- phonu retentions "·ere IG·Oi, i:J-:1() and 71·52. On applying the nnal:y,;is of Yal'iaHl'e technique to the indiYirlual retentions between the tlll'ee g-roups, no sig-nifieau t differences were found.

Siuee the nual,vsis of Ynrian<:e ,;hmYecl no significance for pairings, sigJJificanl'e of trf'atment difte1enl'es were adjuclgt~fl l1y the variation withiu groups only. K o cliffen:nces were fou nrl. This "·as applied to the 1n eli minary experiment with the ;.;ame result.

265

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RAT WEIGIIT. (gm.) Xo. ----- Diet. i and Initial./ Finn!. I Rex. Gain.

f<'ood Total COil· Cain ~umed Food (gm.) (gm.) 'J'AJJT.E .J.. I Ca I Ca in rat in rat at, at begin. encl. (gm.) (gm.)

- I (',, ' g:1in~d i Per-

I

Total f.·om ccntagc P in food Ca . foorl (<rm.)

I

I , p p J>ercnc-p . II Ash . . t t p Cl· p 1n rat In ra . ag~ ecntao-c er- at

I

at gamer! p "' l'entage begin. end. from . 1 of (gm.) I (gm.) foorl gaiJI<"r J femur. (gm.) A 1 "? 60 l4fi 85 294 · 5 · 8783 · :>20!1 · 998~ · + 773 54· 34 · 4696 · 3800 · 7400 · 3600 7fj · 66 51 · 32 4 9 40 110 70 240 . 71iiS . 3+·06

-noo

<l803 53 ·12 . 3827 . zsoo · 5600 · 2soo n-16 4S ·42 7 <f 60 163 103 327 ·97:'\2 ·5180 ·11721 ·fii\35 67·01 -.~214 ·4200 ·8000 ·3ROO 7:!·88 54·.~0 10 Q 58 162 104 333 ·!Hl~2 ·4674-1·1240 ·6!\75 66·20 -.~310 ·3800 ·7000 ·4000 7!\·32 ;'\3·00 13 Q 53 123 70 262 I ·7814 -~fll!i ·8397 ·3782 48·40 ·471R ·3600 6600 ·3000 71·80 46·48 16

o

fiR 127 69 2!J~ · 8 8733 · .3625 1 · 0932 · ;;:107 60 · 76 · 4660 : · !200 · 7600 · 3400 72 · 82 52· 68 19

o

60 128 6R ! 2!J3 ~-8738 ·:>Fi6 11·1:128 ·.>86:? 67·08~·~67:' ·38C:O ·8000 ·4200 89·8911 .'i.~·~!J 1\:l A vemge ...

I

1 1 5D · 56 1 1 7 6 · 0 7 .1) 1 · ()9 ~ ~ ---· I1 ~ 9 GO l+:>. 83 296 · 8823 · .'\209 1· 0338 · .il:l9 58· 13 · 4714 · 3800 7600 <1800 RO· fil .'i2 · 78 5 2 40 I Ofi 6fl 240 · 7154 · 3406 · 7882 · 44 76 62 ·56 · 3822 · 2800 · :iiJOO · 2800 ?:!· 26 :'il ·!'I 8 9. 60 160 100 327 ·9747 ·fil89 1·0971 ·.'i782 59·32 ·ii207 -~200 ·7600 ·:l400 6fl·:'W 51·11 ll Q .'\8 l.'i6 !18 328·7 ·\J7!l8 ·4(i7-1 1·1011 ·6337 04·fl7 ·523-i <lSOO ·7600 ·3800 7:?·60 .'i1·67 l4 <? ;,:~

I

130 77 2flii -7~99 ·-H\15 ·~7()3 . ~178 I -'i2·891 ·4220 ·3600 ·f\800 ·3200 7.'\·82 :)3·33 17

o

:'iS 134 7(; 301 · 8\172 · iifl~;, 1 ·1091 · .1)4()6 60 · f)2 · .J-79~ · -i200 1 ·~lOGO · :1800 79 · :28 .'i2 · Oi~ 20 0 60 I 1:22 62 I 282·61 ·8+24 ·iilf\fl I 06!.'\ I ·:'\149 61·121 --~.iOO ·:lSOO I ·flR()O ·3000 Gfl·661-'>3·5:l Avera~c

I

1 1 69·9-! I 73<W .)2·~~

--c -- - · · -; -;,?-- -- -;:;;;- --

-1"4 ------;;:;:--

2 n6 -l-:- 8820

--:-~209

]:0496

--:-~;7 .'i9 -:;,:;:-

--:- 4726 -:-;sno - ·

~?oo.

- _

~~,0'T

719 4- ;]-:-;;;-

() ';' 40 106 6(i 2~0 ·71.>1 ·3406 ·7684 ·1278 -~9·82 ·3832 ·2ROO ·:i(iOO ·2800 ?:l·Ofi iiO·!IO !1 '!' 60 IM~ 98 327 ·'lH-1 ·5189 1·08:33 ·:>61)4 :'i8·12 ·5221 ·t200 ·7600 ·3400 65·12 ;,Q-27 12 Q 58

I

14+ R6 33:l ·9\1:!3 ·+67+ ·!l247 ·4753 47·89 ·5317 ·38f10 ·6800 ·3000 .'\6·42 -W·2fl 15 '? 53 138 85 ?.6.'i . 789f\ . 46lfl 1 . 0:'\36 . 5fl21 74.98 . 4231 . 3f\00 . 7800 . 4200 9!1· 2() :)0. 38 18-3

I

.'iR i 128 iO :ll11 ·8!16!1 ·.')62.) 1·04:37 ·+832 53·87 ·4806 ·4:WO ·7600 ·3400 70·7-l 54·2R 21 J fiO

I

128 68 :!!):! ·8731 ·:>466 1·0Gl:i ·.')14!) .')8·fJ7 I ·4fi78' -:1ROO ·6800 I ·:3000 fll·l~ l-53·fl:l -~verage ___ ___ _ ___ :)\)·0~ I I I 1 _1_21·5~ fll·O:i

> <1 > H

t

~ -1 f..< ""'

""

~ :; 'f. ~ c: [/l H ~ 5 ~ '--' .... t:j ;.- r

(7)

I'. G. J. LO"C\Y.

~r~n1AHY ANJJ CoNCLUSIONS.

'l'"·o expe1·iment:-; have been earried out with albiuo rats in which the availability of phosphorus in bonemeal, bonemeal ~mpplemenbd with reel oxide of iron and boneash haYe been tested out. In the preliminary experiment th!:' J> was ,;upplierl at a leYel of O·:t3 per cent .. the CafP ratio being 2:1.

From this experiment no ,;ignifieant difterences between the ash percentage,; of the femun; of the rat;; and the phosphorus retentions could he shown.

Low pho::;phoru,; retention:; indicated that the P was supplied 1n exe!:'i-iS of th!:' minimal requirementi-i of tlw nnimals.

In the second experiment P \\·a, c;uppli!:'d at a level of O·l(j per cent., tlw Ca / P ratio lwing 1· 87. 'l'he percentage P retentions were consid?ralJly higher than in the preliminary expt'riment, the average perrentage retentions being 7(i·07, 7=3·:W all(l 7J

·.sz.

^Again^no:;ignifieant <lifferences could he

;;howll by applying ;;tatistical analysi;; to the a;;h pen;entages of the femub aud 1 he P retention,, which illlpli!:'.~ that 11nrlPr the eon<litions of til<"

experiment the avnil:ibilit,v to ''"hit!:' rats of the P in the three supplemeut,-, art> similar.

RE/ERENCES.

D.\ Y, H. G., K V. McCOLLUM (1 93\l). Mineral metabolism, growth o,nd symptomatology of r<tt> on a diet extremely defbent in Phosphorus. Jnl. Biol. Chem., Vol. 130, p. 269.

P[:';KE, C. H., SUBBAROvY, Y. (192.~). The colorimetric determination of Phosphoru~ . ./nl. Bioi. Chem., Vol. 66, p. 37:\ .

. \£ALAN, A. I., DU TOIT, P. ,T. (1932). Studios in Mineral metabolism XXT. A comparison of Phosphatic Supplements for the prevention of aphospho1·osis. 18/h Rept. Dir. Vet. Ser.

and An. Ind. Un. of S.A., pp. 677-702.

:VlcCRUDDEN, F. H. (1911). The determination of Calcium in the p:-esence of l\lagnesium and Phosphates: 'J'he determination of Calcium in Ut·ine. Jnl. Biol. Ghem., Vol. 10, p. 187.

OTTO, .J. S. (11138). The assimilation of Calcium and Phosphorus by the growing bovine.

Onderstepoorl Jou•·nal, \'ol. 10, ~o. 2. pp. 281-311+.

REH~1. P. A:<n WINTERS, J. (1940). 'l'he effect of ferric ch10ride on the utilization of Calcium rwd Phosphorus in the animal bodv. Jnl. -~··utrition, Vol. 19, p. 213.

U'l'LEY, E. AND MACLEOD, F. L. (l93i\). Unpublished data.

WALTNER, K. (Hl27). Uber die Wirknng Grosser mengen Eisens. I. Uber clio Wirkung rles Eisens auf clie Knoekenentwicklnng. Biochem. Zeitschr., Bd. 188, p. 381.

267