THE GROWTH-PROMOTING PROPERTIES (VITAMIN B COMPLEX) … · 710 Vitamin B Complex of Milk procedure was carried out in the manner previously noted. The rice polish was from the same

THE GROWTH-PROMOTING PROPERTIES (VITAMIN B COMPLEX) OF THE CONCENTRATED WATER-

SOLUBLE PORTION OF MILK

BY G. C. SUPPLEE, 0. J. KAHLENBERG, AND G. E. FLANIGAN

(From the Research Laboratories of The Dry Milk Company, Bainbridge, New York)

(Received for pubIication, July 7, 1931)

The multiple character of the water-soluble growth-promoting factors has been well established by the researches of recent years (l-38), and even though only two specific factors are now uni- versally recognized, namely vitamin B and vitamin G,’ the existence of one or more additional factors is suggested by data from various sources.

While many of these studies have involved the use of yeast as the source of these vitamins, the presence of these factors in milk and other food products is generally conceded. Although many of the data regarding the relative abundance of the growth-promoting and antineuritic factors in milk are of an indirect character, the confirmatory evidence from various laboratories leaves no doubt as to their presence. Sherman and Axtmayer (26) have concluded from their studies on the supplementing effect of milk and grain that milk is richer in vitamin G than in vitamin B, whereas, in whole wheat, vitamin B is present in greater concentration than vitamin G. Outhouse et al. (29) have also sub- mitted data indicating that vitamin B is relatively less abundant than vitamin G in cow’s milk. Similar conclusions were reached by Hunt and Krauss (30). The classical work of Goldberger and associates (8, 9, 11, 12) based on field studies and laboratory experiments demonstrated the efficacy of milk in curing and pre-

1 Vitamin B and vitamin G as referred to herein will be used in conform- ity with the recommendations of the Committee on Nomenclature of Vita- min B of the American Society of Biological Chemists, Science, 69, 276 (1929).

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706 Vitamin B Complex of Milk

venting pellagra in man and black tongue in dogs. Johnson and Hooper (39) found that it was necessary to incorporate as high as 30 per cent dry skim milk (spray process) in a polished rice diet in order to prevent polyneuritis in pigeons. This is equivalent to an intake of 6 to 7 gm. of milk solids, or about 75 cc. per day. Supplee and collaborators have recently obtained data (40) show- ing that a water-soluble vitamin concentrate prepared from milk possessed approximately 25 per cent of the antineuritic potency of rice polish, whereas, dried whey possessed only 10 to 12 per cent of the potency of rice polish, or slightly less than one-half that shown by the water-soluble concentrate. These data were obtained with pigeons, by using the curative technique. Norris, Heuser, and Wilgus (41), using the same product in experimental work with poultry, concluded that this concentrate contained another hitherto unrecognized factor necessary for the preven- tion of a particular type of paralysis in chickens and that it also promoted growth.

Inasmuch as the studies which will be reported hereinafter had as their primary objective the determination of the vitamin potency of certain water-soluble milk constituents, a suitable and rea.dily adaptable method was a matter to be given first consideration. Since variations in the biological properties of the casein used in basal rations, even after supplementary purification, ’ have been shown to exist (15, 31, 42, 43), our initial efforts were directed to the development of a method which would be satis- factory for determining variations in different samples of casein as well as for the study of the water-soluble milk vitamins.

EXPERIMENTAL

Part I. Variations in Biological Properties of Different Caseins

In considering the requirements of the method which it was desired to use, it was necessary to supply in the basal ration an amount of vitamin B just sufficient to prevent polyneuritis, and to maintain the animal at constant weight for long periods without permanent impairment of physiological functions. Such requirements imply the addition of a supplementing substance carrying a sufficiency of the maintenance factors in addition to the antineuritic vitamin. If these conditions could be met such growth response as resulted could be attributed to impurities of


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Supplee, Kahlenberg, and Flanigan 707

the various caseins used, or, with a casein of predetermined purity, such a ration could be used for the assay of the growth-promoting factors in other products. Neither yeast, because of its reported variability in vitamin B content, nor its various fractions, as prepared by laborious means in different laboratories, seemed to be suitable for meeting the requirements at hand. Rice polish with its relatively high vitamin B content and apparently low vitamin G potency was considered as a possible supplement. In order to ascertain the merits of rice polish for the purpose under consideration a basal ration of the following composition was used: casein 20 parts, dextrin 60 parts, Crisco 10 parts, powdered agar-agar 2 parts, cod liver oil 2 parts, and Salt Mixture 40* 4 parts. To this basal ration were added varying percentages of a commercial grade of rice polish. The casein used for this series of tests was from a single lot taken from our stock supply, which had been purified some years previous by the following method:

A high grade commercial casein, precipitated by hydrochloric acid, thoroughly washed, and finely ground was leached with 0.1 per cent acetic acid for 10 days. 10 parts of the acidulated water was used for 1 part of dry casein; the leaching water was changed twice dally. Four washings with distilled water followed the acidulated water treatment, the proportions of casein and water being maintained as above. This casein will be designated hereinafter as Type A. Although no evidence has been revealed at this laboratory indicating the inadequacy of this procedure for satisfactorily freeing the casein of growth-promoting factors, the method became open to suspicion after the observa- tions of Chick and Roscoe (15) and of Coward et al. (42,43). These investigators showed that different samples of casein possess variable growth-promoting properties, and that these factors could be removed only after extraction with alcohol and ether following the treatment with acidulated water.

Typical results obtained by incorporating varying percentages of rice polish in the basal ration containing this particular sample of Type A casein are shown in Chart I. In this and in subsequent charts the growth curves of white rats are shown in conjunction with the mg. of daily intake of the test substance calculated on the weekly average basis.

* Steenbock, H., and Nelson, E. M., J. Biol. Chem., 66, 362 (1923)


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The rats on the basal ration unsupplemented with rice polish usually died at the 5th to 6th week without substantial previous gain in weight. Animals receiving as high as 3 per cent rice polish did not show significant growth with this particular sample of casein. At the 4 and 7 per cent rice polish levels a slightly greater rate of growth resulted, although there was no substantial difference in growth at each of these levels. None of the animals receiving rice polish showed symptoms of polyneuritis. Practically all animals receiving 3 per cent or less of rice polish suffered loss of hair, dry skin, and not infrequently scaly lesions on the feet and tail. Extreme pellagrous symptoms as reported by other investigators have not been obtained with this diet. The conditions just described were usually evident at the 4th to 6th weeks. After a certain stage was reached further development of the pellagrous condition was rarely noted and in practically all instances the animal was maintained in substantially the same condition for several weeks. In the light of other investigations this reaction is possibly explained by the existence of a small trace of the antipellagric factor in the rice polish. This explana- tion is further strengthened by the fact that with the higher amounts of rice polish the pellagrous symptoms are less pro- nounced, and at the 7 per cent level they have been but rarely noted.

The results from this series of tests seemed to indicate the feasibility of incorporating 2 per cent rice polish in the basal ration as a supplement for supplying the minimum vitamin B and maintenance requirements. This ration has now been used for nearly 2 years for determining the relative amount of the water- soluble growth-promoting factors in various casein samples, mis- cellaneous products, and for the study of the milk vitamin concentrate with which this report is primarily concerned. It is desirable, however, to record that, although the casein used for the previously described tests was found to be free from accessory factors, later experiences showed that crude casein obtained under the specifications already mentioned and purified by leaching with acidulated water as described could not always be depended upon, Therefore, in order that the variability of different caseins may be better appreciated certain results which have been encountered are recorded at, this time.


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Chart I shows typical results obtained from another lot of casein procured under the same specifications as the lot used for the foregoing series of tests. The acidulated water purification

TYPEA CASEIN TYPE ACASEIN NO.2 WITH WITH RICE POLISH 79 RICE POLISH

W&c\+ NONE

yoc.m+ I p

YOG!% if 3 09

7OG rtl!ht2 K

K

1 YOG h IOT EXTRACTED EXTRACTED WlTHALCOHOL WITH ALCOHOL

8 WKS. TYPE B CASEIN WITH 7+ RICE POLISH

NONE

YOG. cl K YOG

NOT EXTRACTED EXJRACTED WITH ALCOHOL WITH ALCOHOL

CHART I. Variations in the growth-promoting properties of different caseins when supplemented with rice polish.


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procedure was carried out in the manner previously noted. The rice polish was from the same lot previously used. It is to be observed that the degree of growth resulting from the use of this casein supplemented with 7 per cent rice polish was substantially greater than that obtained from the previous lot. This casein after alcohol and ether extraction for a period of 48 hours each gave substantially less growth than before extraction.

Examples of the results obtained from another lot of casein are also shown in Chart I. This was a commercial product manu- factured by the “cooked curd” process, wherein the casein is heated in the whey to a sufficiently high temperature after pre- cipitation with acid to cause coalescence of the curd particles. This type of commercial casein, designated as Type B, was received in a dry, finely ground condition and subjected to the usual purification procedure. This casein gave unsatisfactory results even when unsupplemented with rice polish. It is to be noted that the results are substantially the same, when supplemented with 7 per cent rice polish, before and after alcohol and ether extraction. Failure to obtain greater growth in the presence of 7 per cent rice polish might indicate, in the light of present knowledge, that the impurity in this particular casein was primarily vitamin B.

The results from these tests appear fully to confirm the observa- tions of other investigators to the effect that the acidulated water treatment cannot always be relied upon completely to free all commercial caseins from the growth-promoting factors. These results also indicate the possibility of a selective adsorption and retention of the water-soluble factors by casein prepared by different methods. The meager evidence available accentuates the importance of further basic studies regarding the biological character of caseins used in the basal rations required for the determination of certain vitamins.

Part II. Growth-Promoting Properties of the Water-Soluble Portion of Milk

Since the publication of a brief preliminary report indicating the nutritive character of the concentrated water-soluble portion of milk, further data have been obtained. The product under consideration represents a concentration of milk constituents


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other than the fat, casein, albumin, and lactose, and since there is no evidence which indicates a direct correlation of the water- soluble vitamins with these substances, other than a mechanical

MILK VITAMIN COA’CENTRATE WITH 1.p RICE PaLI H

Yoti &

K

I.S Y- Y.SP* 7rp

CHART II. Growth-promoting properties of the water-soluble milk vitamin concentrate with and without rice polish.

one, it is logical to believe that these factors and other important dietary accessories found in natural milk are contained in the concentrate with which these studies are concerned.


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The gross composition of the concentrate used for these tests, calculated to the dry basis, follows: lactose 52.01 per cent, ash 30.84 per cent, and nitrogen 1.54 per cent. This product was incorporated .in the test rations in the desired proportions by drying on predetermined amounts of dextrin. The basal ration containing casein of predetermined purity was the same as given in “Part I” of this report.

White rats reared in our own stock colony were selected for use at. 40 to 50 gm. of weight, preferably between 40 and 45 gm. of

1p 1 ‘P 3V se

CHART III. Growth-promoting properties of the water-soluble milk vitamin concentrate supplemented by 7 per cent rice polish.

weight. All animals were kept in individual wire cages with screen bottoms. Food consumption records were uniformly maintained, and for convenience and clarity the daily average intake of the supplementing test substances is shown in graphical form on the accompanying charts.

Chart II shows typical results obtained from varying percentages of the water-soluble milk vitamin concentrate with and without 2 per cent rice polish supplement. Those animals receiving 1.5 per cent of this concentrate not only failed to grow but manifested subacute symptoms of polyneuritis a few weeks before


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death. These symptoms were not manifested by those animals receiving the higher amounts. It is to be noted that the milk concentrate unsupplemented by the vitamin B of the rice polish is inadequate for normal and continued growth at a daily average intake of approximately 250 mg., but that normal growth does result when a daily average intake of about 900 mg. is reached. When the basal ration was supplemented with 2 per cent rice polish normal growth was maintained at a daily average intake of

K

YoG. lS”~CCNCENTPATE

7S % CoNCfNTRATE

‘He/* CONCENTRATE

8 WKS. MC-

CHART IV. The effect of the water-soluble milk vitamin concentrate and rice polish as subsequent supplements following suppression of growth.

about 400 mg. All data show that the vitamin B potency of this concentrate is relatively lower than its vitamin G potency. Addi- tional data are also shown in Chart III wherein the rice polish supplement is increased to 7 per cent. With this higher amount of rice polish substantially normal growth was maintained at a daily average intake of between 100 and 200 mg. of the milk vitamin concentrate.

These results show that a comparable growth response may be obtained from varying proportions of each of the different factors


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concerned, and that while vitamin B as well as other factors, must be present, fixed proportions or relationships between each are not necessarily a prerequisite for maintaining a particular rate of growth.

In order further to determine the growth-promoting and antipellagric properties of the water-soluble milk vitamin concentrate, varying percentages of this product were superimposed on the basal ration containing 2 per cent rice polish, as a subsequent sup

FIO. 1. Rat 2342 after 13 weeks on basal ration supplemented with 2 per cent rice polishings. Weight 43 gm.

FIQ. 2. Rat 2842 3 weeks after receiving the water-soluble milk vitamin concentrate furnished at the 13th week (Fig. 1). Weight 122 gm.

plement following suppression of growth; rice polish was also furnished as a subsequent supplement after a decline in weight resulting from the feeding of limited amounts of the concentrate. Chart IV shows illustrative data from this series of tests. During the period of constant weight the mild symptoms of pellagrous character previously described were manifested. None of the animals showed symptoms of polyneuritis. At the 1.5 per cent level the growth response was slight -and the pellagra-like condition was somewhat improved. At the higher levels growth was resumed


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at a rapid rate and the pellagrous symptoms began to show improvement within 3 to 5 days. The complementing effect of rice polish and the milk vitamin concentrate was further illustrated by the prompt growth response brought about by 2 per cent rice polish furnished as a subsequent supplement after decline in weight resulting from limited amounts of the concentrate.

Fig. 1 shows the typical condition of the animals just before the beginning of the subsequent supplementary feeding of the milk concentrate. The weight of Rat 2842 at the beginning of the experimental feeding period was 40 gm. and after 13 weeks during which time it received the basal ration supplemented with rice polish only, its weight was 43 gm. Fig. 2 shows the same animal 3 weeks later at a weight of 122 gm. after it had received a subsequent supplement of 7.5 per cent of the water-soluble milk vitamin concentrate. A similar growth-promoting potency of this substance has since been demonstrated with hundreds of animals. A sample of the concentrate further purified but still containing much inert material has caused a growth response after suppression of growth of as high as 59 gm. per week.

In view of the growth response shown by animals receiving varying proportions of the known or suggested accessory factors, the potential powers for utilization of these accessories during different periods of life raise the question of the specificity of the growth-promoting vitamins. These relationships clearly indicate that nutritional studies dealing with the question of vitamin specificity should take into consideration not only the specific factors but also the vitamin balance of the dietary constituents and the ability of the animal to use the factors existing in such balance. A natural corollary to these considerations is the question of the optimum interrelationship of accessory factors which will permit normal development and continued well being without permanent impairment or disturbance of physiological processes.

Part III. Efect of High Temperatures and Ultra-Violet Radia- tions on the Water-Soluble Milk Vitamin Cuncentrate

The growth-promoting properties of the milk vitamin concentrate with and without a supplement of rice polish having been determined, the stability of these properties at high temperatures


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Vitamin B Complex of Milk

and under intensive ultra-violet ray irradiation was investigated. Similar methods of experimentation have been used to study the

AUTOCLAVE0 MILK VITAMIN CONCENTRATE WITHOUT RICE POLISH

6 WKS. e:g 1 9s z* AUTOCLAVE0 MILK VITAMIN CONCENTRATE WITH ;L fiRiCE POLISH

/.5-Y* y.sp &w lO.SP IS?

&ART 1’. Results obtained from the water-soluble milk vitamin concentrate after autoclaving.

multiple character of the water-soluble vitamins of yeast (10, 11, 20, 22, 24-26, 37).


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After the milk vitamin concentrate was adjusted to pH 9 it was autoclaved for 5 hours at 120’ during which time the pH dropped to 4.5. The autoclaved material was fed with the basal ration unsupplemented, and also supplemented with 2 per cent rice

CHART VI. Results obtained from the autoclaved water-soluble milk vitamin concentrate furnished as a subsequent supplement.

polish, at levels varying from 1.5 to 15 per cent from the beginning of the feeding period, and also as a subsequent supplement after suppression of growth and development of mild pellagrous symptoms. Typical resuhs are shown in Charts V and VI.


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A lower rate of growth, as compared with similar quantities of unheated material, was shown by all animals, irrespective of the amount of test substance ingested. For the groups receiving the autoclaved material supplemented with 2 per cent rice polish from the beginning of the feeding period (Chart V) the rice polish intake was substantially the same with the exception of that of the groups receiving the 1.5 per cent level. In practically all these instances a notch, flattening, or decline of the growth curve ap- pears at a period, or at a stage of development, which substantially coincides with the period of adolescence. In those groups receiving the autoclaved material at the higher levels after suppression of growth the same phenomena are indicated. Pellagrous symptoms were improved in those animals which resumed growth. Those receiving the lower amounts and which did not resume growth showed no improvement in these symptoms. The fact that the daily average intake of the autoclaved concentrate varied from approximately 300 mg. per day to as high as 1400 to 1500 mg. without causing a commensurate growth response in the presence of a relatively constant rice polish intake, indicates in comparison with the data previously recorded, that vitamin G as it exists in milk and as now defined, involves more than a single entity.

These data clearly indicate the thermolabile character of one or more of the growth-promoting factors contained in the water- soluble milk vitamin ooncentrate. The evidence also suggests that the thermolabile factor or factors which exist in this product are utilized by the animal organism particularly at and following the period corresponding to that of sexual maturity. The evidence is such that it cannot be assumed that this factor is vitamin B (antineuritic vitamin) as now defined in its restricted sense, or, if the antineuritic vitamin alone is concerned a specific demand at and beyond the period of adolescence is indicated. The results again emphasize the question of the specificity of the various water- soluble factors in so far as growth requirements are concerned. They likewise bring into prominence the particular requirements of the animal for these factors during different periods of the life cycle. The evidence now available might be interpreted to mean that the requirement for such factors during the period of structural growth is relatively lower than during adolescence.


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Conflicting data have been reported (20, 24) in regard to the destruction of the water-soluble factors by ultra-violet rays. This subject has been given consideration in connection with our studies on the milk vitamin concentrate. The concentrate was irradiated in water solution at 10 per cent concentration, at a distance of 12 inches from a quartz mercury vapor lamp for 10 hours in a layer approximately 2 mm. thick. The material was agitated at 3 hour intervals; water was added as required to replace that lost by evaporation. The irradiated material was fed at various levels supplemented and unsupplemented with 7 per cent rice polish.

w bkl t

6 wG(s.

h

t 7Y 7fi+ 7?4 7$AUTO- Jp+?y RICE IRRADIATED RICE PDLISH CLAVEDAND PDLISH-AUTO-

IRRhDlATED CLAVEDANU IRRAOIATEO

CHART VII. Results obtained from the irradiated and autoclaved water-soluble milk vitamin concentrate.

Certain groups also received autoclaved material as a further supplement. Typical results as recorded in Chart VII show that ultra-violet rays intensively applied as under the conditions of this test have a destructive effect upon vitamins B and G. The animals receiving 7 per cent of the irradiated material showed acute symptoms of polyneuritis before death. Those receiving the same amount of irradiated concentrate supplemented with 7 per cent rice polish showed no greater growth than the animals receiving the same amount of rice polish alone. The animals receiving the concentrate which had been both irradiated and autoclaved provide further evidence of the destructive effect of


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intensive irradiation on the growth-promoting factors. Several similar series of experiments were made wherein different lots of irradiated material were fed at different levels. Inconsistencies in the destructive action of the ultra-violet rays on the growth- promoting properties were observed. Such results indicate that irradiation as applied under the particular conditions of these experiments cannot always be relied upon completely to destroy

R-ILEPDLJSH AS SINGLE ODSAGES SUPPLEMENTING IRRhDIATED OR AUToCLAVED MILK CDNCEIVTRATE

RICE t'OLlSH AS SINGLE DOSAGES AFTER FEfDINGMILKCDNtENTRATE

$!sp Ysp

CHART VIII. Results obtained from single doses of rice polish

the growth-promoting factors. The prodution of toxic substances by irradiation under these conditions does not appear to have been responsible for the irregularities observed. In one series of tests wherein as high as 14 per cent of the irradiated and autoclaved concentrate was fed, the animals maintained substantially constant weight for a period of 8 weeks. At this point 3.6 per cent of non-irradiated and unheated concentrate was superimposed as


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a further supplement. Growth response was prompt, practically all animals gaining at the rate of 15 to 18 gm. per week during the next 5 to 6 weeks. Since the effect of radiant energy of this character is dependent upon the penetrability of the particular material to the rays under consideration, ineffective and irregular results may be obtained as a result of purely mechanical considerations.

Further evidence of the destructive effect upon vitamin B and the growth-promoting factors contained in the milk vitamin concentrate by autoclaving and irradiating is shown in Chart VIII. The graphs represent composite records from thirteen to twenty- seven animals. These results were obtained by feeding variable amounts of the irradiated concentrate supplemented by varying amounts of non-irradiated autoclaved material. In all instances subacute polyneuritis was first observed between 8 and 10 weeks. When these symptoms were first manifested, 1 gm. doses of rice polish were given. The dose was usually consumed within 6 to 12 hours. Improvement in the polyneuritic condition followed the rice polish dosage, such improvement being accompanied in all instances by a slow gain in weight extending over a period of from 1 week to 10 days. Decline in weight then followed over a somewhat irregular period. Upon reoccurrence of polyneuritic symptoms 1 gm. doses of rice polish were again given with repetition of the reactions just described. Chart VIII also shows the records of two of eight animals indicating the effect of single doses of rice polish on mature animals after complete withdrawal of the growth-promoting factors of the unheated and non-irradiated milk vitamin concentrate from the basal ration. In the absence of the accessory factors furnished by the concentrate a rapid decline in weight resulted with early manifestation of polyneuritis. At the first occurrence of these symptoms 1 gm. doses of the milk vitamin concentrate were given without checking the decline in weight or without perceptibly alleviating the polyneuritic condition. 1 gm. doses of rice polish were subsequently fed, resulting in prompt improvement of the polyneuritis symptoms and resumption of growth. After about 1 week, decline in weight again took place accompanied by prompt reappearance of the polyneuritic condition. 1 gm. doses of rice polish were fed on reoccurrence of these symptoms with a high degree of regularity in repetition of the results just described. These results would


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seem to show clearly the specificity of rice polish as a carrier of the antineuritic vitamin as well as to indicate the corelation of this vitamin with other factors necessary for the promotion of growth.

SUMMARY

Data are presented confirming the investigations of other investigators to the effect that acidulated water treatment cannot always be relied upon completely to free all commercial caseins from the water-soluble growth-promoting factors. The results also indicate the possibility of a selective adsorption and retention of the different factors of the vitamin B complex by caseins prepared by different methods. The meager evidence indicates that further studies are needed on the biological character of caseins used in the determination of certain vitamins.

Limited amounts of rice polish supplementing a suitable basal ration were found to supply white rats with sufficient amounts of the water-soluble maintenance factors to permit comparable as- says of the growth-promoting vitamins. Assurance of the purity and suitability of particular lots of casein is prerequisite for deter- minations of this character.

The concentrated water-soluble fraction obtained from milk after the removal of the fat, casein, albumin, and a substantial proportion of the lactose, is shown to contain a high concentration of the growth-promoting and antipellagric factors. This concentrate contains less of the antineuritic principle (vitamin B) than of the other water-soluble growth-promoting factors. The growth response resulting from limited amounts of this milk derivative is increased when additional amounts of vitamin B as found in rice polish are supplied.

Growth induced by the milk vitamin concentrate, either un- suppressed or resumed after suppression, concurrently prevented or alleviated the pellagric symptoms noted in these studies.

Growth may take place even though there are wide variations in the proportions of the accessory factors supplied by the water- soluble milk vitamin concentrate and rice polish. The results indicate the unreliability of predictions as to the amount of any one factor required for normal development, without knowledge of the relative amount of the other factors present; all of the known factors must be present to some degree.


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The autoclaving of a water-soluble milk vitamin concentrate at 120” for 5 hours adversely affects its growth-promoting prop erties.

Intensive irradiation with ultra-violet rays for a period of 10 hours adversely affects the growth-promoting properties of the milk vitamin concentrate. Such treatment, however, cannot always be depended upon completely to destroy the growth- promoting factors. Data are presented which indicate that this irregularity may be due primarily to the inefficiency of the irradiation technique.

Data are presented indicating that accessory water-soluble factors other than vitamins B and G as now defined may be required during different periods of the life cycle, particularly at and following the period of adolescence. Final conclusions as to the existence of such additional factors must await further knowledge of the specificity of the water-soluble factors and the ability of the animal to utilize such factors during the different periods of development.

Such factors as may be concerned in the maintenance of normal development through the period of structural growth as well as during the period of adolescence and beyond, and irrespective of their final identity and classification are, according to these studies, present in the water-soluble fraction of milk. Data are presented indicating that such factors are thermolabile at the high temperatures used in these experiments.

We are indebted t,o Miss 0. D. Dow and Dr. R. C. Bender for technical assistance in connection with certain phases of this work.

BIBLIOGRAPHY

1. Emmett, A. D., and Luros, G. O., J. Biol. Chem., 43, 265 (1920). 2. Funk, C., and Dubin, H., Proc: Sot. Exp. Biol. and Med., 19, 15 (1921). 3. Heller, V. G., J. Biol. Chem., 56, 385 (1923). 4. Willaman, J. J., and Olsen, A. G., J. Biol. Chem., 55, 815 (1923). 5. Levene, P. A., and Muhlfeld, M., J. Biol. Chem., 67,341 (1923). 6. Kinnersley, H. W., and Peters, R. A., Biochem. J., 19,820 (1925). 7. Hauge, S. M., and Carrick, C. W., J. Biol. Chem., 69, 403 (1926). 8. Goldberger, J., Wheeler, G. A., and Syden-Stricker, E., Pub. Health

Rep., U. S. P. H. S., 36, 648 (1920).


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9. Goldberger, J., and Tanner, W., Pub. Health Rep., U. S. P. H. S., 40, 54 (1925).

10. Smith, M., and Hendrick, E., Pub. Health Rep., U. S. P. H. S., 4l, 201 (1926).

11. Goldberger, J., Wheeler, G. A., Lillie, R. D., and Rogers, L. M., Pub. Health Rep., U. S. P. H. S., U, 297 (1926).

12. Goldberger, J., and Lillie, R. D., Pub. Health Rep., U. S. P. H. S., 41, 1025 (1926).

13. Lair-d, C. N., Am. J. Hyg., 6, 201 (1926). 14. Chick, H., and Roscoe, M., Biochem. J., 21, 698 (1927). 15. Chick, H., and Roscoe, M., Biochem. J., 22,790 (1928). 16. Chick, H., and Roscoe, M., Biochem. J., 23,498 (1929) ; 24,105 (1930). 17. Hassan, A., and Drummond, J. C., Biocha. J., 21, 653 (1927). 18. Palmer, L. S., and Kennedy, C., J. Biol. Chem., 74, 591 (1927); 75,619

(1927). 19. Kennedy, C., and Palmer, L. S., J. Biol. Chem., 76, 607 (1928). 20. Kennedy, C., and Palmer, L. S., J. Biol. Chem., 83,493 (1929). 21. Williams, R. R., and Waterman, R. E., Proc. Sot. Exp. Biol. and Med.,

26, 1 (1927); J. Biol. Chem., 78, 311 (1928). 22. Williams, R. R., Waterman, R. E., and Gurin, S., J. Biol. Chem., 83,

321 (1929). 23. Hunt, C. H., J. BioZ. Chem., 79,723 (1928). 24. Hogan, A. G., and Hunter, J. E., J. BioZ. Chem., 78,433 (1928). 25. Reader, V., Biochem. J., 23. 689 (1929). 26. Sherman, H. C., and Axtmayer, J. H., J. BioZ. Chem., 76, 207 (1927). 27. Daniels, A. L., and Brooks, L., Proc. Sot. Exp. BioZ. and Med., 26, 161

(1927). 28. Macy, I. G., Outhouse, J., Graham, A., and Long, M. L., J. BioZ. Chem.,

73, 189 (1927). 29. Outhouse, J., Macy, I. G., Brekke, V., and Graham, A., J. BioZ. Chem.,

73, 203 (1927). 30. Hunt, C. H., and Krauss, W. E., J. BioZ. Chem., 79,733 (1928). 31. Aykroyd, W., and Roscoe, M., Biochem. J., 23, 483 (1929). 32. Daniels, A. L., Giddings, M. L., and Jordan, D., J. Nutr., 1,455 (1929). 33. Eddy, W. H., Gurin, S., and Keresztesy, J., J. BioZ. Chem., 87, 729

(1930). 34. Guerrant, N. B., and Salmon, W. D., J. Biol. Chem., 89, 199 (1930). 35. Williams, G. X., and Lewis, R. C., J. BioZ. Chem., 89, 275 (1930). 36. Sherman, H. C., and Sandels, M. R., J. Nutr., 3, 395 (1931). 37. Chick, H., and Copping, A. M., Biochem. J., 24,932 (1930). 38. Hunt, C. H., and Wilder, W., J. BioZ. Chem., 90,279 (1931). 39. Johnson, J. M., and Hooper, C. W., Pub. Health Rep., U. S. P. H. S.,

s4, 2037 (1921). 40. Supplee, G. C., Kahienberg, 0. J., Flanigan, G. E., and Dow, 0. D.,

J. Dairy SC., (1931) in press.


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41. Norris, L. C., Heuser, G. F., and Wilgus, H. S., Poultry SC., 9, 133 (1930).

42. Coward, K. H., Key, K. M., and Morgan, B. G., Biochm. J., 23, 695 (1929).

43. Coward, K. H., Key, K. M., Morgan, B. G., and Cambden, M., Bio- chm. J., 23, 913 (1929).

44. Supplee, G. C., Dow, 0. D., and Flanigan, G. E., J. Dairy SC., 11,420 (1928).


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FlaniganG. C. Supplee, O. J. Kahlenberg and G. E.

WATER-SOLUBLE PORTION OF MILKOF THE CONCENTRATED

PROPERTIES (VITAMIN B COMPLEX) THE GROWTH-PROMOTING

1931, 93:705-725.J. Biol. Chem.

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THE GROWTH-PROMOTING PROPERTIES (VITAMIN B COMPLEX) … · 710 Vitamin B Complex of Milk procedure was carried out in the manner previously noted. The rice polish was from the same