OF THE GUINEA-PIG. CCII. VITAMIN C REQUIREMENTS

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CCII. VITAMIN C REQUIREMENTS OF THE GUINEA-PIG. BY SYLVESTER SOLOMON ZILVA.1 Frm the Divisin f Nutritin, Lister Institute, Lndn. (Received 27 June 193.) VAN EEKELEN et al.

1 CCII. VITAMIN C REQUIREMENTS OF THE GUINEA-PIG. BY SYLVESTER SOLOMON ZILVA.1 Frm the Divisin f Nutritin, Lister Institute, Lndn. (Received 27 June 193.) VAN EEKELEN et al. [1933], Hess & Benjamin [1934] and Jhnsn & Zilva [1934] have shwn that human beings d nt excrete ascrbic acid r excrete it nly t a limited extent when it is ingested by them unless they are fully " saturated" with the vitamin. The last-named wrkers were at the same time able t shw by means f detailed quantitative experiments that certain individuals may be s depleted f the vitamin as t take a cnsiderable time t reach the cnditin f saturatin when cnsuming daily dses f 1-15 mg. f vitamin C. Yet such individuals nt nly fail t display any symptms even f latent scurvy but appear t be in perfect bdily and mental vigur. Similar results have since been btained by a number f wrkers. Sme f these are, hwever, inclined t attribute t this lack f vitamin C "saturatin" a clinical significance which in the pinin f the writer is nt warranted by the existing knwledge f the subject. Experiments n guinea-pigs are given belw which thrw sme further light n the matter. The chief aim f this investigatin was t ascertain the relatinship f the intake f ascrbic acid t the vitamin C cntent f the "selective" rgans and ther tissues f these animals, t their susceptibility t scurvy, t the urinary excretin f ascrbic acid and t their general weli-being. Strage f vitamin C and susceptibility t scurvy. That guinea-pigs f abut 3 g. maintained n a gd diet with cabbage ad lib. under favurable hygienic cnditins shw little individual variatin in susceptibility t the disease is well knwn t wrkers in experimental scurvy. The tissues f these animals, especially the " selective " tissues such as the adrenals, anterir lbe f the pituitary, the liver, the small intestine etc. cntain vitamin C in quantities which cannt be raised even by further administratin f high dses f ascrbic acid either per s r by injectin. In ther wrds, when the tissues f grwing guinea-pigs are supplied with vitamin C t their maximum capacity, the animals vary little in the time taken by them t succumb t scurvy. The fllwing experiments were designed in rder t btain mre detailed knwledge cncerning the influence f the bdily stre f vitamin C in the guineapig n the time f survival n a scrbutic diet. Fr this purpse chsen animals, as used fr custmary vitamin C testing, were placed n the scrbutic diet emplyed in this labratry. During the first 1 days the varius grups received per s daily dses f -5, 1, 2, 3, 1 and 2 mg. f ascrbic acid respectively. At the end f this time the daily dse was discntinued and the animals were allwed t remain n the scrbutic diet until they succumbed. It is seen frm Fig. 1 that the time taken by grup 1 (daily dse -5 mg.) t succumb t scurvy after receiving the last dse f ascrbic acid (average time abut 2 days) was Member f the Scientific Staff, Medical Research Cuncil. ( 1419 )

2 142 S. S. ZILVA ~~~~~~~~~~~~~~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ C C;) ~~~~~~~~~~~~~~~~~~~~ ~ ti~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~c LO PR- *M

3 VITAMIN C REQUIREMIENTS OF THE GUINEA-PIG 1421 less than that taken by the cntrl grup 9. The animals in grup 2 als died f the deficiency earlier than the cntrl grup f animals, but the difference here is less marked than in the case f grup 1. The remaining grups appear t the writer nt t have shwn any significant deviatin in their behaviur n the deficient diet frm that f the animals which cnsumed very generus supplies f vitamin C in the frm f cabbage during the perid preceding the deprivatin f the vitamin. It is f interest t nte that when the guinea-pigs were depleted f vitamin C previus t the perid f dsage with 5 mg. f ascrbic acid the time taken by the animals t succumb t scurvy (grup 7) was almst the same as when n previus depletin tk place (grup 1). Furthermre, a dse f.5 mg. administered daily after exhausting the animals f their vitamin C stre by maintaining them n a scrbutic diet fr 1 days was sufficient t prevent the prductin f macrscpic lesins after 37 days *hen the animals were killed by chlrfrm (grup 8). The imprt f these results will receive cmment in a subsequent sectin when they will be cnsidered in cnjunctin with the ther infrmatin btained in this investigatin. The influence f the quantity f ascrbic acid cnsumed n the cncentratin f the vitamin in the tissues. The riginal aim was t establish the minimum daily dse f ascrbic acid necessary t be cnsumed per s by the guinea-pig in rder that the animal shuld excrete it in appreciable amunts in the urine and t crrelate this dse with the cncentratin f the vitamin in the tissues. This culd nt be ascertained by direct experiment, since it was fund that the guinea-pig had t cnsume extremely high dses f ascrbic acid befre any appeared in the urine. Recurse was, therefre, made t injectin after which, as has been shwn already by the writer [Zilva, 1935, 1, 2], urinary excretin f the vitamin quickly fllws. Analyses f the tissues f a number f guinea-pigs which received single graded injectins were therefre carried ut and cmpared with thse f grups f animals receiving similar dses per s. All the injectins, unless therwise stated, were perfrmed intramuscularly (femral muscle) withut an anaesthetic. The injected animals were previusly depleted f vitamin C by maintenance n a scrbutic diet fr days but the ther grups received their dse rally fr 15 days whilst subsisting n a scrbutic diet withut being previusly depleted. Details f the methd f analysis have already been described in a previus cmmunicatin [Zilva, 1935, 1]. It is seen frm the figures in Table I that, cnsidering the limitatins f the methd, the injectin f a single dse f ascrbic acid brings abut in the tissues f the guinea-pig a cncentratin f the vitamin similar t that fund when the animal receives rally an equal dse daily fr 15 days whilst n a scrbutic diet. Cnsequently the necessary cncentratin f ascrbic acid in the tissues fllwing injectin which permits its excretin in the urine can be reached by the same dse given per s fr 15 days. It is further seen that a dse f 5-8 mg. was required by the guinea-pig n a scrbutic diet befre even a trace f the vitamin culd be detected in their tissues and finally that the highest cncentratin apprximately equal t that btained by the injectin f 5 mg. was nly attained when dses f abut 2 mg. were emplyed. Results btained by Jacbsen [1935], Girud et al. [1935], Fx & Levy [193] and De r [193], in cnnexin with the depsitin f ascrbic acid in the tissues agree in principle with thse given abve. Owing, hwever, t the different experimental prcedures adpted by these wrkers, especially by the frmer, direct cmparisn f the quantitative data is made difficult.

4 1422 S. S. ZILVA r-. - Z 4.J d -,.r ti C. O Or N Iru c GN1- : _-_-i lz Q 4.,a CL) C.) r. S.4 z - ti.'. ;.4 E x P-11 C COC C i iii O 1sl C1) C.) ) + c E-- + EH + I:i c3 E-- " H ce > >, - c -, H - )- ) F*- 2 e C O. 2: 9 CS C 2: O., C; C 1 P2C C O O (=-4 (= *. ( 1: N: C C O 2:12: O1 C I-d 1 t1 O ti 2 N CO1O C. " ) O r c3~ < = L 2: XC1: c I c c) c C,: - C-C1 tlo lf -4O 2:C f IC De s (D CDc 2:. u c C -' b r 999: 11 1: O 2 C li2 2: 2 : j -- O 1 - b N c 1 1N - S 2:.1- _ e1 O O C < C: 1 1 O UO C> * aq, 2: N ) O C2 9 g in 1 1- =11 4Q <e D. G *: ~ ltl t Ut: U: l - _- 4-2: 2:2:2: 2: N1 N CN- N 9 e1m 1 m bo 1 = c~ C.) t C > C CCeec q) P. P. - e

5 VITAMIN C REQUIREMENTS OF THE GUINEA-PIG 1423 :r1 1 1CO1C O n CO CO O O O c:~~~~~~~~~~~~ucm1 cccel cl cl CIMcc Li m m sc 1D 1 N,'q I-L N11N CL-~L Li lx11 CO 1 1 C r CO CO cl IHIIII I: c HHIIH ± E+qca+ + + ± C CO- CcN1t X C - -4 Cr O CDCO C Cn C 1 ci'c 1 1 O 1 11 CO 1. CACO CO11O 1 1NccC1 CO c dq es_s m_ N. : m ItI I?1,?s c c v 1CO - A A~~~~~~~~~~~~c 1 A - A l A Aa 1_ O *d O t O -_ t 1t- O tsc - O t ce < + r r c:, ~~~~~~~~~~~~ -s; - : 9999 C C - -- r _ O. c,) C. c C c C e~.- CD 71 ~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~~~~~~O I c; c c; ' C 1 ' t- OCO - _ N e~~~~~~~~~c O CC c3 O Cd - -- N ) O N t O ON O O O CO ,. O-O.~ _ O C OOOOOO ~~~C C CO COCOCO mesg CO CO CO CO CO CO CO 1Xcsc 111 n ) b Q~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

6 1424 S. S. ZILVA ~ bo W r-r-4 lilm im c 4 t~~~~~~~~~~~~~~~x CD-ti ti- - 4 X,.4.. i L III I in L ± ++~~~~~~~~ ax~~~ ±+g + +s 99 v 4 $ E -B -^. c C _ - 4 r- S - - r - i -Q* QqIgP t d ~~~~~~~~~~~~C X * 1 ~~~~~ _m _ q CO *C- -C COCOC*O CO N> c c O O _ -- - O x C t m_q1 xm -4. N_ C> e r _ -4 r Oc ~~~~~ ~ ~ ~ ~ ~ ~ ~~*4 ~ ~ ~ ~ ~ - e I * - ~~~~ _L _ -H N _ O N in 4 IM u~~~~~c C a t a^ 3 ^ ^: t : ^ ^ *\ I~~~~~1- g O S g = R u ^ ^ ^ = :: eq~~~~~~~ m s> Xsst cp m n ~(:> t- = -d -.1 e: X m cq r- t1 91

7 VITAMIN C REQUIREMENTS OF THE GUINEA-PIG 1425 The influence f the quantity f ascrbic acid in the tissues n its excretin in the urine. Reference was made abve t the fact that extremely high dses f ascrbic acid must be administered t guinea-pigs per s befre it appears in the urine. This is illustrated by the fllwing details. Guinea-pigs previusly depleted f vitamin C by subsisting fr 5 days n a scrbutic diet failed t excrete any ascrbic acid in the urine after receiving 9 daily dses f 5 mg. The administratin f as much as 3 mg. t animals nt even previusly depleted failed t cause any appreciable excretin. Only when the dse was raised t 5 mg. did the vitamin appear in the urine and then in a cmparatively small amunt, namely 8 mg. Similarly a dse as high as 1 g. was fllwed by the excretin f as little as 3 mg. Euler [1935] als failed t find appreciable excretin by the guinea-pig after similar dses given per s. As seen frm Table I, ascrbic acid is much mre readily excreted by the kidney after it is injected. It was fund in fact that the vitamin began t be vided in the urine after a single injectin as sn as its cncentratin in the tissues f the animal apprached the maximum. In these experiments this was achieved when a daily dse f 2 mg. was cnsumed by the guinea-pigs fr 15 days r after a single injectin f the same dse in vitamin C-depleted animals. It is t be nted that cmparatively little ascrbic acid is excreted in the urine when 2 mg. are injected but the quantity excreted increases with the rise in the dse. The amunt f the vitamin vided is, therefre, cntrlled nt nly by the cncentratin in the tissues but als by the size f the dse injected. If the dse is t small n significant quantities f the vitamin will appear in the urine. Vitamin C depletin f the tissues and prlnged grwth f guinea-pigs. As a pint f interest in cnnexin with the prblem f the influence f vitamin C strage n the welfare f the guinaea-pig, it is apprpriate t recrd here the histry f these animals cvering the majr part f their life while subsisting n vitamin C supplies which, althugh fully prtective, d nt permit f their strage in significant quantities in the tissues. A number f such guinea-pigs were examined in this cnnexin and a few representative cases are described here in a summarized frm. All the animals t be discussed were females since the main purpse f the experiment t which they belnged necessitated the chice f this sex. The sex factr des nt, hwever, affect the present issue. These guinea-pigs were kept n a scrbutic diet ffered ad lib. which cnsisted f ats, bran, middlings, fish meal, barley meal and autclaved fiull-cream milk made up frm a dried pwder. Besides the basal diet the animals received daily 1 ml. f decitrated lemn juice which was apprximately equivalent t 5 mg. f ascrbic acid. B 1. This guinea-pig was under bservatin n the abve diet frm 28 July 1933 t 8 Nvember Its initial weight was 35 g. and it reached a maximum weight f 75 g. after 313 days. The animal was killed by stunning and bleeding at the end f 47 days and its tissues were examined fr the presence f ascrbic acid. N significant quantities f the vitamin culd be detected. Macrscpic signs f scurvy were nt fund at the pst-mrtem examinatin. B 77. This guinea-pig was under bservatin n the abve diet frm 31 July 1933 t 7 July Its initial weight was 31 g. and it reached a maximum weight f 97 g. after 221 days. The animal was killed by stunning and bleeding at the end f 45 days. N significant quantities f ascrbic acid were fund in

8 142 S3. S. ZILVA its tissues and n macrscpic signs f scurvy were fund at the pst-mrtem examinatin. B 87. This guinea-pig was under bservatin n the abve diet frm 7 September 1933 t 4 May 193, i.e. fr 99 days, when it died f pneumnia. N macrscpic signs f scurvy were fund at the pst-mrtem examinatin. Its initial weight was 34 g. and it reached the maximum weight f 985 g. after 49 days. B 99. This guinea-pig was under bservatin n the abve diet frm 15 January Its initial weight was 255 g. It is still alive and grwing, having reached the weight f 875 g. The abve typical experiments shw that the animals culd survive fr lng perids and reach bdy weights similar t thse attained by fully "saturated " guinea-pigs subsisting n a diet cntaining cabbage ad lib. withut having any appreciable stre f vitamin C in their tissues. The influence f vitamin C depletin in guinea-pigs n the cmplement f their serum. A number f years ag it was shwn by the writer [Zilva, 1919] that guineapigs existing n a scrbutic diet and receiving lw dses f the vitamin, insufficient t prtect them frm scurvy but high enugh t prlng the duratin f the disease fr several mnths, when immunized with a typhid rganism yielded sera with as high ambceptr and agglutinin titres as thse btained Table II. Wt. f guinea Cmplement dilutin pig Diet g. 1/12 1/1 1/24 1/32 1/48 1/4 1/9 1/128 1/192 5 days n scrbutic diet trace -,,1~,, trace -,,,, trace trace days n scrbutic diet trace trace - Mixed diet trace - 7 days n scrbutic diet i trace - Mixed diet days n scrbutic diet trace trace - Mixed diet days n scrbutic diet Mixed diet days n scrbutic diet Mixed diet 35 i trace - 1 days n scrbutic diet trace Mixed diet trace trace 19 days n scrbutic diet trace - -,,9,9, trace - Mixed diet trace - 2 days n scrbutic diet trace trace - Mixed diet trace trace - 7 days n scrbutic diet trace trace Mixed diet days n scrbutic diet trace trace - Mixed diet trace -

9 VITAMIN C REQUIREMENTS OF THE GUINEA-PIG 1427 frm animals receiving cabbage ad lib. Nr was there any diminutin in the cmplement activity f the bld f these animals subsisting n the restricted diet fr prlnged perids. Recently Marsh [193] asserted that in guinea-pigs depleted f vitamin C fr 7 days r less the cmplement, as determined by a standardized haemlytic serum, either disappeared r suffered reductin. N experimental details were given. Table II gives data f cmplement titratins btained with sera frm guinea-pigs which have been deprived f vitamin C fr 5, 7, 1, 19 and 2 days respectively. It is seen, as wuld have been expected frm the writer's earlier wrk, that n significant difference between the titres f the sera f these animals and thse f cntrl animals receiving a mixed diet with cabbage ad lib. culd be bserved. I am indebted t Dr D. B. Steabben fr checking the readings f the abve titratins. DISCUSSION. The minimum prphylactic daily dse which is capable f prtecting grwing guinea-pigs f abut 3 g. subsisting n a vitamin C-deficient diet frm macrscpic scrbutic lesins lies in the regin f 1 mg. [Zilva, 1932; Hirst & Zilva, 1933; and thers] and accrding t Gettsch [1928] and thers abut duble this quantity is required t bviate the develpment f micrscpic lesins in the pulp f the teeth f these animals. The abve results shw that nly traces f the vitamin are fund in the "selective" tissues f guinea-pigs, when a daily dse f 5-8 mg.-a dse 2-3 times as great as that which prevents the prductin f micrscpic lesins-is given fr 15 days. Furthermre, t attain the maximum cncentratin in the tissues such as is btained by the injectin f 5 mg. f ascrbic acid in a vitamin C-depleted animal, ten times the prtective dse f 2 mg. is necessary. It was fund in additin that n administering the prtective dse (2 mg.) t guinea-pigs n a scrbutic diet fr 1 days, the time taken by these animals t succumb t the disease was nt significantly less than that taken by guinea-pigs which received a daily dse f 2 mg. during the preliminary perid and even by guinea-pigs which cnsumed cabbage ad lib. previus t being placed n a deficient diet. Yet in bth the latter grups the cncentratin f ascrbic acid in the tissues was at a maximum befre the vitamin was withheld frm the diet. The accumulated vitamin C in the bdy f the guinea-pig therefre des nt act as a stre in the true sense f the wrd. The difference between the dse necessary fully t prtect the guinea-pig frm scurvy and the ne which brings abut the maximum "saturatin" f its tissues with the cnsequent eliminatin f a part f it in the urine, is therefre great. N evidence culd, hwever, be btained which indicated that guineapigs receiving daily dses ranging frm the minimum prtective dse t that necessary t bring abut the "saturatin" f their tissues with the vitamin, r even t that necessary fr discrete traces f ascrbic acid t appear in the " selective " tissues, deviated frm their " saturated" fellws in any discernible feature. If guinea-pigs in this zne f " unsaturatin" are in a cnditin inimical t their well-being it is f a character t subtle t be detected by the methds emplyed in this investigatin. Slight micrscpic changes in the teeth f guinea-pigs can be bserved after they have subsisted fr 7-1 days n a scrbutic diet [Zilva & Wells, 1919], by which time we nw knw that their tissues have lst the best part f their vitamin C, but as was seen tissues f animals receiving dses capable f preventing these micrscpic changes cntain ascrbic acid in minimum amunts and cnsequently very marked depletin f

10 1428 S. S. ZILVA guinea-pig tissues f vitamin C is n indicatin f the presence f even these micrscpic lesins. Great cautin must be exercised in interpreting the bearing f the abve results n human beings. Obviusly systematic experiments such as thse described cannt be carried ut n man. The nly infrmatin available in this respect are data recrded by a number f wrkers in cnnexin with the urinary excretin f ascrbic acid-results which are nt easy t crrelate. The cmparisn is made difficult by the fact that in determining the balance between the intake and utput f the vitamin, the amunt f ascrbic acid nt accunted fr varies with the magnitude f the ingested dse, the experimental subject and even with the same individual at different times under similar dietary cnditins [cf. Jhnsn & Zilva, 1934]. Nevertheless, frm the available evidence it may be cncluded in general that in human beings suffering frm declared scurvy the degree f "saturatin" with vitamin C' is lwer than that f the mst "unsaturated " subjects wh are free frm the disease and in gd and apparently nrmal health. This fact emerges particularly frm the investigatins f Schultzer [193] and Jhnsn & Zilva [1934]. The bservatins f Archer & Graham [193] in the writer's pinin als pint in the same directin. There is s far, hwever, n evidence, clinical r labratry, that such "unsaturated" subjects are in a wrse state f health than "saturated" persns. The present investigatin carried ut n animals mre susceptible t scurvy than man als failed t disclse any significant departures f the ne type frm the ther. It was in fact fund that " unsaturated" guinea-pigs receiving daily dses f vitamin C just sufficient t prevent the develpment f macrscpic and micrscpic scrbutic lesins succumbed t the disease in almst the same time after the daily dse was discntinued as did the fully " saturated" guinea-pigs. It is als significant t nte in this cnnexin that Schultzer's patients remained "unsaturated" fr a cnsiderable time after the disappearance f the scrbutic symptms, in spite f receiving such high dses as mg. per day. There seems therefre t be in human subjects als a wide margin f "unsaturatin" which has n bvius detrimental effect n the health f the individual. T apply t this zne f " unsaturatin" such expressins as "hypvitaminsis C ", "latent r ptential scurvy", "vitamin C sub-nutritin", "subacute scurvy" etc.-expressins which imply ill-defined pathlgical cnditins-is nt justified by ur present state f knwledge. Even if sme subtle inimical change be assciated with this particular state f "unsaturatin" the use f such expressins can nly tend t bscure the issue. It is apprpriate t add that the failure n the part f an "unsaturated" subject t excrete ascrbic acid in the urine after the administratin f a single dse, even a high ne, des nt yield any infrmatin as t the degree f his "unsaturatin ". SUMMARY. An ral dse f abut 5 mg. f ascrbic acid is necessary fr grwing guinea-pigs (f abut 3 g.) subsisting n a mixed diet befre the vitamin is excreted in the urine. In cntrast t this, excretin is brught abut by the injectin f a very much lwer dse. Graded dses f ascrbic acid were injected int vitamin C-depleted guineapigs and it was fund that demnstrable traces f the vitamin appeared in the 1 The expressin "saturatin" is used in the sense defined by Jhnsn & Zilva, i.e. an animal is said t be "saturated" when upn ingesting a cnstant daily amunt f ascrbic acid it excretes a mre r less cnstant daily amunt f the vitamin in the urine.

11 VITAMIN C REQUIREMENTS OF THE GUINEA-PIG 1429 tissues nly when a dse between 5 and 8 mg. was emplyed. The injectin f 2 mg. f the acid prduced a cncentratin in the tissues equal t that bserved when 5 mg. were injected and this was cnsidered the maximum cncentratin attainable under the experimental cnditins described. Similar results were btained when crrespnding dses were administered per s fr 15 days t guinea-pigs n a scrbutic diet. Ascrbic acid first appeared in the urine when 2 mg. were injected, i.e. when the maximum cncentratin f the vitamin in the tissues was reached. As the dse was increased the amunt f ascrbic acid excreted als rse. The time taken by guinea-pigs t succumb n a scrbutic diet after having previusly received fr 15 days whilst n this diet 2 mg. f ascrbic acid (the prtective dse) daily was abut the same as that taken by animals which were ffered cabbage ad lib. during the preliminary perid. Previus "saturatin" f the tissues therefre des nt delay the fatal terminatin f the disease. Guinea-pigs n a scrbutic diet receiving daily dses f 1 ml. f decitrated lemn juice (abut 5 mg. f ascrbic acid) were bserved t live fr a number f years during which time they attained very high weights, althugh the tissues f such animals cntained nly minimum amunts f vitamin C. The cmplement activity f guinea-pig bld suffers n reductin when the animals are depleted f vitamin C r even when they are in a scrbutic cnditin. Thanks are due t Messrs Hffmann La Rche, Ltd., fr a gift f ascrbic acid. REFERENCES. Archer & Graham (193). Lancet, i, 71. De r (193). Hppe-Seyl. Z. 24, 179. Euler (1935). Sartryck ur Sven8k kem. Tidskr. 47, 1. Fx & Levy (193). Bichem. J. 3, 211. Girud, Leblnd, Ratsimamanga & Rabinwicz (1935). C.R. Sc. Bil., Paris, 12, 33. Gettsch (1928). Quart. J. Pharm. 1, 18. Hess & Benjamin (1934). Prc. Sc. Exp. Bil., N.Y. 31, 855. Hirst & Zilva (1933). Bichem. J. 27, Jacbsen (1935). Skand. Arch. Physil. 72, 259. Jhnsn & Zilva (1934). Bichem. J. 28, Marsh (193). Nature, Lnd., 137, 18. Schultzer (193). Acta med. scand. 88, fasc. II-iv, 317. Van Eekelen, Emmerie, Jsephy & Wlff (1933). Acta brev. neerl. Physil. 3, 18. Zilva (1919). Bichem. J. 13, 172. (1932). Nature, Lnd., 129, 943. (1935, 1). Bichem. J. 29, 112. (1935, 2). Bichem. J. 29, 23. & Wells (1919). Prc. ry. Sc. B 9, 55.

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