Onde'l'stepoO'I't J (!'l.lrnal of V eterinary Science and Animal Indust1'y, Volume 11, N mnber 1, hdy , 1938.
Printed in tile
r
nion 0f South ~Hrica by the Go,·crnment Printer, Pretoria.Sulphur Metabolism. VI.-The Eff ect of Va riou s Levels of Fats and Prote ins in th e Ration on the Toxicity of Elementary Sulphur . .
By J . H. KELLERMANK, Section of Bioe;hemistry, Onderstepoort.
LEWIS
ANDL EWIS (1927) haYe found that the ine;orporation of one per cent . elementary sulphm in t h e milk powder-starch diet of Shennan and Merrill (1925) and in the low protein diet of Osborne and Mendel was definitely toxic .
\\~itha few exceptions, the liYer,;
of the rats -n·hi ch died showed
amarked peripheral zonal n ecrosis.
Hcmever, these authors found t h at the rats on t h e
Osborne-~£endelc liet were m ore resistant to the toxic effects of sulphur than were those on the
Sherman-~£enillration . 'l'he former diet is rich er in fats a nd proteins than the l atter and t h e authors expressed t he opinion that th e lo"·er toxicity of sulplnn in the
Osborne-~fencleldiet might be due to its higher fat a nd p rotein contents. Their suggestion is t h at the fat probably form s a coating around i.he sulphur particles which either preY ented t hem from coming into iutimately clo:;e contact with the intestin al mucosa or the bacterial action on t h em i s thereby r educed. Furthermore, the h igher protein l evel might leacl to a better state of nutrition and h ence a greater resistance to t h e toxic action. This supp osition, a s far as the writer is awa r e, has n eYer b een put t o the test, and it was t h e object of the following experiments to determin e its validity.
ExrERHlE:t\T;\J..
Young rats of t h e \Yista r strain ''er e used in these experiments . They were equally diYid ed amongst the Yarious groups with r esp ect to litter, sex and -n·eight. Each animal \Yas kept in a separa te cage on a r aisecl scr een bottom. In addition to t h e chs tille cl -n·ater, to
\l·hi ch the animals al ways h a d hee access, each rat was fed daily its particular ration ad lilnhnn \Yith recorded foo cl consumption for t h e exp erimen ta l period. o ,,·ing to t h e slightly acid nature of tlie yeast extract on starch , to he d escribe d later, t h e diets rapidly developed a rancid odom:. In order, therefore, t o preY en t rancidity in these cli ets, tb e yeast Pxhad was \Yithh eld from the bulk of the cliet ancl intimat ely mixecl with each clay's suppl y as practised by Gallup nnd R eder (1935) . The composition of th e ration s is g iven in Tahl e I.
The basal diets contained nll the di etary Pssentials known. The Yitamin B complex was given in the form of a n extrart. prepared from hre-n·er s' yeast accor ding to the m eth od of Itter, Orent ancl
107
SULPHUR :METABOLISM.
TABLE
I.
Compos· ition of Ration s in P e1·centage by l1 Tl eight.
Low High
l
Protein.
I
Protein.- - - -_ _ _ _ ....!...__ __ _ __ __ _ _
" Vitaminized starch" ... . 10 10 Dextrinized starch ... . 44·7 lli Casein ................ . 10 40 Sucro e .... , ... . 12 12 Salt 40 ... . 4·5 4·5 AgaL' .... .
Cocl-Ii,·er oil. ... · .... .
2·5 2·5
1 1
·Elementary sulphur ... . Lard ... .
3 3
Butter fat ... . 12 12 1-cystine ... . 0·3
Lo"·
.I<' at.
10 10
49 43·6
20 20
12 12
4·5 4·5 2·5 2·5
2 2
3 2·4
- -- --
I
High Fat .
10 lO
31·4 26
20 20
12 12
4·5 4·li 2·5 2·5
2 2
3 17·6 20
JicCollum (1935) . Thi s ex tract was eYaporated on dextrinized starch subsequently (1esignatecl " Yitaminized starch " . One gram of
thi~sta rch was equival ent to 0 · 5 g. of yeast. The composition of Salt 4U w as similar to that of Steen bock :mel X elson (1923) a,; modified by K eenan and ot hers (19aJ) . HoY\·evPr , in order to obtain a sulphm- free salt mixt ure the m agu esium a nd manganese sulphates were r eplaced by equiYalent amo unts of their carbonate forms and the CuS0
4· 5R
20 Ly cupri(' acetflte. In a ddi tion to lard the effef·l s of butterfat, b eef drippin g (:YJ.P. 43-49° C. ) and olive oil
011the toxi city of elem entary sulphur wer e stu(liecl. The ration s conta ining- th ese fats were identical in
pver~-r espect to the lard ration excep t that th e latter \Yas replaced by an equivalent amount of these variou s fats.
Furt hermore, all the high fat diets "·ith and withou l the aclclition of _ elementary sulplnu w·e re marle equicaloric by replacement of some of the st:lrch by isorlynamic q uantities of fat.'Y< Similarly, the lcm fat diets, '"ith ancl '"ithout a c1dec1 sulphur, \vere made isocaloric and t h e same applied for th e low aucl hig·h protein ration;;. After the rnts hnd hrrn on e:s:perintr nt for eigl1t \\'eeks the)· '"ere killed
b~-a blow on thr h end a nd various org<m s imm ediately remo1 ·ed and pre- sen ·ecl in a formalin solution for histopa t hological examination.
The results obtained on the lo1Y nnd high protei n ranons are presen ted in Tables II , III, nncl IV. T able II gives th e original results, Table Ifl tl1 e mPans ancl Tnble IV t he analvsis of Y nrianre.
From Table IV it is clear that the growth-promoting value of the
10\Y
protein ratiou n·a s
significant!~-greater than that of the hi gh protein one ; th e probability P being ·01. T his observation substan - tiates, therefOl'e, the r esu lts of Osborne, Jiendel and F erry (]9 19) , Mitchell
(1920-24),St. John am ! oth er s
(1934),Chic k anrl cO-\Yorkers (1935) and Basu, Kath and :Mukherpee (1907) . Howenr , th e growth- promoting valu es for ma 1 rs ancl females were not
siguificantl~clifferent and there " ·as no interaction of se:s: with th e protein cont ent of the ration.
*Tho energ~- Yalues of the diets were calculated on the bilsis of 4·0 Cal.
per grnm for the earboh~·drates and cnsein. and of 9·0 Cal. per gram for the vartons fnts and oils.
108
J. H. I~ELLElDL\XN.
'l'ADLE
II.
1'he gTmcth-p1·onwting ralues of sulp lwr SU J 71Jlem ented rations low and h1 'gh in 11rotein co ntent as det enm. n ed by th e method of Osbome, Jl end el and F e1·ry (1919) .
PERIOD : 8 .WEEKS.
Total
I
ProteinI
Sex. Food consumed.
c-onsumed.
I
Hation.
! P.
g. g.
Male :363 30·86
Low Protein (8·5 per cent.) .. I
:\Iale 266 22·52
l\Ialc 3!)6 3::!·66 Female 371 31· ;)4
Female 322 27·37
Female 292 24·82
:\Iale 316 107·4
:Hale 417 141·8
High Protein (34 per cent.) ....
:\[ale 298 101·4
Female 321 109·1
·temale 302 102·7
Female 283 96·23
'l'ADLE
III.
Jf ean Growth-p romoting ra liteS :
~~~--~====
Low Protein ... , ... , ... . High Protein ... .
TOTAL ...•.••....••... . . • ... . . •.. . .•.
'1
1.·\DLEIV.
::\Iales.
1·9 0·64 1·27
Analysis of V ariance.
D.F.
s.s.
]I[.S. S.D.Lowyrotein vs, high pro-l 4·6501 4·6501 2·156 tom
::\Iales vs. females ... 0·0884 0·0884 Interaction ... .
I
·weight Ratio inCI·ease. i "/P (or
G-P
\\". value).
- - - - - g.
69 2·24
27 1·20
76 2·26
72 2·28
41 1·50
34 l. 37
64 0·06
106 7·75
59 0·58
66 0·60
45 0·44
40 0·41
.females. Total.
1·72 J. 81 0·48 0·56 J . 1 1·19
Logo S.D.
I z.
- --- - 0·7682 1·6896
Error ... .
I
8I o t· ·~ - ~~~84 I
o-ooo4"v
0·1582 0·398 1-0·9213I
_ ) _ _ _
TOTAL .. . . . .... . ll
I
5· !l9G7l=i==J~====I=~I===
From Wisher's (1932) Table of "z":
For n1 = 1 n, = 8 Hence this value of z is significant at P
109
atp = ·0.5 atp = ·01 z = 0 · 8365 1 · ZlOG . OI.
SULPHUR :\1ETADOLISM.
The results obtained on the lo11· and high fat n1tions are g'lYen m Tables \" and VI.
TABLE
Y.
Effect on Efficiency (J'uot,ient of add inr; ;j l)("l' cell
t.
El em en/a '"!J S1tlplwr to Synthet'ic Rations of Forio11s Fat and l)rotem Conte11t. Period8
Weelcs.Ration.
Low fat ... . Low fat ... . Low fat ... . Low fat ... . High fat (lard) ... . High fat (lard) ... . High fat (lard) ... . High fat (lard) ... . High fat (butter) ... . High fat (butter) ... . High fat (dripping) ... . High fat (dripping) ... . High fat (olive oil) ... . High fat (olive oil) ... . All males ... . All females ... . All low fat diets ... . All high fat diets ... . Low fat diets ... . Low fat diets ... . High fat diets ... ..
High fat diets ... .
Supple- mented with.
Sulphur Sulphur
Sulphur Sulphur Sulphur Sulphur Sulphur Sulphur Snlphur Sulphur
Sulphur
+
SulphurSulphur
+
SulphurNumber\
of Sex.
Rats.
3 3 :3 3 6 6 3 3 3 3 3 3 3 3 24 24 12 36 6 6 12 24
i\1ean Gain.
143·5 98 102
46·8 107·7 85·5 66 55·7 82 66 83 64·7 72·3 59·3 93·8 68 97·6 74·2 120·7 74·4
!)6·6 68·6
:\[ean total food.
670·5 673·5 554 383·6 402·6 395·7 35:'5 306·3 347 324·3 352·3 331·2 312·7 313·5 427·7 389·7 570·4
3~-1-l
672 468·8 399·1 330·3
i\Iea n efficiency quotient.
3·2 5·41 4·65 6·29 2·88 3·88 4·4 5·46 3·45 4·62 3·63 4·91 3·87 5·:!5 3·62 4·06 4·89 4·10 4·30 5·47 3·38 4·45
Table V g1ves t he mean values and Table YI the anal ysis
nfvariance of the efficien ey quotients . The data in th e l atter table
sho>~·
t hat t h e effiriency quotien ts, calcula ted arrording t o t h e lll ethorl of Palmer aml Kennedy ( 1929)* of the males \Yere ,;ign ificantly b etter than those of the fem ale,; . The probabi lity P "a s · 01 " ·hich su bstan- tiates the result;, of Pa lmer and Kenne dy (198 1). On t h e high fat r atioll s effic ien cy quotients \Yere obtaine<l that were
significantl~yR uperior (P = ·01) to t l10se on t h e lo''" fat ration s. This result might be expected in view of the great differen ce in calmic value bebYeen t h e bYo types of diet. ..Within both low nml high fat rations the nddition of el ementary sulphur sig nifica ntly
lowe~'edthe animals' efficie ncy to utilize their food; t h e proba bility P being · 01. The lmYer food con sump tion on the sulphu r rations might h ave contributed to t h e inferior utilization of food inasmu ch as Palmer ancl Kenned y (1931 ) found that a restri ction of food intake l o" ·ered the index of food utilization. However, th e efficiency quotie nts on th e butterfat, lard , dripping aml olive oil rations \Yere not significantly d ifferent , and t h e sam e " ·as true fo r the interacti on of sex with the Yarious fats .
* 'J'he dry matter only, not the digestible d1·y matter, was used in these calculations.
110
J. H. KELLElti\L\:\"K.
TABLE
VI.
Analysis of llm--irmce of E.fjicienry Quot·ients.
===;=======
1 D.T•'.
!
S.S. i\I.S. S.D. J Loge S.D.i z.
---~· ~~----~----~--
::\Iale vs. female ... . 21·6008 21·6008 4·648 1·5364 2·0540 Low fat vs. high fat...
T
\\'ithin lm,- fat: Sulphur 1 vs. no ~.
\\'ithin high fat: f:>. vs.
no f:>.
"'5li486
5·6486 4·09:)0 4·0950 9·10:2:2 !)·102:22·377 0·8658 1·3834
:2·024 0·7051 1·2:27 3·017 1·1041 1·627
\\'ithin S. butter vs. lard 3 :2·6819 0·8940 0·946 - ·0565 0·4621
1·s. dripping 1·s. oliYe oil
Between all fats ... . lnterndion of sex with
fats
Trmttments ... . Error ... .
TOTAL . . ..
6 :21· :5'277 6 1·8009 13 44·9294 :>4 12·077:{
47 57.0067 From Fisher's (193:2) Table of z:
:3.5879 1·894 o-aoo:2 0·548 3·4561 1·859 0·3552 0·596
0·6387
I
0·6:201- 0 517(i
1·1563
1·1377
at P = O·:'i at P = ·01
fo•·n1 1, n2 :30, z 0·7141 1·0116
n1 3, n2 30, z 0 · 5632 0 · 7531
n1 6, n2 30, z 0·44:20 0·6226
n1 1:2, n2 30, z 0 · 3691 0 · 52:24
A comparison of the z ,·alues from the abo>e tabk, with tbC>se criterions indicate that nil, except the" Butte•· vs. laecl vs. dripping v~. oli,·e oil" z, aee significant at P = · 01.
On t h e whol e, th e histopathological changes noted in the livers of rats fed the various diets, with and \l·ithout the addition of elementary sulphur, did not vary much from those seen in t he liYers of normal animals. The 1nost common obserYations were fatty changes which Yarie d hom 8light to faidy adnnce<l and diffuse . The kidneys of the animal s on the l ow and high prote in rations were al so examined . However, except for slight fatty changes, these organs did not sh o11· a n y constant pathol ogical changes, and one is therefore led to the conclusion that el em entary sulphur has no dele- terio us effects on t h e structure of liYers and kidneys, which i s most probably also true for a ll other organ s of rats as long as the animals are feel a ration that contains :.1ll the essential ('Onstituents for growth.
S
'C1UIA 11 Y.J. 'l'he extent to which Yario us leYels of fats an d proteins in rations could influe n ce the rffP-ets of elementary sulpJmr on rats as judge d from g-rowth , food util ir.ation and p:.1thologieal lesions, "·as sh ld ied.
2. 'l'h e grmdh-promoting value (gain per unit protein ronsumed) of the low protein ration w:.1s significantly greater th a n that of the high protein diet.
3 . On t h e same ration the males utilized their food significan tly better th an the fema les.
111
SCLI'IlT:R SlELUJULIS~\1.
4. \Yitltin Loth lO\\' a!Ul high fat rations lhe auditi on of elerneutar_,.
~ulphursig nifica ntly lo\\·ered the animals ' eHicieucy of food utilization.
o. hr espediYe of the fat and rnotein contents of the diets the liYers and kidneys of the sulphur fe!l rats,
ascompared 11·it h those of the control gro up s, !l id
uotexhibit any con stant pathological changes.
Ac
1\:KO IIT.EnG El\IEXTS.The \ni ter "·elcomes this
opportunit~-of rel:o rding his t han k::;
to
Dr~.K. C. A. S(' hulz and A. D. Thoma s for Jnaking the hist o- p:!tho lng·i,·:tl ex :tntin:Jtion s :nul to
])r-_U. B . L a ure nce for
makingth e sta t ist ic-al ana.lys is.
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