15
Critical Review VITAMIN D MILK--A R~SUM]~ ~AMUEL ~R//EDMAN, ~V~.D. ]~OSTON, MASS. INTI~ODU CTION DVANCES in our knowledge of vitamins, especially the fat-soluble vitamins, have been outstanding in the reMm of nutritionM prob- lems. Less than a decade has passed since the first efforts toward augmenting the natural vitamin D content of milk were initiated. During these few years great strides have been made in both the com- paratively simple problem of producing what is now termed "vitamin D milk" and in the more complex one of determining to what physical or physicoehemical changes this addition to our antirachitic armament owes its potency. The rapidity with which these advances have been made, the never-ending succession of theory evolved, and facts deter- mined, make a complete knowledge, understanding, and proper evalu- ation of the subject somewhat difficult for the practicing clinician. Consequently, it was felt that a rSsum6 of the work that has led up to oar present vitamin D milk and its clinical application might be of value. Cow's milk, the chief dietary substance of artificiallyfed infants, has been endowed with a nmnber of virtues by a kind Pvovidence, bu~ it does exhibit a few deficiencies;one of thes~ is a relative lack of the antirachitie factor, vitamin D. Consequently, rickets has always been an extremely common condition in infancy and childhood and even in recent times, in spite of our newer knowledge of nutrition and increas- ing number of antiraehitic medicinal agents, has been and is fairly prevalent due either to the failure to offer antirachitic substances to babies or to the offering of insufficient amounts of these substances Thus, a milk in which the. vitamin D content is s~ffficiently high to af- ford complete protection against rickets is, a priori, distinctly advan- tageous inasmuch as it represents an automatic form of prophylactic therapy. Efforts to augment the natural vitamin D content of cow's milk have thus far included : I. Direct irradiation of the milk. II. Feeding of vitamin D concentrates to cows. III. Irradiation of cows. IV. Direct addition of vitamin D concentrates to the milk. I. DIRECT IRRADIATION OF hIILK In 1924, Alfred Hess ~ announced to the American Pediatric Society his discovery that antirachitic properties could be imparted to cer- tain substances by exposure to ultraviolet irradiation. The value of 678

Vitamin D milk—A résumé

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Page 1: Vitamin D milk—A résumé

Critical Review

V I T A M I N D M I L K - - A R~SUM]~

~AMUEL ~R//EDMAN, ~V~.D. ]~OSTON, MASS.

INTI~ODU CTION

DVANCES in our knowledge of vitamins, especially the fat-soluble vitamins, have been outs tanding in the reMm of nutr i t ionM prob-

lems. Less than a decade has passed since the first efforts toward augment ing the na tu ra l vi tamin D content of milk were init iated. Dur ing these few years great strides have been made in both the com- parat ively simple problem of producing what is now termed "v i t am in D mi lk" and in the more complex one of determining to what physical or physicoehemical changes this addition to our antirachitic armament owes its potency. The rap id i ty wi th which these advances have been made, the never-ending succession of theory evolved, and facts deter- mined, make a complete knowledge, unders tanding, and p roper evalu- at ion of the subject somewhat difficult fo r the pract ic ing clinician. Consequently, it was fel t tha t a rSsum6 of the work tha t has led up to oar present v i tamin D milk and its clinical applicat ion might be of value.

Cow's milk, the chief dietary substance of artificially fed infants, has been endowed with a nmnber of virtues by a kind Pvovidence, bu~ it does exhibit a few deficiencies; one of thes~ is a relative lack of the antirachi t ie factor, v i tamin D. Consequently, r ickets has always been an ex t remely common condit ion in in fancy and childhood and even in recent times, in spite of our newer knowledge of nut r i t ion and increas- ing number of ant i raehi t ic medicinal agents, has been and is fa i r ly prevalent due ei ther to the fai lure to offer ant irachit ic substances to babies or to the offering of insufficient amounts of these substances Thus, a milk in which the. v i tamin D content is s~ffficiently h igh to af- ford complete pro tec t ion against r ickets is, a priori, dis t inct ly advan- tageous inasmuch as it represents an automatic form of prophylac t ic therapy.

Efforts to augment the natural vi tamin D content of cow's milk have thus far included :

I. Direct i r radiat ion of the milk. II . Feeding of vi tamin D concentrates to cows.

I I I . I r radia t ion of cows. IV. Direct addition of vi tamin D concentrates to the milk.

I. DIRECT IRRADIATION OF h I I L K

In 1924, Alf red Hess ~ announced to the American Pedia t r ic Society h i s discovery tha t ant i rachi t ic proper t ies could be impar ted to cer- tain substances by exposure to u l t rav io le t irradiat ion. The value of

678

Page 2: Vitamin D milk—A résumé

CRITICAL R E V I E W 679

l ight r ays in the t r ea tmen t of r iekets had sugges ted to him the prob- lem whether exposure of iner t substanees to ul t raviolet rays could en- dow them with antiraehit ie poteney. Consequently, various fluids were subjected to irradiatio~aY Co.ttonseed oil was exposed in ~ Pe t r i dish for one ho.ur a t a distance of one foot f rom a mercury vapo.r quartz lamp and was then fed in amolmts of 0.1 and 0.25 e.c. dai ly to ra t s on a s t anda rd rachi t ie diet. Control animals were given nonir radi - a ted oil. I t was found tha t the oil t h a t had been i r rad ia ted acquired proper t ies which pro tec ted the ra t s aga ins t r i cke t s while the eontrol animMs developed the disease in the usual manner . Similar resul ts were obtained when linseed oil was i r radiated. The rats which were protected showed a serum inorganic phosphorus content of 6.84 milli- grams per cent in comparison with 3.22 mil l igrams per eent in the raehit ie eontrols. I r r ad ia ted minerM oil, in contradist inction to cotton- seed and linseed oil, failed to offer any protection. At that time, t Iess felt i t was too ear ly to discuss the nature of the change brought about in these fluids whieh impar ted to them the ant i raehi t ie p(>teney.

I tess a, ~ then proceeded to. investigate whether the same results could be obtained in growing plants, whether s imilar differences could be demonstra ted be tween vegetables and p lan t s g rown in the da rk and those g rown in the l ight and subjec ted to it~radiations f rom a m e r c u r y lamp. These problems were subjeeted to exper imenta l analysis. Whea t was g rown in the l a b o r a t o r y both in darkness and in l ight wi th i r rad ia t ion given daily for one hour. The whea t was then fed to rats, in dai ly amounts of ten grams, in addi t ion to the rout ine raehi t ic diet. The r a t s receiving the whea t g rown in darkness (et iolated) de~eloped rickets while those ingesting the i r rad ia ted wheat were protected.

Similarly, and to rtfle out any r61e or fae tor tha t differences in growth might play, ord inary plucked lettuce leaves were i r radia ted and

�9 fed in s imilar amounts to. rats. The same resul t was obtMned as with wheat : the i r radia ted leaves prevented rickets while the nonirradias lettuce fai led to offer protection. Titus, an ant i rachi t ic factor was pro- duced, by irradiat ion, both in vi t ro and in the growing plant..

Hess" work was closely fol lowed by tha t of S teenboek ~, c who demon- s t ra ted tha t growth-promoting and bone-calcifying propert ies could be impar ted to r a t s ' rat ions by exposure to the quartz mercury vapor lamp. When the rat ion was i r radia ted at a distance of about two feet for ten niinutes, it produced the same eff'eet as when the animals were i rradi- ated direetly. This act ivat ion took place whether the rat ion was ir- radia ted in an open dish or in a quartz flask but not in a brown glass bottle. The po tency was not subsequent ly des t royed by subjec t ing the food to a vaeumn, hea t ing a t 96 ~ C. for for ty-f ive minntes or allow- ing to s t and fo r twen ty - four hours. ( I t was l a t e r demons t ra ted t ha t i r r ad ia ted olive oil could s tand fo r ten months wi thout losing its potencyL) The previous observa t ions of Goldblat t and So.ames were also confirmed when it was demons t ra t ed by feeding exper iments tha t tissue such as lung', liver, or muscle t aken f rom direct ly i r r ad ia ted animals had ant i raehi t ie propert ies . F u r t h e r m o r e , such tissue could also be endowed direct ly wi th g rowth -p romot ing and bone-eale i fying proper t ies b y exposure to u l t ravio le t light. Pr 'oteet ion against r ickets in the animals reeeiving the i r r ad ia ted substanees was not only judged by clinical s t andards but was confirmed by histologie sections s and by chemical analysis of bones. The femora of control animals showed

Page 3: Vitamin D milk—A résumé

680 T H E J O U R N A L O]~ ~ P E D I A T I ~ I C S

an ave rage ash content of 47.2 per cent while those of the animals receiving i r rad ia ted ra t ions had an ave rage ash content of 53.9 pe r cent2

The n u m b e r of substances capable of ac t iva t ion b y i r rad ia t ion was fu r the r amplified and included such diverse mater ia ls as :9, no, 11 vege- table oils, wheat , corn, flour, yeast , egg yolk, but ter , p lants and vege- tables, animal tissues, and wha t is of especial interest fo r our immedi- ate discussion, milk. Thus a firm founda t ion was laid for f u r t he r exper imenta l and clinical work.

The knowledge tha t exper imental animals could be protected against and cured of rickets by the ingestion of i r radia ted food, including milk, was soon pu t to clinical applieation. CowelP ~ was probably the first to give a clinical repor t on the feeding" of i r radia ted milk to. infants. Three young" children, f rom one and a ha l f to two. and a ha l f years of' age, with active rickets were the first human subjects. They were given a diet whose only ant i raehi t ie value lay in the daily ingestion of a~ p in t of milk. The two infants who received i r radia ted milk (exposed for' twenty minutes at a distance of three feet f rom a mercury vapor lamp) showed definite improvement a t the end of four weeks. The thi rd infant , who acted as a control and received noni r rad ia ted milk, showed no. change. K r a m e r a3 followed short ly thereaf te r with another more careful elinieal analysis. I r rad ia ted milk was given to eight raehit ic infants. I ( r a m e r ' s method o.f i r radiat ion consisted of pour ing the milk into a large shallow dish and i r radia t ing with a mercu ry vapor lamp at a distance of two feet fo r not more. t han two hours. I I e f e l t then, however , t ha t the t ime was p robab l y excessive and could be reduced to ten or t w e n t y minu tes ! (This emphasizes the differences in technic of i r rad ia t ion of a few years, ago and of the. p resen t day which will be discussed short ly.) In the chi ldren rece iv ing the i r radia ted milk, heal ing of rickets was demonstrable by the th i rd week and was usually well marked Ly the four th week. The phoslJhomls content of the blood was raised, reaching a normal level about the four th week of t reatment . Control patients, who were on the same diet save for lack of act ivat ion of the milk, showed no improvement . Gyhrgy ~4 gave the first German repor t on. the efficacy of i r rad ia ted milk in the t r ea tment of rickets. I r r ad i a t ed milk was given to eighteen children with florid rickets with marked im- pro.vement in sixteen, judging by clinical, blood, and x-ray findings.

Some of the more impor tan t references to articles tha t followed la ter are appended. 1~-~5 Most of the repor ts at tested to both the prophylac- tic and curative efficacy of i r radia ted milk al though m a n y investigators found it to. be a somewhat slower curat ive agent than viosterol2 a-Ss I n Germany, especially, due to the na tu ra l ly low vi tamin D content of the milk produced there, a good deal. os invest igat ive work has been done with this antirachit ie agent and an abundance of clinical repor ts has emanated f rom tha t country.

Wi th the clinical value of this type of milk definitely established, fur- ther advances in this count ry have consisted chiefly in improving meth- ods of irradiat ion. Much of this work has been done by Supplee and his a.ssociates. ~9-4~ A ful l analysis of the physics, energy quotients, and ro.eehanieal difficulties involved would be unnecessary for a medical dis- cussion. Suffice it to say tha t the wave lengths o.f the rays which have pro~ed efficient in the i r radia t ion of milk and other substances are of

Page 4: Vitamin D milk—A résumé

CaIT~CA~ aEVIEW 681

the magni tude of 300 ~ or 3,000 A. l~ays above 313 t~t~ in length are of pract ical ly no value. I t makes but little difference whether the source of the ul t raviolet rays is in the form of an are (such as carbon or mag- nesitum) ]amp or a quartz mercu ry vapo.r lamp provided comparable amounts of energy are used. Supplee has shown that the degree of ant irachit ic potency impar ted to the milk bears a direct relationship to the amount of energy (in ergs) applied up to a certain limit, beyond which f u r t h e r increases in ene rgy appl ied increase the v i tamin D con- ten t but little. The efficiency of the reac t ion is p robab ly grea tes t dur ing the first few seconds of exposure. The present method of ir- r ad ia t ion consists of un i fo rmly exposing the milk in the form of a mov- ing film which receives rays f rom carbon arc lamps at constantly chang- ing angles of incidence, varying" f rom 0 to 90 ~ The thickness of the film of mi lk is about 0.4 ram. and the t ime of exposure does not exceed s ixteen seconds. Each cubic cent imeter of mi lk receives about two and 0he-half million ergs of r ad ian t energy. The resul t ing fo rma t ion of v i t amin D represents a l a rge p e r c e n t a g e of the m a x i m u m obtain- able, ye t the exposure is so shor t as not to cause detectable changes in tas te or smell or v i tamin A content. Seholl, in Germany, has devised a sys tem of i r r ad ia t ing the mi lk in the presence of an a tmosphere of carbonic acid gas, in order to avoid changes in taste and smell, bu t wi th the presen t short exposure, this seems unnecessary.

W h a t chemical, physical, or physicochemical changes are b rough t about by the i r rad ia t ion of milk or other substances tha t enable them to assume ant i rachi t ic properties.~ H o w can we correlate two such u t t e r ly different fac tors as v i t amin D and u l t rav io le t rays? The an- swers to these questions, while not as yet fu l ly complete, have been buil t up on a series of pa ins tak ing exper iments evalua ted b y the clear logic of several br i l l iant workers .

It m a y be recal led tha t Hess in his ear ly exper iments was able to endow a la rge num ber of seemingly he te rogeneous substances wi th ant i rachi t ie powers. The question immedia t e ly arose whether the ir- r ad ia t ion pe r se was the pr ime fac to r in the act ivat ion. I t was known that ozone was formed when the ul t raviolet lamps were used and in order to rule out this substance as the causat ive agent of ant i rachi t ic potency, ozonated wa te r was fed to rachi t ie rats . There was no im- p rovemen t in thei r condition. Similarly, hyd rogen peroxide migh t be fo rmed but it, too, was found to be devoid of a n y ant i rachi t ie value. I t was then shown, by f rac t ioniza t ion of vege tab le oils, as Zucker 43 had prev ious ly demons t ra t ed in cod l iver oil, t ha t the active i r radiable pr inciple was present only in the nonsaponif iable por t ion of the oil. 4~ This pr inc ip le was shown to be a s~erol, p r e s u m a b l y cholesterol. Rosenheim and Webs t e r ~5 also found tha t cholesterol could be acti- va ted and endowed with ant i rachi t ic proper t ies . Substances r ich in cholesterol, such as skin, could be rendered h ighly antirachit ic . ~~ 4~ Tha t the resul t ing change was p robab ly a s imple chemical or physieo- chemical one r a the r t han ~ complex biologic process was a t tes ted to by the fac t t ha t de tached skin, obviously en t i re ly depr ived of connec- t iou wi th nervous or c i rcu la tory system, could be i r rad ia ted and ren- dered ant i rachi t ic . 1~ In the p l an t world, where there is no cholesterol, it was demons t ra t ed t h a t the sterol, cor responding to cholesterol in the animal kingdom, is phytosterol .

Page 5: Vitamin D milk—A résumé

682 T]-IE J O U R N A L OF PEDIATIr

Cholesterol, it seemed then, was the p r i m a r y substance which in some way was changed dur ing the process of i r rad ia t ion and, wi th this metamorphosis , assumed ant i raehi t ie propert ies . Cholesterol, it may be stated, is an opt ica l ly act ive unsa tu r a t ed sterol whose struc- tural formula, while not definitely settled, is probably represented by :~7

H/V~e.. C H~. C llZ. ~ Hp. C HMe2,

FlO

Of particular interest is the presence of the hydroxyl group and the double bond between two carbon atoms. Tile presence of both of these has been shown to be necessary for activation by irradiation. Thus, compounds in which the double bond is saturated by hydrogenation, i.e., dihydrosterol cannot be activated. I~ 4s, 49 Nor can compounds in which the hydroxyl group is replaced by other groups, i.e., cholesterol chloride, ~~ be act ivated. (S t r ic t ly speaking', this is not absolu te ly t rue as the esters of cholesterol, such as cholesterol acetate, have been ac- t ivated, but this is p robab ly due to the fac t tha t the esters are very easily hydrolyzed, thus restoring' the h y d r o x y l group. TM 52)

However , the work was still by no means, completed. I t was found by Hess and Windaus ~a and Rosenheim and Webster '5~, 5~ that highly purif ied cholesterol lost its power of assuming ant i raehi t ie ac t iv i ty on i r radia t ion. They felt , then, tha t there was some. con tamina t ing substance closely associated with cholesterol to which the l a t t e r owes i ts act ivi ty. This had been prev ious ly suspected b y Zueker '~a and by Steenbock. 9 I t was found tha t when this contaminat ing substanee was separa ted and i r radia ted , it had a po tency at least 500 t imes tha t of i r r ad ia ted cholesterol25 In the meant ime, Hess and Windaus, ex- perimenting ' wi th a large number of cholesterol derivat ives, found tha t only ergosterol was capable of' being rendered ant i raehi t ic by, ir- radiation.a6, ~7

As m a y be suspected, the con tamina t ing substance in t imate ly asso- ciated with cholesterol and ergosterol are one and the same. I t seems at present tha t e rgos tero l is the only substance capable of assuming ant i raehi t ie proper t ies dur ing the. process of i r radiat ion. All sub- s tances owe their power of becoming ant i raehi t ie fol lowing i r radia t ion to the con tamina t ing presence, in minute quantit ies, of ergosterol , which appa ren t ly is the p recurso r of v i t amin D.

Ergos te ro l is a ster'ol closely re la ted to cholesterol but containing three unsa tm.a ted double bonds and m a y p robab ly be represen ted by the fo rmula :,7, ~s

Me/CHMe..CH ~ C H. CliMe. CHMe~,

Exact ly Mtat happens dur ing the process o.f i r radiat ion is not yet definitely known. Oxidative changes are rMed out by the fac t tha t i r radia t ion is equally successful in produeing antirachit ic potency in an atmosphere of nitrogen. Probably no chemical change per se takes

Page 6: Vitamin D milk—A résumé

C lgITICAL R E V I E W 6 8 3

place as following irradiat ion, the melt ing point, optical rotation, and composition according to analysis remain unaltered.59 There has, how- ever, been demons t ra ted a definite a l t e ra t ion in spect ra l absorp- tionSO, sl, 62 Hess 63 fe l t tha t some isometr ic change took place on ir- radiation. Wejdling', G4 believing' t ha t the crystall ine s t ructure of er- gosterol m igh t be changed in the process of act ivat ion, fe l t tha t the change should be demonstrable by measuring" the change in the distance between atoms i n v a c , w by means of x-rays. He found, instead, tha t at the moment of strongest ac t iva t ion no c rys ta l l ine s t ruc tu re was ]eft, t ha t as soon as ergosterol was. t r a n s f o r m e d by i r radia t ion into the vitamin, the atoms lost tlheir r egu la r a r rangement . This may explain why once the substance is overexposed to u l t raviole t i r radiat ion it loses its act ivi ty and can never be reactivated.

To recapi tu la te : substances, including" milk, which can be rendered antirachit ie by i r radiat ion owe this p rope r ty to the presence of vary ing contaminat ing amounts of ergosterol, which is the forerunner of v i tamin D. I t is the only substance which has thus f a r been definitely shown to be capable of activation. Precisely what physical or physicoehemical change takes place on i r radia t ion is not yet known.

II. F E E D I N G OF V I T A M I N D C O N C E N T R A T E S TO C O W S - - ~ Y E A S T ~ M I L K

In the ear ly stages of the work on vitamins, F u n k advanced the sug- gest.ion tha t the secretion of these factors in milk was controlled by their presence in the food of the lactat ing animal, t towever, i t was not until a number of years had passed before a t t empts were made to in- crease the v i tamin D content of cow's mi lk by the a d d i t i o n o f large amounts of v i tamin D to the cow's diet.

In 1924, Lesn~ and Vigliano. 65 added 500 g~a. of cod l iver oil dMly to a cow% feed. When tes ted exper imenta l ly , the milk produced showed increased ant i rachi t ic potencyl exhibit ing' good cura t ive and p ro tec t ive powers aga ins t r ickets. The fol lowing year, W a g n e r GG could demons t r a t e no increase in the antira.chitic po tency of milk of cows fed f rom 50 to 300 gin. of cod l iver oil daily. Gotding et al., 6~ however, fe l t t ha t the addi t ion of 8 ounces of cod liver' oil dai ly to a cow's win te r ra t ion did raise the ant i rachi t ic po tency somewhat. The addi t ion of 2: ounces dai ly had no demons t rab le effect.. 6s Fa i lu re to in- crease the v i t amin D content of cow's mi lk b y the addi t ion of cod l iver oil to the feed m a y well be expla ined b y the fac t tha t such large. doses as a re necessary decrease m a r k e d l y the f a t moie ty of the mi lk in which the ant i raehi t ic f ac to r lies.

Similar exper iments were being' conducted abou t the same t ime on humans. Hess fed cod l iver oil to both p r e g n a n t and lac ta t ing women in an a t t e m p t to supply addi t ional v i t amin D to the. young. The resul ts were disappointing" and Hess concluded tha t r ickets in the offspring could not be p reven ted by improv ing the mo the r ' s diets e i ther dur ing p regnancy or lactat ion. 6','~~ Weech ~1 pe r fo rmed a simi- lar expe r imen t on a g roup of l ac ta t ing mothers (colored) and thought tha t while the adminis t ra t ion of cod l iver oil to the laeta.ting mother is not a sa t i s fac to ry method of prevent ing ' r i cke t s in the young, a cer- ta in amoun t of addi t ional ant i raehi t ic subs tance does pass into the milk, e f fee t ing a rise in the serum calc ium-phosphorus p roduc t in the in fan t and lessening x - r a y evidences of the disease. Oe r s t enbe rge r ' s ~2 efforts to increase the v i t amin D content of the mi lk of wet-nurses b5

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684 T H E J O U R N A L OF P E D I A T I ~ I C S

feeding them a ha l f ounce of cod l iver oil dai ly resul ted in failure. Thus we see tha t the addi t ion of v i t amin D in the fo rm of cod l iver oil to the diet of l ac ta t ing a n i m a l s - - b o t h bovine and h u m a n - - p r o v e d , en the whole, unsuccessful.

However , with the in t roduct ion, wi th in a few years, of such highly po ten t v i t amin D concent ra tes as i r r ad ia t ed ergosterol and i r r ad ia ted yeast , success seemed more imminent . Euf inger et al. 7a were the first t o - i nc r ea se sa t i s fac tor i ly the v i t amin D content of h u m a n mi lk by feeding vigantol ( i r rad ia ted ergosterol) to lactat ing mothers. Af te r only a few days of this s u p p l e m e n t a r y feeding, both the eolost rum and mi lk became highly ant irachi t ic . Similarly, Gers tenberger '74 gave 15 rag. of i r radiated ergosterol daily to. wet-nurses. Raehitie infants who received f rom one pint to one qua r t of this milk daily showed rap id healing. He fel t t ha t a dai ly dose to l ac ta t ing mothers of f r o m 2 to 3 rag. of i r rad ia ted ergosterol would be adequate to give the i r milk good ant i rachi t ie proper t ies .

Wach t eF ~ was the first to feed i r r ad ia ted yeas t to cows and the reby n~larkedly increase the v i t amin D content of the milk produced. His work was closely fo l lowed by the inves t igat ions of Steenbock and his eoworkers . 76 Steenbock fed 200' gm. of i r r ad ia ted yeast dai ly to cows. The mi lk produced showed high ant i raehi t ic po tency as measured by feed ing exper iments wi th rats. Whi le the f emora of groups of ra ts cn o rd ina ry milk showed an ave rage ash content of f rom 34 to 37 per cent, the bones of the animals on " y e a s t " milk showed an ash content of 52 per cent, demonstra t ing the marked calcifying proper t ies of the " y e a s t " milk. When the daily amount of i r radia ted yeast was reduced to 10 gin., the resul t ing bu t te r f a t of the milk produced was na tura l ly much less calcifying but was still superior' to the but te r fa t produced on an unsupplemented diet. Steenboek felt tha t this amount of i r radia ted yeas t p roduced about the same effect as the dai ly ingest ion of 6 ounces of cod l iver oil.

Thomas and MacLeod 77 conducted similar experiments on twenty-one cows divided into seven groups. One group was left on an unsupple- men ted diet as a cont ro l ; the n e x t t h ree groups were given, respec- t iveIy, 10,000, 30,000, and 60,000 r a t uni ts of v i tamin D in the fo rm of i r rad ia ted yeast ; and the last three groups were fed 15,000, 45,000, and 135,0'00 ra t units of v i t amin D in the fo rm of i r r ad ia ted ergosterol . The bu t t e r fa t s of the i r mi lks were then compared by feeding to ra ts on a raehit ic diet and the antiraehit ic potencies measured by the line test. The relat ive potencies of the b u t t e r f a t s are shown in Table I.

TABLE I

RELATIVE, ANTIRACIIITIO I~AT' U'NITS FED

POTENCY 0]~ BUTTER I~AT

Control 10,000 units as irradiated yeast 30,000 units as irradiated yeast 60,000 units as irradiated yeast 15,000 units as irradiated ergosterol 45,000 units as irradiated ergosterol

135,000 milts as irradiated ergosterol

1 2 8

16 2 4

16

Krauss ' , Bethke's , and Monroe 's 7s experience, with i r radia ted er- gosterol only, proved to be similar. A s tudy of the antirachit ic poten-

Page 8: Vitamin D milk—A résumé

CI~ITICAL I~]~VIEW 685

eies of the b u t t e r fa ts of mi lk p roduced wi th increas ing adminis t ra t ion of i r r ad ia t ed ergosterol showed the fol lowing compara t ive values :

CII.ITICAL ])ALLY LEVEL 01~ B. l~.

RAT U N I T S 01~ VITAMIN D (TO Pt~0D:UCE DEFINITE EVI- RAT U N I T S FER

:VED TO COWS DAILY DENCES Ot~ HEALING IN GM. Ol~ B. F. THE RAT)

0 600 rag. 0.17 7,500 350 rag. 0.29

15,000 200 rag. 0.50 100,000 60 rag. 1.67 200,000 40 rag. 2.50

Thus, we see t ha t according to the i r figures, the ant i raehi t ic po tency was increased a p p r o x i m a t e l y f if teen t imes by the dai ly adminis t ra t ion of 200,000 uni t s of v i tamin D as i r r ad ia t ed ergosterol . J udg ing f rom Thomas and ]V[acLeod's resul ts and those of H a r d e n b e r g h a n d Wil- son, ~9 which fol low short ly, these are r ea l ly r a t h e r conservat ive in- creases.

The l a t t e r inves t iga tors fed i r r ad ia ted yeas t and i r r ad ia ted ergos- terol as s u p p l e m e n t a r y v i tamin D offerings. The ergosterol was fed in da i ly amoun t s of 100,00'G and 200,000 r a t un i t s and the yeas t in dai ly amoun t s of 30,000 and 60,000 ra t uni ts wi th the comparat ive in- creases in antirachit ic potency as shown in Table I I .

TABLE II

RAT U N I T S OF ' AMOUNT Ol~' B. 1 ~. RELATIVE

SUPPLEI~ENT I~ED VITAMIN D CONTAINING FED DAILY 1 RAT UNIT POTENOIES

Control . . . . . . 8.0 gm. 1 Irradiated ergosterol 100,000 0.5 gm. 16 Irradiated ergosterol 200,000 0.25 gm. 32 Irradiated yeast 30,000 0.5 gm. 16 Irradiated yeast 60,000 0.25 gm. 32

I r r ad ia t ed yeast seems to be the supplement of choice as it has proved to be about three times as effective as i r radia ted ergosterol on the. basis of the number of ra t units fed.

Clinical experiences wi th yeas t milk ( including in this t e rm milk produced with either yeast or ergosterol suppIements) have, with rare exception, been excellent al though Gers tenberger ' s ~~ repor t on tile clini- cal use of the milk produced in K r a u s s ' feeding experiments seemed somewhat disappoint ing. Two raehi t ic in fan ts who were. given one pint of this milk daily (plus one p in t o.f o rd ina ry skimmed milk and sufffeient carbohydrate to meet their calorie needs) showed rather' slow healing. The bones of the two. infants we.re not completely healed at the end of ten and eleven weeks, r espec t ive ly ; ' the blood calcium levels became normal only after' the tenth week, and the serum phosphorus had not a t ta ined a normal value by the end of the experiment. Gersten- berger felt, f rom comparat ive clinical experience with cod liver oil, tha t a p int of this fortified milk contained slightly less than half a teaspoon- ful of cod l iver oil equivalence.

Other r epor t s have definitely shown this mi lk to have excellent pro- teetive and curat ive powers. Hess and his eowo.rkers 81 repor ted elini-

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cal resul ts on a group of 102 infants , m a n y of them colored or' I ta l ian, who were given milk f rom cows fed 100,000 and 2.00,000' units of ergosterol and 30,000 and 60,000 uni ts of i r r ad ia ted yeast. This m i lk , as we have. seen, is s ixteen and th i r ty - two times as potent as o rd inary milk, and assays 80 and 160 uni ts of v i t amin D, respect ively, to the quart . All but the weake r yeas t mi lk p roved quite sa t i s fac to ry f rom both the eura t ive and pro tec t ive s tandpoint . Two repor ts by Wy- mane2, sa have been quite favorable . Kramer"s and Cdt t leman 's *~ ex- 1)erienee with the use of a yeas t mi lk t r ea t ed so as to contain only 55 and 40 units, respect ively, of v i t amin D was eminent ly sa t i s fac to ry f rom the curat ive s tandpoint .

III. IRRADIATION OF COWS

The efficacy of exposure of the lactating animal to, light rays as a means of increasing the vitamin D content of its milk has been both opposed and upheld. Luce.SS, s6 was one of the first to investigate the relative influence of diet and exposure to sunlight on the growth-pro- mot ing and antiraehit ic propert ies of milk produced by cattle. She found that milk f rom pasture-fed cattle had a definitely higher anti- rachit ic value than milk f rom cows tha t were stall-fed in the dark. However, it was found impossible, by means of sunlight alone, to raise the antirachit ie value o.f the milk if the diet of the cows was deficient. She concluded, therefore, tha t exposure to sunlight plays o.nly a minor, subsidiary rSle in the p roduc t ion of v i t amin D factors, in the milk. Chick and Roscoe, s7 however, expressed the opinio.n, following experi- menta l work, tha t while the diet was the imper t an t factor in the pro- due t ion of v i tamin A, the degree of insolation played the more impor- t an t r61e in the determinat ion of the ant iraehit ic potency of the milk produced. Supplee and Dow ss found tha t summer milk contained some- what higher amounts of v i tamin D than winter milk. AgMn, it is diffi- cult to evaluate the rSle p layed by the amount of sunlight and the type of food ingested dur ing these two seasons, t towever, none of these opinions are of much aid in determining the extent of increase of vita- min D tha t might be expected f rom direct i r radiat ion of the animal by means of a mercury v a p o r or carbon arc lamp, inasmueh as the sun ' s rays when they str ike the earth are of but meager potency in the ul t ra- violet range.

Steenbock *s proeeeded to i r radia te an animal directly and used for this purpose a lacta t ing goat. The ha i r was elipped closely, and the animal was i r radia ted daily. I t was found that, while previously a dai ly ingestion of 12 e.e. of its milk was necessary to cause incipient ealcimn deposit in exper imenta l animals, following i r radia t ion (as ear ly as four days af ter i r radia t ion was begun) only 2 c.c. were required to produce the same effect.

L a t e r and similar exper iments wi th cows, however, produced, en- t i re ly negative results, s~ Cows were exposed dMly for one hour a t a distance of f rom 20 to 30 inches f rom quar tz mercury vapo r iam.ps, var ious regions such as head, baek, and udders being i rradiated. No demonstrable increase in the v i tamin D content of the milk was ob- tained. We are at a loss to explain this negative result as pract ieal ly all other investigators have been able to effect an increase in the artti- raehit ic potency of the milk by direct i r radiat ion of' the laetatir~g ani-

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mat. Thus, Go.wen et at. 9z repor ted tha t definite curaZive powers were impar ted to cow's milk by exposure of the an imal ' s back to the ul t ra- violet lamp for as short a period as fifteen or th i r ty minutes daily. Falke~flleim 92 was also successful in rais ing the vi tamin D content by means of direct i r radiat ion of the animal. W a g n e r and his eoworkers 9a i r r ad ia t ed cows daily for one hour at a distance of 50 era. f rom the tdtraviolet lamp. The milk thus produced was given to eight infants, six of whom were entirely protected against rickets and two, a pa i r of twins, began to show ear ly craniotabes at the end of three months. I t was fel t tha t the milk was sa t is factory for o rd inary routine prophylaxis against rickets.

A t t empts to raise the v i tamin D content of breast milk by i r radia t ion ef the lac ta t ing mother have also proved successful, t tess 94 i r radia ted a lacta t ing woman every other day for a month. Previously, 25 c.c. of her milk fed to rachitic rats daily had fai led to cause any improvement in thei r condition. Following irradiat ion, the same quant i ty of milk caused definite improvement and raised the phosphorus content of the blood of exper imental animals f rom an average of 1.98 rag. pe r 100 c.c. to 5.61 rag. pet" 100 e.e. (The phospho.lals content of the milk itself was ~:ot found to be increased.) Hess pointed out tha t there was an increase in the nonsaponifiable f ract ion of the milk. Gerstenberger ~2 and Lesn~ and Dreyfus-Sge 9s have repor ted equally successful experiments.

The latest repor t on the i r radia t ion of cattle comes f rom Mitchell and his groupY s Carbon arc lamps were a r ranged in the stalls so that each cow's udders were exposed on one side to the lamp for fifteen minutes daily. The lamps were at a distance of about two feet f rom the animals and were in this o.rder:

Cow: L a m p : Cow: Cow: L a m p : Cow: Cow: Lamp: Cow.

Biologic assay of the milk f rom i r radia ted animals showed the anti- rachit ie potency to. be approx imate ly 22 ra t uni ts of v i tamin D per quar t as compared with 5 rat. units of v i tamin D per quar t for ord inary milk. Feeding experiments with twenty-one infants over a six- to eight-month period showed tha t this milk had as good protective powers against rickets as direct ly i r radia ted milk.

IV. DIRECT ADDITION OF VITAMIN D CONCENTRATE TO TttE 3/IILK

This, the latest, method of increasing the v i tamin D potency of cow's milk has been reported by ZuekerY ~ A concentrate of cod l iver oil of high potency and pu r i t y furnished in the fo rm of a "150 D " p repara - tion, i.e., a p repara t ion 150 times as potent as s tandard cod l iver oil as measured by the Steenboek line test, is di luted in the propor t ion of one p a r t in 12,000 par t s of milk. This procedure results in a milk which contains approximate ly 150 units of v i tamin D to the quar t or the equivalent of about three teaspoonfuls of cod liver oil. The in- corporat ion of the concentrate can be done by homogenization, par t ia l homogenization, or by a th i rd method not involving homogenization. There is no separat ion of the concentrate front the creanl by any of these methods. The milk has been f requent ly assayed on rats with excellent results f rom both the curative and protect ive standpoint. Invest iga- tio~ by Barnes 9s with the use of the concentrate in infants has shown it to be a highly effecti~e antiraehit ic agent, effecting, in rachit ie in- fants, a r e tu rn to normal of the serum calcium and phosphorus' and the

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x-ray findings within two weeks. One hundred and fifty units of vi tamin D per quart has been selected as the amount which has seemed from clinical experience with cod liver oil to be a good prophylactic standard.

D I S C U S S I O N

What should be stressed in a comparison of the relative advantages and disadvantages of these various methods devised for increasing the antirachit ie potency of cow's milk ?

Ear l ie r objections to direct ly i r radia ted milk were largely due to errors of technic in irradiation. Thus such disadvantages as changes in taste and smell and destruction of vi tamin A have been shown to be definitely attributable to o~erexposure to the ultraviolet rays. With the present exposure of only sixteen seconds, there is no detectable change in palatabil i ty or vi tamin A potency29 Supplee and Dow 1~176 have demonstrated a slight bu t measurable loss in vitamin C potency in ir- radiated fluid milk but not in i r radia ted dried milk. Inasmuch as antiscorbutic factor's are added rout inely to the infant ' s diet, this slight loss of vi tamin C is of practical ly no importance. This method, of course, does require additional appara tus and is applicable chiefly to centers with large pasteurizing plants. In smaller communities, the installation of such appara tus would hard ly be commercially and finan- cially advisable. However, as i r radia ted dried milk it may be shipped t~, all parts of the count ry and kept for several months without any loss in antirachitic potency.

In its favor is the fact tha t no special feeding or supervision of cattle is necessary. The milk is shipped as usnal into the pasteurizing plant and the additional process of i r radiat ion is easily and quickly performed at a cost of only a f ract ion of a cent per quar t of milk. Any Qbjection to the method because it involves fu r the r handling of the milk is met with the answer tha t fa r f rom the introduct ion of any contaminating process, the irradiat ion has been actually shown to lower markedly the bacterial count. TM lOl, lo2 The milk, following irradiation, has a vitamin D content of approximately 50 to 55 rat units which has seemingly proved ample for both curat ive and protective purposes.

The production of yeast milk involves special supervision and feeding of cattle and, because it is difficult to follow closely, due to the long experimental work involved, the potency of a milk supply whose vi tamin D content is dependent on feeding operations carried on over scattered areas, is at present and probably for the near fu ture limited to super- vized dairy farms such as are now able to pu t certified milk on the market. These farms are, of necessity, l imited to areas supplying large urban districts such as New York, Boston, and Philadelphia. At pres- ent, the method is a good deal more expensive than the direct irradia- t ion of milk. Krauss feels tha t the feeding method is a more nas procedure for increasing the vitamin D content of cow's milk. No fur- ther handling or t rea tment of the milk is necessary. No untoward effects on the cattle have been observed with continued feeding of the yeast or ergosterol.

Hess, in a comparison of the two milks, i.e. " y e a s t " and irradiated, was inclined to place the yeast milk second in point of efficacy becallse, in clinical experiments, the same results seemed to be obtained with i r radia ted milk, which contains only about 50 units of vi tamin D to the quar t as with the yeast milk which contains 160 units of vitamin D

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CRITICAL R E V I E W 6 8 9

to the quart . However, K r a m e r s~ has shown tha t yeast milk t reated so as to contain only 55 and 40 units, respectively, of v i tamin D to the quar t is as efficacious as a curat ive agent as i r radia ted milk containing the same number of v i tamin D units.

The direct i r radia t ion of animals again involves special appara tus . Once installed, however , the actual process of i r rad ia t ing an animal for fifteen minutes a day does not seem too involved. Close supervision, as required in supplementa ry feeding, does not seem necessary. We have again, however, the same objection as wi th yeast milk, namely, the difficulty in following closely the potency of a milk, whose v i tamin D content, in this case, is dependent on i r radia t ion operations carr ied on over widely scat tered areas. A little more clinical evidence as to its efficacy, moreover, seems indicated. The method resulted, according to Mitchell, in a milk having an ant i rachi t ie potency of only 22 units of v i tamin D per quart, which is only about four t imes tha t of ord inary milk. Whe the r this will be sufficiently protect ive under all types o f circumstances remains to be corroborated.

The four th method seems very simple and does no% depend on special, controlled feeding" of cows or on elaborate appara tus . Small pasteur- ization plants can car ry out the procedure equally as well as large ones. The potency of the milk is definitely known beforehand; one does not have to wai t for several weeks of animal exper imenta t ion to evaluate the potency. The chances for error are minimal as the whole procedure involves only the addit ion of a standardized, previously assayed con- centrate. While the procedure is probably more expensive than direct i r radia t ion of milk] the price nevertheless is less than that of ord inary milk plus cod l iver oil. The method does not have to be, restr icted to larger centers of populat ion but can be used wherever there is a pasteur- ization plant .

There remains, finally, a question which has as yet not been satis- factor i ly answered. Hess was one of the first to place it before us. W h y are fewer units of v i tamin D in the f o I ~ of v i tamin D milk suf- ficient to protect against r ickets than in the fo rm of other ant irachit ic agents, such as cod l iver oil or viosterol ? I-Iess s~ compared the potency of yeast milk with tha t of cod l iver oil and of viostero~ in Table I I I .

TABLE, Ill

INTAKE OF COW IV[ILK (1 LIT%R) IX T~R1ViS O1~ VIOSTEROL COD LIVER

i x RA~ ~NITS ~ A ~ ~ I T S (D~0PS) 0IL ( ~ . ) Ergosterol 100,000 80 1 6 Ergosterol 200,000 160 2 12 Yeast 30,000 80 1 6 Yeast 60,000 160 2 12

He drew the conclusion tha t yeast milk is more comparable to cod liver oil than to viosterol, for while 12 gm. of cod l iver oil (correspond- ing to one l i ter of the 160 D milk) would be a f a i r daily prophylact ic dose for infants, 2 drops of viosterol would be highly inadequate. Hess then f u r t h e r demonstra ted tha t one l i ter of milk, 15 e.c. o~ cod l iver oil, and 10 drops of viosterol are each sufficient as a daily dose to protect the average in fan t f rom rickets. I f these figures are transposed into ra t units, we find the comparat ive potencies are : 160:200:830 or in the

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rat io of 1:1.25:5.2. The difference between the yeast milk and viosterol is striking, tha t between tile milk and eod l iver oil not so prononneed. However, Kram er has shown that as little as 40 units of v i tamin D in the fo rm of yeast milk daily is suffieient as a curative agent in riekets. This would make the rat io : 40:200:830 or approximate ly 1:5:21. Simi- la r results have been recorded for i r radia ted milk, and numerous clinieal repor ts attest to the efficacy of i r radia ted milk containing only 50 units of v i tamin D per quart . As we have seen, the direct i r radia t ion of cattle results in a milk which contains only 22 units of v i tamin D per quart , and yet the milk has seemed to be an efficient protect ive agent. On reviewing earlier reports , we see tha t MacKay and Shaw TM 1~ effeeted healing of rickets in fr.om two to three weeks with as little as 3I/2 ounces of i r radia ted dried milk or 5 ounces, of i r r ad ia ted condensed milk dai ly! This amount of milk probably did not contain even 20 units of v i tamin D. Thus, we have extensive clinical evidence to. the effect tha t f a r fewer units of v i tamin D in the fo rm of v i tamin D milk are required t.o protect against or heal rickets t han is the ease for other' antirachit ie agents.

Watson l~ has given us an interest ing discussion o.f this problem, t t e states in p a r t : "Aecor 'd ing to official biologic tests, the increase in the amount of antiraehit ie p rope r ty in milk brought about by i r radia t ion is, f rom the laboratory point o.f view, tr if l ing in comparison with the large amount of v i tamin D present in var ious i r radia ted medicinal sub- stances. On the other hand, we have the s t r iking fac t tha t i r radia ted milk has been shown to cure r ickets in cases which have failed t~ re- spond to the prolonged adminis t ra t ion of i r radia ted commercial p repara - tions in common use. W h a t is the explanat ion of this. anomaly ? I t is possible tha t i r radia ted medicinal sabstanees may steadily lose their curat ive powers, milk having the advantage of being consumed in the fresh state. I t is equally possible tha t i r radia ted milk possesses some p rope r t y of value which is not determined by the present laboratory test for v i tamin D. There is the fu r t he r possibili ty of a tendency to e r r in thinking the results obtained f rom experiments in rats under l abora tory conditions are capable o.f fa i r ly r igid applicat ion to. clinical conditions in the human subject. I t is conceivable tha t the milk acts merely as a carr ier of some specific energy impar ted to. i t by suitable irradiat ion, this added p rope r ty only coming into action in the tissues. I n this event, i t would be d e a r tha t i ts action is not the same as in the case of i r radiated ergosterol where a definite physical ehange is effected in the ergosterol by the i r radia t ion process ." Watson believes we must come to,.the conclusion tha t some vital p rope r ty has been added to the milk which is not represented by the present labora tory test for v i tamin D. He proceeds : " I n nature , v i tamins are found only in the vegetable kingdom. Vitamins in animal tissues and in milk a r e derived f rom the vegetable foods consumed. The vi tal energy of the sun strikes the cells in the plant, and in those cells the solar energy is t rans formed into. a chemical vi tal energy which promotes the nonna l growth and develop- ment o f the tissues in the vegetabIe kingdom. The act ivated tissues hold some of the original vi tal energy which init iated the chemical changes. Those tissues are in tu rn consumed by animals or man, to whom tha t energy is in t u rn t ransmi t ted as a so.-called v i tamin . . . . Thus solar energy is t r ans formed in na ture and by artificial means (ul- t raviolet lamp) into chemical energy. , I t is appa ren t tha t between the

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chemical energy, as it is stored up in the p lan t or substance, and the sunlight energy, which starts the process, there mus t exist a very close kinship in nature , if not indeed iden t i ty . "

I~EFERENCES

1. Hess, A. F.: Proc. Am. Pedlar. So% June 7, 1924 (abstract, Am. J. Dis. Child. 28: 517, 1924).

2. Hess, A. F., and Weinstock, M.: .Proe. Soe. Exper. Biol. and Med. 22: 6, 1924. 3. Hessj A. ~., and Weinstock, M.: Ibid. 22" 5, 1924. 4. Hess, A. F., and Weinstock, M.: J. Biol. Chem. 62: 301, 1924. 5. Steenbock, II.: Science 60: 224, 1924. 6. Steenboek, H., and Black, A.: J. Biol. Chem. 61: 405, 1924. 7. Stecnbock, }I., and Black, A.: Ibid. 64: 263, 1925. 8. Steenbock, H , and Nelson, ~ . T.: Ibid. 62: 575, 1925. 9. Steenbock~ tI., and Danlels, A . L . : J . A. Iv[. A. 84:: .1093, 1925.

10. Hess, A. F~., and Weinstock, HI.: J . Biol. Chem. 64: 181, 1925. 11. ]:Iess, A. F.: J . A . 3el. A. 8~t: 1910, 1925. 12. Cowell, S . J . : Brit. M. J. 1: 594, 1925. 13. Kramer, B.: Am. J. Dis. Child. 30: 195, 1925. 14. Gy5rgy, P. : Klin. Wchnsehr. 4: 1118, 1925. 15. MacKay, II. M. M., and Shaw, H . F . : Brit. 3/I. J. 2: 344, 1925. 16. GySrgy, P. : Jahrb . f. Kinderh. 111: 201, 1926. 17. Hott inger, A.: Sehweiz. reed. -Wchnsehr. 56: 170, J926. 18. OySrgT, P.: ~21in. Wchnschr. 5: 747, 1926. 19. MacXay~ H. M. M , and Shaw, tI . F'.: Lancet 1: 8, 1926. 20. Daniels, A. L., Pyle, S. I., and Brooks, L.: Proc. Soc. Exper. Biol. and Med.

23: 821, 1926. 21. Gillern, K., and tIussa, V.: Wien. reed. Wchnsctm 77: 1686, 1927. 22. t~ohr, F., and Schhtz, O.: Klin. Wehnsehr. 6: 848, 1927. 23. Gillern, K., tIussa, V , and Schirman~ M . A . : Wien. reed. Wchnsehr. 78: 793,

1928. 24. Scheer, E:.: Miinehen. reed. Wchnschr. 75: 642, 1928. 25. Watson, C., and Finlay, T . Y . : Lancet 2: 704, 1929. 26. Bratusch-Marrain, A., and Siegl, J . : Wien. klin. Wchnschr. 42: 1279, 1929. 27. DeSanctis, A. G, Ashton, h O, and Strlngiield, O . L . : Arch. Pediat. 46: 297,

1929. 28. Bamberger, P.: Deutsche reed. Wchnschr. 55: 1547, 1929. 29. Schhnen, J . : Ibid. 55: 1629, 1929. 30. Bras A., and Siegl, J . : Arch. f. Kinderh. 89: 20], 1930. 31. Andresen, O.: Ibid. 90: 65~ 1930. 32. Reichhuber, X.: Wien. klin. Wehnschr. 44: 765, 1931. 33. Essig, B.: Mfinchen. reed. Wchnschr. 78: 273, 1931. 34. Scheer, I~.: J . State NIed. 39: 607~ 1931. 35. Hess, A. F., and Lewis, g . M . : J. A. 2r A. 99: 647, 1932. 36. Niermann, M., and Winter, 3/f.: Arch. f. Kinderh. 91: 69, 1930. 37. Brnhl, I~.: Med. Kiln. 26: 509, 1930. 38. Ran, H., and Gruber, A.: Miinchen. reed. Wchnsehr. 78: 274, 193]. 39. Supplee, G. C., Dorcas, M. J., and IIess, A . F . : J . Biol. Chem. 94: 749, 1932. 40. Supplee, G. C, t Ianford, Z. 3/I., Dorcas, M. J., and Beek~ IL I-I.: Ibid. 95:

687, 1932. 41. Supplee, G. C., Beck, H. H., and Dorcas, M. J . : Ibid. 98: 769~ 1932. 42. Supplee) G.C. : Am. J. Pub. Heal th 23: 225, 1933. 43. Zucker, T. F'., Pappcnheimer, A. M., and Barnet t , M.: Proc. Soe. Exper. Biol.

and Med. 19: 167, 1921. 44. Hess, A. 1%, and Weinstock, 1~.: J. Biol. Chem. 63: 297, 1925. 45. ~osenheim, O., and Webster, T. A.: Lancet 1: 1025, 1925. 46. Hess, A. F'., Weinstock, 1VL, and I lehnan, F . D . : J. Biol. Chem. 63: 305, 1925. 47. Heilbron, I. M., Simpson~ J. C. E., and Spring, F. S.: J . Chem. Sot., June,

626,1933. 48. Hess, A. F., Weinstock, ~ . , and Shermanj E.: J. Biol. Chem. 67: 413, 1926. 49. Webster) A., and ttill , L.: Brit . M. J. 1: 956, 1925. 50. Bills, C. E., mad McDonald, F . G . : J. Biol. Chem. 72: 13, 1927. 51. Rosenheim, O., and Webster, T . A . : Biochem. J . 20: 537, 1926. 52. Rosenheim, O., and Webster, T . A . : Ibid. 21: 197, 1927.

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53. Hess, A. F., and Windaus, A.: Prec. Soc. Exper. Biol. aad Med. 24: 369, 1927. 54. Rosenheim, O., and Webster~ T . A . : J. Soc. Chem. Ind. 45: 932, 1926. 55. Rosenheim, 0., and Webster, T . A . : Biochem. J. 21: 389, 1927. 56. Hess, A. ~., and Windaus, A.: Prec. Soc. Exper. Biol. and ?led. 24: 171, 1926. 57. Hess, A. ~., and Windaus, A.: Ibid. 24: 461, 1927. 58. Heilbron, I. M., Morrison, A. L , and Simpson, J. C. El: J. Chem. See., March,

302, 1933. 59. Hess, A. t~.: J. A. 3/I. A. 89: 337, 1927. 60. Hess, A. ~., and Weinstoek, M.: J. Biol. Chem. 64: 193, 1925. 61. Sehlutz, F. W., and Niorse, hi.: Am. J. Dis. Child. 30: 199, 1925. 62. Heilbron, I. ~ . , Nature, E. D., and Morton, Ir A.: Biochem. J . 21: 78, 1927. 63. Hess, A. t0.: Acta ~aediat. 11: 1, 1930. 64. Wejdling, K.: Ibid. 11: 77, 1930. 65. Lesn6, E., and Yig]iano, 3/I.: Compt. Rend. Acad. d. Sc. 179: 539, 1924. 66. Wagner, R. J., and Wineberger, It . : Ztschr. f. Xinderh. 40: 295, 1925. 67. Gelding, J., Soames, K. NI., and Zilva, S. S.: Biochem. J. 20: 1306, 1926. 68. Gelding, J., and Zilva~ S. S.: Ibid. 22: 173, 1928. 69. Hess, A. t~., and Weinstoek, M.: J . A . 2r A. 83: 1558, 1924. 70. Hess, A. F. , and Weinstock, /eL: Am. J. Dis. Child. 27: 1, 1924. 71. Weech, A . A . : Bull. Johns Hopkins Hosp. 40: 244, 1927. 72. Gerstenberger, H. J., Hartman, J. J., and Smith, D. Ig.: California and West.

3/led. 27: 40, 1927. 73. Eufinger, H.: Wiesbade, H., and Focsaneanu, L.: Xlin. Wchnschr. 8: 826,

1929. 74. Gerstenberger, H. J., and Russell, G. 1~.: Acta Paediat. 11: 32, 1930. 75. Wachtcl, M.: lKii•chen, med. Wchnschr. 76: 1513, 1929. 76. Steenbock, H., Hart , E. B., Harming, :F.~ and ]~[umphrey, G. C. : J . Biol. Chem.

88: 197, 1930. 77. Thomas, B. H., and ]~iacLeod, F. L.: Science 73: 618, 1931. 78. Xrauss, W. E., Bethke, R. M., and Monroe , C . F . : J. Nutrition 5: 467, 1932. 79. Hardenbergh, J. G., and Wilson, L. T.: Am. J. Pub. Health 23: 230, 1933. 80. Gerstenberger, H. J., and Goresh, A . J . : J. lgutrition 5: 479, 1932. 81. Hess, A. F., Lewis, J. 3/I., 3s F. L., and Thomas, B. tI . : J . A . 3s A.

97: 370, 1931. 82. Wyman, E. T., and Butler, A. M.: Am. J. Dis. Child. 43: 1509, 1932. 83. Wyman, E. T.: New England J. Med. 209: 880, 1933. 84. Kramer, B., and Gittleman, J . F . : Ibld. 209: 906, 1933. 85. Luce, E. IV[.: Biochem. J. 18: 716, 1924. 86. Luce, E. ~V[.: Ibid. 18: 1276, 1924. 87. Chick, tI., and Roscoe, 5[. H.: Ibid. 20: 632, 1926. 88. Supplee, G. C., and Dew, O .D. : J. Biol. Chem. 73: 617, 1927. 89. Steenbock, H., Hart, E. B., Hoppert, C. A., and Black, A.: Ibid. 66: 441, 1925. 90. Steenbock, H., Hart , E. B., Riisi~g, B. NL, Hoppert, C. A., and Basherov, S.:

Ibid. 87: 103, 1930. 91. Gowen, J. H., Murray, J. M., Gooch, 1Vi. E., and Ames, F. ]3.: Science 63:

97, 1926. 92. Falkenheim, C., u W., and Kitsch, W.: Xlln. Wchnschr. 5: 2071, 1926. 93. Wagner, t~. J., Bruek-Biesok, U., and Pirquet, C.: Ibid. 6: 952, 1927. 94. ttess, A. ~., Weinstock~ M., and Shermall, E.: J . A . M . A . 88: 24, 1927. 95. Lesn6, E., and Dreyfas-S~e, G.: Nourrisson 19: 90, 1931. 96. Mitchell, J. M., Eiman, J., Whipple, D. u and Stokes, g. : Am. J. Pub. t tealth

22: 1225, 1932. 97. Zucker, T. F.: Ibid. 23: 10, 1933. 98. Barnes, D . J . : Am. J. Dis. Child. 46: 250, 1933. 99. Supplee) G. C., and Dew, O.D. : J. Biol. Chem. 75: 227, 1927.

100. Supplee, G. C., and Dew, O.D. : Am. J. Dis. Child. 41: 1353, 1931. 101. Nabarro, D., and Hickman, J. O.: Lancet 1: 127, 1930. 102. tIickman, J . O . : J. State Med. 38: 476, 1930. 108. Watson, C.: Trans. ~r163 Soc. Edinburgh 45: 91, 1931.