A Study of Halibut-Liver Oil I. With Respect to Its Vitamin Potency, Physical Constants, and Tolerance : I D. . EMMETT AKD 0. D. BIRD,Parke, Davis and Company, Detroit, RIich. AiYD
C. SIELSEN AKD H. J. CANNON, Abbott Laboratories, North Chicago, Ill. m H I S project was undertaken for the purpose Of i n v e s t i g a t i n g certain practical possibilities of producing a high grade halibut-liver oil. rich in vitamins -A and D. The study included: (1) ways and means Of and
1
Halibut-lirer oil was made conimercial1.y. 6.y . specially devised processes. .Yumerous samples were assayed for the vitamin A content by bofh aninid and colorimetric methods. There was a definite correlation. The potency was 100 10 110 times that of u 400 f o 500 unit-gram cod-liver oil, uhich is twice that wreaiouslv reDorted. The
of the animals in any one group, a gain in weight of a t least 15 grams within a period of 35 days under the conditions of growth and diet specified in this assay. The maximum weight must be attained at the end of the test and the eye condition must be corrected by an amount of cod liver oil not to exceed 3 times the minimum growth factor (4).
I n carrying out these assays on the halibut-liver oils, two control check groups of rats were in varying mays; (4) the tolerance coristants are included. always run p a r a l l e l . I n the of the oil given in massive doses. negative control, the rats were The present paper is a report continued on the basal vitamin '1-free diet after they of progress with particular reference to the last two phases. I n 1929 Schmidt-Nielsen (12) reported their findings on were ready for the correctile treatment, and then given comparing the T-itamin A content of various fish-liver oils. orally a n equivalent amount of the vitamin-free oil which The results showed that halibut-liver oil was exceptionally mas used as the diluent for the samples under test. In potent in vitamin A, having around 25,000 units per gram. the standard control group, the rats, when ready for corThe determination of the vitamin D potency was not reported. rective treatment, were given orally a definite amount of I n the present studies, the authors endeavored to confirm a cod-liver oil which had been previously standardized. these observations, to ascertain the vitamin D value and to I n this way, it was possible to evaluate the potency of the determine the possibilities of adapting production conditions fractions to as definite a degree as this type of procedure will permit. to the use of this oil in human nutrition. COLORTMETRIC METHOD. The colorimetric method used EXPERIMENTAL PROCEDURE was carried out essentially according to the procedure recently I n making the assay for \-itamin A, recommended by the British Pharmacopeia Commission VITAMIN-1POTENCY. the biological method was taken as the main criterion. -1 (11 ) . This is a modification of the original Carr-Price method modification of the antimony trichloride colorimetric method ( 3 ) . I n addition, the authors adopted the suggestion of of Carr and Price (3) was also used as guide. Korris and Church (8) in regard to dilution of the oil prior The bio-assay procedure followed was based essentially to color reading in the Lovibond tintometer. They have upon the U.S. P. method (9). However, as this has been shown that, if the dilution is such that a low color value is obmodified in certain particulars to meet several criticisms, tained, the intensity of the blue color produced with antimony such as the defects in the basal diet, the plan followed was trichloride not only approaches more nearly a linear function that given in the report of the vitamin committee of the of the oil taken, but also runs more nearly parallel to the American Drug Manufacturers Association (4). activity determined by the bio-assay method. In the present The vitamin A-free hasal diet was: determinations all readings were made a t dilutions which gave 5% between four and seven blue units. As a matter of fact, most Casein (purified). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 of the dilutions were adjusted so as to give five blue. Cornstarch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6; Hydrogenated oil (vitamin-free). . . . . . . . . . . . . . . . . . . . . . . . . . . VITAMIND POTENCY. As there has been no official E. S. P. Dried yeast (vitamin B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Salts (McCollum's No. 1851 . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 method for vitamin D, the bio-assay used on the halibut-liver Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 oil was that given in the report of the vitamin committee of the Vitamin D (viosterol) equivalent t o 3% cod-liver oil American Drug Manufacturers Association (4). There was, The conditions as to technic, such as source, age, handling, however, a n additional precaution taken in these serial testsfeeding, weighing, and treatment of the young rats, mere namely. besides having the usual negative control group of carried out by the two laboratories in essentially the same rats during the treatment period, another group was given a manner ( 4 ) . definite amount of a solution of irradiated ergosterol of a The weight criterion was followed in deciding the vitamin d predetermined potency. I n other words, this group served as potency throughout the project. Both of the above citations a standard and made it possible not only to evaluate better the (./t, 9) refer to gain in weight as the basic index: variations in potencies from time to time but also to make This assay (vitamin A) is based upon the estimation of the indirect comparisons with cod-liver oils which had been tested minimum amount of cod liver oil necessary to meet specific in the same way. growth-promoting requirements in a standard test animal (9). The rickets-producing diet used was that of Steenbock and The vitamin A potency of cod liver oil shall be expressed in units per gram of oil, the unit to be t,he minimum daily amount Black ( I S ) . The ?vlcCollum "line test" procedure (5) was employed. (in milligrams) of cod liver oil required to cause, in sixty per cent 10?3
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 24, No. 9
mg. t 2 m g ) , the halibut-liver oil in Figure 2 had 50,000 units of vitamin A per gram. The approximate range in vitamin A value of halibut-liver oils, collected during 1931, is given in Table I. Expressing these potencies in relation to the standard cod-liver oil (500 units per gram), it is seen that the halibut-liver oils were from 75 to 125 times as rich in vitamin A as the cod-liver oil. If the averages of the two respective series of data from laboratories X and Y are compared, the halibut-liver oils were 98.0 and 100.3 times as rich in vitamin A as cod-liver oil, or, on a grand average basis, 99.2 times. TABLE I. VITAMIN A CONTENT OF HALIBUT-LIVER OIL (Measured bv bio-assav method) X--. ~ ~ -LABORATORY ~ Ratio Units C. L. 0. Units per to per Sample gram H. L. 0." Sample gram 38,861 1:lOO 1 38,881 110 1-4 39,791 110 4 39,801 100 7A 39,841 100 7E 39,861 100 7B 40,021 110 8B 40,231 75 8E 40,491 100 8G 41,141 I1 100 41,431 IIIA 100 42,171 85 IIIB 836,869 85 836,869 841,606-7 95 841,606-7 841,747 100 841,747 Average 49,000 98.0 50,166 Cod-liver oil to halibut-liver oil. 7
Y ~
L
-
~
Ratio C. L. 0. to H. L. 0.5 1:lOO 100 125 100 100 75 100 100 90 125 110 100 100 80 100 100.3
Q
1. RELATIVE GROWTH RESPONSEOF RATSKEPT ON A comparison of the variations in vitamin A assay of the VITAMINA-FREE DIET AND THEN SUPPLEMENTED DAILY same samples of halibut-liver oils by laboratories X and Y is WITH VARYING AMOUNTS OF HALIBUT-LIVER A N D COD-LIVER also presented in Table I. These data include the last three OILS Exoept for negative control, first part of initial preliminary period is oils-836,869 to 841,747. The values agree very closely.
IGURE
omitted (from laboratory Y).
VITAMIN A POTENCY (BIo-ASSAY)
A comparative assay' was carried out to ascertain how halibut-liver and cod-liver oils would influence the rate of growth if given daily in varying or graduate amounts to rats which have manifested the required degree of vitamin A deficiency. Thus, knowing the effects which are produced when a potent cod-liver oil is given to rats under these conditions, the question arose as to whether halibut-lirer oil would act in a similar way. I n Figure 1 the curves give very definite evidence that both fish-liver oils reacted in the same way in stimulating recovery from a lack of vitamin A, the rate of response varying in direct proportion to the quantity of oil consumed. The next point was to ascertain more specifically the ratio, in vitamin -4potency, between cod-liver oil of known biologic value and halibut-liver oil. Accordingly, the standard for cod-liver oil was selected a t a range of 400 to 500 vitamin A units per gram. The former figure has been accepted as the minimum by the Council of Pharmacy and Chemistry of the American Medical Association (1) and by the American Drug Manufacturers Association ( I O ) . The latter figure (500 units per gram) is the one that has been generally adopted by leading distributors of cod-liver oil as the more desirable value of reference. I n Figure 2 the curves for the cod-liver oil group a t the 2-mg. daily dose level, and those for the halibut-liver oil group a t a dosage level which was one-hundredth as much or 0.02 mg., ran nearly parallel with one another. The rats in the negative group continued to decline, developed ophthalmia, and died. As the standard cod-liver oil, a t the level of 2 mg. per day per rat, had a unit value per gram of 500 (1000 1 In the followmg discussion, the various data from each research laboratory will be referred to as X (Parke, Davis and Company) and Y (Abbott Laboratories)
I
I
I
FIGURE2. GROWTH CURVEOF RATS FED VITAMINA-FREE DIET,SHOWIXG CURATIVE EFFECTOF HALIBUT-LIVER AND COD-LIVER OILS WHENGIVENYOUNGRATSIN EQUIVALENT POTENCYDOSESOF 0.02 AND 2.0 MG., RESPECTIVELY, PER DAY (From laboratory X)
September, 1932 S'ITAhfIN
INDUSTRIAL AND ENGINEERING CHEMISTRY
d
POTENCY (COLORI3fETRIC
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METHOD)
A series of experiments was carried out with the modified Carr-Price colorimetric method on some of the same samples of halibut-liver oil that were assayed on rats. The results are given in Table 11,expressed in bio-assay units, blue units, and calculated vitamin A units per gram. It was found that the color given by the antimony trichloride with halibut oil was not the same as that for the cod-liver oil. Instead a definite blue-green was produced. TABLE11.
VIT.4MIN
CONTENT O F HALIBUT-LIVER OIL*
(Measured by bio-assay a n d colorimetric method?) COLORIMETRIC METHOD DE"1AT1oN* BIO-ASSAY Blue Bio-assay !La SAMPLE NETHOD units value a
38,861 38.881 39;791 39,801 39,841 39,861 40,021 40,231 40.491 41;141 41,431 42,171 836,869 841,606 841,607 841,747
Average
...
mn
48,900
* From laboratory X.
514
Factor used:
48,878
-0.43 +0.06 +0.14 +0.05 +0.05 f0.05 +0.13 -0.27 +0.05 +0.14 +o. 10 -0.01 -0.12 +0.05 -0.16 +0.05 0.12
- = 95.1 rat units per 1blue unit.
Morton and Heilbron ( 7 ) reported the predominating spectral band of the cod-liver oil and antimony trichloride complex a t 606 mp, while with concentrates of such oils the band shifted to 620 mp. Brode and Magill (2) also found for codliver oil that the major spectral band was a t 608 mp, and that the intensity was directly proportional to the potency of the oil. These same investigators (publication in press) tested the same samples of halibut-liver oil which are included in the present report. The observed dominant spectral band was a t 622 my, vhich corresponds to what Morton and Heilbron found for concentrates of cod-liver oil. It is thus seen that halibut-liver oil is different from cod-liver oil not only in respect to the blue color produced with antimony trichloride, but also in the spectral band formed when the oil is combined with this reagent. Therefore, inasmuch as Sorris and Church (8) found the Carr-Price method more applicable to concentrates of cod-liver oil than to the oil itself, it may be possible that the colorimetric method is also adaptable quantitatively to halibut-liver oil. A comparison of the data in Table I1 shows the correlation between the bio-assay values and those calculated from the blue unit values. The ratio of the average bio-assay value to that for the blue units is 48,900 t o 514, or 95.1 t o 1. That is, the factor for converting blue units into U. S. P. units is 95.1. The respective calculated potency values for the various oils are given in column 6. With the exception of oils 38,861 and 40,231, the deviations are small. The average of all values indicate that, by using the factor 95.1, one can obtain values within 12 per cent of the bio-assay potencies. As a matter of fact, there are often variations greater than this by the bio-assay method itself. Whether this degree of correlation will hold in general is being studied further, but the present findings are a t least suggestive. VITAMIS D POTENCY The fact that halibut-liver oils were found to be so rich in vitamin A led to the study of its antirachitic vitamin D activity. The results, obtained by applying the same technic as is used on cod-liver oils and viosterol, are given in Table 111. The potencies varied between 2000 and 3333 units per gram.
FIGURE 3. GROWTH CURVESOF RATSFEDNORMAL DIET AND 200 MG. E.4CH OF HALIBUT-LIVER OIL, C O M P A R E D WITH CORRESPOXDING RATS WITHOUT TREATMENT
I t is thus seen that halibut-liver oil is a very potent natural source of vitamin D, much in excess of cod-liver oil. At the same time, in halibut-liver oil the vitamin A content, in proportion to the vitamin D content, is much higher than in cod-liver oil. TABLE 111. VITAMIND CONTENT U F HALIBUT-LIVER 011 (Measured by McCollum line test, 6) L4BORATORY LABORATORY Y Unitsn per Units" per Sample gram Sample gram 39,791 2666 1A 3333
x
40,021 40,491 41,701 41,711 41,141 41,431 41,721 42,171 42,872
a
Expressed on
2666 40,721 41,211 2666 2666 841,606 2666 841.607 2666 2133 2666 2666 2395 of d a i l j corrective dose per rat
2000 2000 2000 2000
(4)
PHYSICAL CONSTANTS In Table IV the results are given for some of the physical constants of several samples of halibut-liver oil. The U. S. P. specificztions for cod-liver oil are included for purposes of comparison. TABLE IV. GENER \L PHYSICAL CONSTANTS OF HALIB UT-LIVER OIL
SAMPLE
(Preliminary report from laboratory Y) SP GR.AT NONSAPONIFIABLE SAPONIFICA- IODINE 25' C RESIDUE TION N O . NO. % ._
15-11 19,647 841,747 841,606 841,607
Average U. S. P. codliver oil
0.927 0,928 0.928 0.928 0.928 0.928 0.918 t o 0.927
7:44 7190
..
7.67
Not more than 1.5
...
179 193 185 185 186 180 t o 190
124 120 119
126 118 121 140 t o 180
The respective data for the specific gravity and saponificstion number are almost identical for the two types of oils.
I Pi D U S T R I A L A4N Il E N G I h E E R I S G C H E M I S T R 1-
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The results for nonsaponifiable residue are definitely higher than the corresponding values of cod-liver oil. This may be partly due to methods employed in preparing the halibut-liver oil which are different from those used for cod-liver oil. The iodine number of halibut-liver oil is lower than that for cod-liver oil. This is a desirable feature as it suggests
HAUBUT~IVER OIL
-
d 46mgmper ddyfpJaoxA) & 92 MgH.perddy ~ 6 0A)0 ~
that halibut-liver oil is less easily oxidized than cod-liver oil, which favors a greater stability of the vitamins in the former product. The free fatty acid determinations on numerous samples of halibut-liver oil showed a rather wide variation, apparently depending upon the method of extraction used. The average values ranged from 1.2 to 1.8 cc. of 0.1 N alkali per gram.
TOLERAWE TO M.4SSIVE DOSES Having presented data on the vitamin A and D potency and on the physical constants of halibut-liver oil, it is important to ascertain data relative to the possible nutritional disturbances arising from large doses of the oil. Two series of experiments were carried out. I n one experiment (laboratory Y) rats were fed on a normal diet. Some were given supplemental treatment for 100 days of 200 mg. per day per rat of halibut-liver oil having a potency of 50,000 vitamin A units and 2000 units of vitamin D per gram. Others, serving as controls, received no halibut-liver oil. I n Figure 3, comparing the corresponding growth curves of the the male rats, there was no appreciable difference between the control rats and those receiving the daily massive doses, equal, respectively, to 10,000 times the vitamin A and 400 times the vitamin D requirements for curing the induced symptoms of vitamin starvation. Likewise, the treated female rats grew a t a normal rate which is always lower than that of the male. The excessive dosage of halibut-liver oil did not seem to disturb the well-being of the rats in any way. This was verified by killing and making autopsies of the animals. There were no evidences of abnormalities.
i-01. 24,
so. 9
In the second experiment (laboratory X) young rats were placed on the vitamin A-free diet soon after weaning. When they began to show indications of the vitamin A deficiency, they mere all placed on treatment with a halibut-liver oil having 50,000 and 2666 units of vitamins A4and D per gram, respectively. For the first 2 weeks each rat received orally per day 2300 times the minimum corrective dose of vitamin h and 124 times that of vitamin D; then the daily dose was doubled for the following 36 days. The average weight of the rats was 94.3 grams a t the beginning of the treatment period, 155 grams a t the end of the first 15 days of treatment, and 220.5 grams a t the close of the test. The net average gain per rat was 126.2 grams for the 51 days, making an average gain of 2.5 grams per day. The curves are given in Figure 4. At the end of the experiment, the rats were killed On autopsy, there were no apparent pathological conditions. The nonsaponifiable fat of the combined livers from the six rats was carefully tested by the colorimetric method for its vitamin h value. The yield was 0.87 gram of fat with a blue S. P. units unit value of 500, which is equivalent to 47,550 6. per gram or 83 grams of cod-liver oil. The average amount of vitamin A stored in each rat’s liver would be equivalent to 13.9 grams of cod-liver oil. Figured on the basis of 2 mg. of cod-liver oil per day, this would be sufficientto last a rat about 20 years. This is in agreement with Moore’s observation (6) where he fed rats large amounts of carotin and found correspondingly large quantities of vitamin A in their livers. Calcium and phosphorus were determined on the composite samples of the blood and compared with similar samples from normal untreated rats. The values were, respectively, for the calcium, 11.20 and 11.32 mg. per 100 cc. of serum; and for the phosphorus, 4.8 and 4.9 mg. per 100 cc. of whole blood. It is therefore evident that these massive doses did not produce hypercalcemia or hyperphosphatemia. These two experiments on the administration of massive doses of halibut-liver oil to rats, fed normal and vitamin Afree diets, give evidence of no untoward effects. I n fact, the rats gained a t a rapid rate and apparently performed normally in every way. SUMJl.4RY
AND
CONCLUSIONS
1. -4comparison of the vitamin A and D assays of halibutliver and cod-liver oils, made by two independent laboratories using the same methods on the same oil samples, shows that the results obtained were sufficiently close to justify quantitative deductions. 2. The vitamin A potencies of halibut-liver oils, as determined by the bio-assay method, were in close agreement with the values for the corresponding oils when measured by the modified Carr-Price colorimetric method. This fact gave added confidence to the vitamin A data. 3. The vitamin A potency per gram of oil ranged from 37,500 to 62,500 units with an average for the thirty samples of 49,583 units. Compared with the standard 500 gram-unit cod-liver oil, the halibut-liver oil was from 75 to 125 times more potent than cod-liver oil. This shows that halibut-liver oil is an extremely potent source of vitamin A. 4. The antirachitic vitamin D potency per gram of oil varied from 2000 to 3333 daily units, averaging 2479 units. This is much greater than for cod-liver oil. I n fact, as a natural source of vitamin D, halibut-liver oil occupies a very high place. 5. From the standpoint of physical constants, the specific gravity and saponification number of halibut-liver oil were essentially the same as for cod-liver oil. The free fatty acid value and nonsaponifiable residue were higher in the halibutliver oil than in the cod-liver oil. The iodine number of the halibut-liver was lower, however, indicating that halibut-
September, 1932
I N D U S T R I l L AND E S G I N E E R I N G CHEMISTRY
1077
liver oil contains less unsaturated fatty acid, and therefore be taken with impunity and accepted as a valuable accessory mav be expected to oxidize less easilv than cod-liver oil. to human nutrition. 6. Tolerance tests on rats gave evidence that halibut-liver LITER.4TUKE CITED oil produced no undesirable effects. K h e n the young rats (whether on a normal diet or a vitamin A-free diet) were 4 m . Med. .-issoc., "Xew and Nonofficial Remedies, 1931," p. 259. given, over periods of 50 to 100 days, as much as 10.000 Brode and Magill, J . Bid. Chem., 92, 87 (1931). times the daily vitamin X and 400 times the daily vitamin D Carr and Price, Biochem. J . , 20, 497 (1926). requirements to correct the induced deficient symptoms, they Holmes, J . A m . Pharm. Assoc., 20, 588 (1931). grew rapidly and del-eloped no apparent indications of any McCollum et al., J . Bioi. Chem., 51, 41 (1922). Moore, Biochem. J . , 25, 275 (1931). pathology. The level of blood phosphorus and serum calcium Morton and Heilbron, Ihid., 22, 987 (1928). remained a t normal. The excess of vitamin A was itored to a Norris and Church, J. Biol. Chem., 85, 477 (1930). large degree in the liver as a nutritional reserve. Pharmacopeia of the United States, 10th Decennial Rev., p. 7. Halibut-liver oil, as far as the authors are aware, is the 469, Lippincott, 1928. Proc. Am. Drug -Wig. Assoc., p. 281 (1931). richest knoim natural source of vitamins A and D. The high Rept. of Brit. Pharmacopeia Comm., March, 1931. vitamin d content of this oil is particularly valuable because Schmidt-Nielsen and Schmidt-Kielsen, Biochem. J., 23, 1153 this vitamin exerts a direct influence on the growth and de(1929). \-elopment of the young, and may be a n aid toward the estab- (13) Steenbock and Black, J . B i d . Chem.;64. 263 (1925). lishment of better resistance of the body to infections in genR E C E I ~ E 4pril D 7, 1932 Presented before t h e Division of Medicinal eral, ~ ~the tolerance ~ h ~ , Chemistry a t t h e 83rd Meeting of the American Chemical Society, New Orto~halibut-liver oil~by the experimental animals-even in massive doses-indicates that it can leans, L a , x i a r c h 28 to 4pril 1, 1932
Optical Activity of Commercial Caseins S. P. GOULD,Bureau of Dairy Industry, Department of Agriculture, Washington, D. C.
S
EVERXL i n v e s t i g a t o r s
tions or p h y s i c a l tests. The The spec$c rotations of fifteen commercial of this paper thought author h a v e d e t e r m i n e d the caseins and a Hammarsten casein have been that differences in specific rospecific rotation of pure determined us a means of establishing the tation might furnish informacasein, but whileeach apparently molecular identity of these samples. .'2lost of tion to e x p l a i n these differh a s obtained r e p r o d u c i b 1e the differences found among these values for inences in coating characteristics, r e s u 1t s , there is considerable difference between the values particularly adhesive strength. dicidual caseins appeared to be due to experiIt was a l s o c o n s i d e r e d that r e p o r t e d . Much of this dimental dificulties. The average value for the the effect of using an excess of v e r g e n c e may be due to the hydrochloric acid group agreed reasonably well the p r e c i p i t a t i n g a c i d , of difuse of strong alkali as a solwith that f o r the lactic acid caseins; that of the v e n t , w h i c h causes hydroylferent acids, or of time and sulfuric acid group was distinctly less. I t is sis, or to other fundamental temperature of heating in the c h a n g e s in the casein, which process of manufacture might believed that the differences observed are not would bring about a variation b e s t u d i e d by this method. suficiently large to indicate appreciable deAt the same time, the desirain the rotation. Long (3) decomposition of the casein molecule in any sample termined the specific r o t a t io n bility of using i t as a regular except in the case of the sulfuric group. No o f H a m m a r s t e n casein, b u t analytical procedure to detercorrelation between adhesive strength and specific his results are not comparable mine the purity of commercial with those of the author since caseins and their s u i t a b i 1i t y rotations could be established. Further exhe used mostly strong alkalies for specific industrial purposes periments showed that a n excess of the precipitatas solvents, whereas the writer could be appraised. ing acid changed markedly the specific rotation employed sodium acetate of casein; also that overheating as high as 90" C . solution. Zaykowsky (5) has XETHODS USED during drying did not aflect noticeably the optical investigated v e r y extensively the optical activity of various When Hammarsten c R s e i n activity. a m o u n t s of p u r e casein disdissolved in any of the comsolved in alkaii, acid, and salt solutions of different concen- mon solvents was used, no difficulty was experienced in obtrations. He concluded, among other things, that the specific taining solutions clear enough to polarize. However, in rotation varies with the concentration of the casein and the the case of the commercial caseins, although they were diskind of solvent employed. Hewitt ( 2 ) reports the value solved in a large number of acid, alkaline, and neutral solvents, [n]E& = - 105' for casein but, since it is not clear what few were found which gave clear solutions. Filtration, solvent was used, a comparison is not possible. Csonka and when first tried, proved such a slow process as to be impracHorn (1) have studied the effect of sodium hydroxide on pro- tical. Later it was found that the addition of Filter-Cel teins, including casein, and report the specific rotations of increased enormously the speed of filtration. In selecting the cleavage products. These rotations show a decrease of a solvent, it was important to choose one which would not about 50 per cent from the usual values for the unchanged cause appreciable hydrolysis under the conditions of the casein. experiment. Zaykowsky's results (6) indicated that 10 per Unpublished work on fifteen commercial caseins has cent sodium acetate solution would meet this requirement; brought out differences in paper-coating characteristics which it was therefore chosen for this work. '0 far have not been accounted for by analytical determinaThe following procedure was adopted for all the caseins:
