MOLECULAR DISTILLATION' Examination of Natural Vitamin D K. C. D. HICKMAN AND E. LEB. GRAY Eastman Kodak Company, Rochester, N. Y.
RECEKT paper (9) conExamination by distillation and rat to determine the temperature of assay of the fractions of calciferol shows maximum elimination appeared firmed the fact that vitain fish to be to add the calciferol to cod A and that it has a simple elimination curve. liver oil distillates similar to oils partly in the free state and partly in combination as esters Cod liver Oil has a complex curve indicatthose which had first been used ing two chief vitamins, two present in to find the elimination maximum with the fatty acids in the oil. lesser quantities and traces of two more. of cod liver vitamin D. This Vitamin A was revealed as a for spearfish and white sea procedure offered the attractive single entity, and evidence of a The second growth-promoting subfeature that it seemed to provide bass are different from each other and a direct comparison between the stance was not forthcoming, even from cod liver vitamin D- The lowest two vitamins. If calciferol were after repeated distillation. The boiling vitamin D is probably devoid of identical with the cod liver vitavitamin D of cod liver oil, on the min D, then the curve of the side chains on the C1,atom of the cholane other hand, showed definite indilatter would be elevated in procations of complexity, which was nucleus. portion to the number of units expected after the many papers of calciferol added without being published on the multiple nature shifted in position. If the vitamins evaporated a t different of vitamin D. The investigation of this vitamin was continued by means of molecular distillation and the method of temperatures, two maxima or a distorted maximum would result, and the relative positions of the true peaks could then elimination curves described previously (6, 8). Although the task was commenced four years ago without be calculated. As it turned out, the distillations followed neither of these aid, generous help has since been received from workers discourses, and the work was interrupted until the carrier-oil tinguished in this field. A. Windaus supplied a crystalline specimen of vitamin D3dinitrobenzoate, and A. L. Bacharach technic had been evolved. Distillation from synthetic glycerides eventually gave the true calciferol curve. The supplied pure calciferol; C. E. Bills contributed the liver oils, other than cod, on which the distillations were carried earlier experiments are reported, however, because they reveal some reactions of calciferol which have features of inout. It is largely from Bills's writings (9, S , 4 ) that the backterest. ground for the investigation was acquired. The earliest solvent for calciferol was a distillate comprisThe elimination curve of the natural vitamin A esters in fish oils is complex and is safely judged to be so because it ing the first 5 per cent evaporated from Norwegian cod liver oil in a large continuous still. After partial removal of the can be compared with the simple curve given by the free cholesterol by chilling, it was designated CLO-1 and was set vitamin. The free vitamin A elimination curve is shaped aside as stock material for a series of distillations. This strictly in accordance with theory; its form and position are series, carried out in the cyclic still, yielded fractions of repeatable and readily matched with those of various dye fairly uniform bulk a t temperatures ranging from 70" to indicators. The distillation curve of natural vitamin D, or the mixture 250" c. The f i s t experiment was made without calciferol. The of antirachitic substances occurring in fish oils, cannot be examined with the same certainty as vitamin A unless a t fractions were examined by one-rat multilevel assays, and the potencies were plotted against the temperature of distillation, least one chemically pure variety of vitamin D can be shown t o give the simple theoretical elimination curve. In terms giving the approximate elimination curve for cod liver of the present investigation, the potencies of the samples of vitamin D (experiment 1, Figure 1). vitamin D distillate, examined by rat assay, should lie along A distillation was carried out on another portion of fraction CLO-1 to which had been added a quantity of calciferol a predicted distillation curve. Since two supposedly pure vitamins were available-namely, the calciferol and the SO that the natural vitamin D and the calciferol each contributed equal rat potencies. The sample of CLO-1 convitamin D3-the first step was to examine the behavior of tained approximately 100,000 U. s. P. units of natural vitamin these substances on distillation. D, and 2.5 mg. of calciferol were added. The elimination curve of this mixture is shown as experiment 2, Figure 1. Elimination Curve of Calciferol The cod liver oil maximum a t 150" C. was Dreserved. and a The first experiments with calciferol were carried out in new maximum, presumably that of calcifeiol, appeared a t 1934. No constant-yield (I) carrier oils had yet been com140' C. The whole curve was narrower and the limbs were pounded, nor had their need been recognized. The only way steeper than for cod liver oil vitamin D alone, whereas for a 1 This paper completes the series whioh was started in September, 1937 mixttm having separated maxima, the reverse should be the case. The over-all yield in rat units was less than 60 per cent ( 1 , 6 , 8, 8). 796
JULY, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
of the original potency, which cast doubt on the value of the experiment. The next variation (experiment 3) consisted of distilling a portion of fraction CLO-1 with a large excess (25 mg.) of calciferol. The true maximum of calciferol should have developed essentially free from distortion caused by the relatively small quantity of natural vitamin D also present; the maximum a t 140" C. should have persisted and that a t 150" C. should have been absent. The actual curve, however, when reduced to a scale comparable with the others, showed little evidence of either maximum. A peak a t 140" C. is discernible, but a new maximum has appeared a t 160" C. with a well-defined subsidiary peak a t 190" C. A possible inference from experiment 2 is that t8hecalciferol reacts with natural vitamin D to produce a varied assortment of heavier antirachitic bodies. Experiment 3, on the other hand, suggests that calciferol can suffer internal rearrangement without interaction with the constituents of the oil or that it reacts with provitamins or sterols; the supply of natural vitamin D is insufficient, presumably, to alter the whole quantity of calciferol used. An attempt was made to produce a solvent for the calciferol free from vitamins. The solvent (fraction CLO-1) was heated for 12 hours a t 200" C. in the absence of air to destroy vitamins A and D (verified by animal assay), and was then completely distilled to free it from too volatile and nonvolatile products of pyrolysis. The center fraction, having distillation characteristics similar to the original CLO-1, contained no free fatty acids and was deemed suitable for use as a vehicle for the distillation of more calciferol (5 mg.). The elimination curve was again complex but did not resemble previous curves. The work was continued with the aid of constant-yield oil and distilled diluent oil of high boiling point, both of which were free from vitamins and almost free from sterols. With this solvent the curves of Figure 1, from experiments 5, 6, and 7, were obtained. EXPERIMENT 5. Five milligrams of calciferol were dissolved in a mixture consisting of 105 cc. of constant-yield oil (boiling point, 70" to 200' C. at 1p) and 105 cc. of ure diluent oil (boiling point, 210" t o 260" C. at l p ) , and were &gassed in the cyclic still at 50" C. overnight. Distillation was carried out with the distilland and preheated to 50" C. before reaching the column, using 5" C. temperature and 10-minute time intervals. EXPERIMENT 6 was similar to 5 except that the distilland was degassed overnight at room temperature. EXPERIMENT 7 was similar to 6 except that the distilland was not preheated before admission to the column. Also, the apparatus, column, and distributing gage were scrupulously cleaned before use.
The three experiments tell a connected and, apparently, a conclusive story. The points on the low-temperature ascending limbs of curves 5 and 6 are superimposable within the limits of experimental error. The limbs rise with a steepness very nearly appropriate to the quantity of calciferol originally added to the distilland. At 140" C. with the sample which received the longest thermal preexposure, and at 145" to 150" C. with the sample which was degassed in the cold, the calciferol was converted into a higher boiling antirachitic agent. As the supply of original calciferol was suddenly diminished in the middle of distillation, the elimination curve exhibits a sharp break, only to rise again as the maximum of the new substance is approached. From experiment 7 we obtain for the first time the true elimination curve of calciferol; it has a single maximum and a simple form. The area included accounts for 95 per cent of the original vitamin. The points on the ascending limb are parallel to those of experiments 5 and 6, but they do not superimpose on the latter exactly because no loss has occurred to the vitamin during the degassing period.
797
8-
c-
110
t
170
130
140
150
I / \c
zot
160
170
190
180
\
E X P T
7
EXPT
4-
1 110
120
I I I I K\ 130 140 I50 160 I70 180 190 TEMPERATURE O F D I S T I L L A N D , OC.
FIGURE1. ELIMINATION CURVES OF MIXEDAND CALCIFEROL
OF
PURE
It is significant that, whereas the over-all yield of vitamin in the distillate is only 60 per cent in experiment 5, it is over 100 per cent in experiment $. Evidently the new antirachitic substance(s) is more potent to rats than calciferol, or it is produced in more than one molecular equivalent. SUMMARY.Calciferol can be distilled unchanged from sterol-free oil. It yields an elimination curve of theoretical shape for a substance having an exceptionally high latent heat of evaporation. The maximum is a t 146", compared with diethylaminoanthraquinone a t 151 " C. Calciferol can undergo pyrolysis in oil solution a t 140" to 160' C. to form antirachitic substances of less volatility. Criteria for a Single Antirachitic Substance It is now possible to lay down tentative criteria by which to recognize a single antirachitic substance: The distillation curve should become simpler as the thermal exposure is decreased, approximating a single-peak curve for distillations involving the least exposure. The points on the low-temperature limb should be repeatable within the limits of assay. The steepness of the limb should be appropriate to the quantity of vitamin originally present in the distilland. Redistillation of fractions corresponding to any part of the curve should yield a new curve which, when adjusted to the same height as the old curve, is similar in shape. If redistillations of head and tail fractions give dissimilar curves, there is evidence of more than one vitamin. The distillation should be considered untrustworthy i f less than 80 per cent of the original potency is recovered in the distillates.
Vitamin
DD
The sample available consisted of a minute quantity of t h e dinitrobenzoate of vitamin Da. It was a year old when used.
798
INDUSTRIAL AND ENGINEERING CHEMISTRY
before it is accepted. Therefore, together with the most, probable points on the vitamin D curve, the assay limits outside of which the potencies c a n n o t l i e were included. These limits were chosen distinctly wider than the assay indications. This precaution makes it possible to assume with certainty that, wherever the limits of two curves fail to overlap, a difference of distillability of the vitamins has been demonstrated, and more than one vitamin is represented by the two curves.
FIGURE2. ELIMINATION CURVESOF VITAMIN D,
The sample was hydrolyzed with a 20 per cent solution of potash in 50 per cent ethyl alcohol. The saponification mixture was heated on the steam bath for one hour. After cooling, it was extracted three times with low-boiling petroleum ether, the extract was washed free of soaps, and the petroleum ether was evaporated. The residue was taken up in a mixture consisting of 100 cc. of constant-yield oil and 100 cc. of pure diluent oil. The oil solution was assayed biologically and proved to contain a total of 200,000 rat units. It was divided into two parts which were then distilled, forming experiments 8 and 9 of Figure 2, A and B, respectively. The curves were highly unsatisfactory. Since the lower limbs coincide approximately, it is apparent that decomposition is occurring a t higher temperatures. The curve of Figure 2A accounts for 50 per cent recovery, and that of Figure 2B for 80 per cent, so that we can construct a probable curve, (9a, Figure 2B) for 100 per cent recovery. This places the highest point a t 168" C. For a peak a t such a high temperature, the potencies of the fractions taken a t 115" C. are far in excess of what is plausible for a single substance. It must be concluded that the material contained more than one kind of vitamin D a t the time the experiments were started. This does not dismih the likelihood that the crystals were a single substance when they were first prepared by A. Windaus.
VOL. 30, NO. 7
I
t
I*
-
/\
Norwegian Cod Liver Oil
C. E. Bills has continually warned that Norwegian cod liver oil is likely to contain more than one species of fish, so that an investigation of this oil will not yield conclusive information about the antirachitic vitamin in the livers of Gadus morrhua. Nevertheless, since a wide range of fractions was available from the distillation of many tons of the Norwegian oil, and since the oil continues to be a medicinal favorite, completion of the investigation seemed justified; it was borne in mind, however, that any complexity found in the vitamin D might arise from the mixture of species. About 70 per cent of the vitamin D in cod liver oil is present in ester form, making it desirable to saponify the oil before distillation. This is advisable because the ester of a light fatty acid and a heavy vitamin D might distill a t the same temperature as that of a heavy fatty acid and a light vitamin D, and a poor separation would result. Accordingly, sufficient oil to yield about 200,000 U. S. P. units of vitamin was saponified, extracted with ether, and incorporated with the mixture of constant-yield and diluent oils, as described for vitamin DO.The mixture was distilled under the usual conditions,. yielding the elimination curve (experiment 10) shown in Figure 3A. The existence of more than one vitamin D in fish oil is such a n interesting and, a t the moment, controversial subject that the evidence for complexity should be well established
FIGURE3. ELIMINATION. CURVESOF ANTIRACHITIC SUBSTANCES IN CODLIVEROIL ELIMINATION CURVEFOR WHOLE FIGURE4. AVERAQED CODLIVEROIL
JULY, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
Experiment 10 on saponified cod liver oil was repeated exactly in experiment 11 (Figure 3B). Considering the difficulties in technic, the curves are sufficiently similar. I n each curve there are inflections discernible in the other, but only the central dip is obviously common to both. Although a t first, four to six inflections, and hence vitamins, seem to be present, this central dip a t 150" C. casts discredit on them all because it does not appear likely from Figure 10 of the earlier paper (8) that maxima, situated as closely as these, can show a depression between them. Further distillations indicated, however, that this conclusion is wrong. The two curves and their limits were combined in Figure 4 t o give t,he master curve for whole cod liver oil. The distillate fractions from which the curves were constructed were mixed together in three lots and reexamined.
0-
6-
1-
zI I10
100
I20
I 130
I
I
140
150
I
I 160
I10
180
4-
3-
337-
I5-
799
I n experiment 12 the fractions distilling between 100" and 130" C. were combined with constant-yield oil and distilled. The fractions between 130" and 150" C. were blended appropriately and redistilled in experiment 13, and the fractions between 150" and 190" C. in experiment 14. The curve from the low-boiling fraction (experiment 12) is shown in Figure 5 . The potencies a t 120" to 130" C. are higher and those a t 170" to 180" C. lower than the potencies of the corresponding fractions of the whole cod liver oil. Only one maximum appears. When the low-boiling curve is superimposed on the master curve (as in Figure 6), the change in the vitamin distribution is clearly seen. The peaks a t 140" C. coincide satisfactorily, although the breadth of the maximum from the low-boiling vitamin is broader than would be expected for an undistorted peak. It must be confessed, too, that the assays from 130" to 160" C. were poor; and it is possible that the potencies a t 150" and 155" C. were assessed a t too low a value. The elimination curve for the high-boiling fractions (redistilled in experiment 14) is given in Figure 7. The subfractions showed no potency below 120" C., and the high-boiling maximum a t 160 " C. coincides with the higher maximum of the master curve (Figure 8). An even more convincing demonstration of the separation which the two redistillations achieved is shown in Figure 9 where the highboiling and low-boiling curves are superimposed. Figure 10 records the most probable curves, without the assay limits, of all the distillations of cod liver oil. In Figures 8 to 10 we can count the vitamins by the peaks and points of inflection. The curves suggest that two chief vitamins (marked 1and 2) are present in nearly equal unitage, the combination accounting for 70 to 80 per cent of the rat potency of the oil. Certainly two other vitamins are present, KO. 3 occurring in substantial amounts. The maximum a t 125" C. on the high-boiling curve of Figure 7 and the paddingout of the low-boiling curve (Figure 5) are satisfactory evidence of vitamin 4. For the lowest and highest boiling members (5 and 6), there is less convincing proof. The increased potency of subfractions taken off at 190" C. during redistillation of the high-boiling main fractions may be due to an alteration of the lower boiling vitamins, an alteration of the kind shown by calciferol, I n that case, however, it would be expected that redistillation of the low-boiling main fractions would reveal some of these changed vitamins also. It shows none. Furthermore, the curves are exactly repeatable, which the anomalous calciferol curves are not. The evidence, therefore, is distinctly in favor of the existence of traces of a vitamin boiling 38" C. higher than calciferol in cod liver oil. The case for vitamin 5 rests on different grounds. At 90" to 110" C. it is improbable that the main vitamins have suffered any thermal decomposition. The existence of vitamin 5 is suggested by the abnormally gentle slope of the elimination curve a t 105" C. and by the fact that this temperature corresponds with the smallest antirachitic molecule that is likely to be found containing the cholane nucleus.
b5-
"Rat' ' and "Chicken" Vitamin D's
LI-
I 100
FIGURE 5.
110
120 I30 140 150 IC0 170 TEMPERATURE OF DISTILIANC,°C.
1 6 0
IS0
REDISTILLATION OF LOW-BOILINQ COD LIVER OIL FRACTIONS
F I G U R6. ~ COMPARISON OF LOW-BOILING FRACTIONS WITH WHOLECODLIVEROIL FIGURE7. REDISTILLATION OF HIGH-BOILING COD LIVER OIL FRACTIONS
In 1930 Massengale ( I O ) , working in Bills's laboratory, reported that irradiated ergosterol of potency sufficient to cure rickets in rats was devoid of curative properties for chickens. This was confirmed by Mussehl and Ackerson (11) and by Hess and Supplee ( 7 ) who, with other workers, established beyond doubt that calciferol and the natural vitamin D's have different physiological actions. Bills and co-workers (4) later examined the relative rat-chicken poten2 The quantity of each vitamin present is, of course, inversely proportional to its potency to the rat.
INDUSTRIAL AND ENGINEERING CHEMISTRY
800
10
VOL. 30, NO. 7
-
8-
6-
5
4-
E4 z -
3 ? IO,
-HIGH
j:'
TUNA-
$ 2
p:
110
120
I I I I I I 130 140 150 180 170 180 T E M P E R A T U R E O F D I S T I L L A N D , OC.
I
FIGURE 11. VITAMIND ELIMINATION CURVESOF LIVER
OILSFROM WHITESEABASSAND SPEARFISH FIGURE 12. REDISTILLATION OF HIGH-AND LOW-BOILING TUNAOIL FRACTIONS 10
-
, 8-
the assays of the fractions were excellent, and the curves (superimposed in Figure 11) leave no doubt that the vitamins distill a t different temperatures. The sea bass gives an unusually pure curve, suggesting that there is a preponderance of a single "chicken" vitamin in this oil.
LOW F R A C T I O N S
6-
4-
Albacore V i t a m i n D 100
110
I20
130
I40
150
I60
IlD
IS0
T E M P E R A T U R E OF D I S T I L L A N D , OC.
FIGURE 8. COMPARISON OF HIGH-BOILING FRACTIONS WITH WHOLE.COD LIVEROIL FIGURE 9. ELIMINATION CURVESOF HIGH-AND LOW-BOILING FRACTIONS OF CODLIVEROIL FIGURE 10. VITAMIN D ELIMINATION CURVESOF CODLIVER OIL
cies of a vast range of fish oils and showed that some oils are more effective, rat unit for rat unit, than cod liver oil for the chicken; others are less effective. Many oils measured in rat units are less potent to chickens than cod liver oil, but only a few are more potent. Of these, white sea bass (Cynoscion nobilis) is unique in being three times more active (4). Liver oils which are correspondingly less active come from the albacore (Thunnus germo) and from the spearfish (Tetrapturus mitsukurii). It was decided to compare the distillation curves of the vitamins from these opposite types of oil. The vitamin D of tuna liver is so unstable to heat that consistent curves could not be obtained., The comparison was, therefore, made between the white sea bass and. the spearfish. Fortunately,
During the distillation from a constant-yield mixture of saponified long-finned tuna liver oil, 20 to 40 per cent of t h e vitamin D was lost and the elimination curve was seldom reproducible. However, when the head and tail fractions of the surviving vitamins were redistilled, the curves shown in Figure 12 were obtained. They suggest that the oil holds a. preponderance of a single vitamin, probably the one isolated by Brockmann (5) and identical with the vitamin DB of Windaus (IZ?), accompanied by smaller amounts of higher and lower boiling members.
N a t u r e of the V i t a m i n s Since this work involved no chemical investigation, remarks concerning the constitution of the vitamins are necessarily speculative. From the formulas established for calciferol (IS), vitamin Dy,and other antirachitic bodies (14), it is not unreasonable to suppose that the many marine vitamins possess the same essential cholane nucleus and that variations are largely confined to the side chain attached ta the seventeenth carbon atom. The variants would thus b e homologs and isomers of one another. The writers previously found from a study of the amino-. anthraquinone dyes that methylene groups in a side chain contribute approximately 4.5' C. each to the temperature of evaporation (Figure 13). If the elimination maximum of
JULY, 1938
INDUSTRIAL AND ENGINEERING CHEMISTRY
E L I M I N A T I O N CURVES OF P I L O T DYE5 :
110
130
150 I10 190 TEMPERATURE OF
210 230 OISTILLhND,
250 -C
FIGURE 13. EFFECT OF METHYLEKEGROUPSON EVAPORATION TEMPERATURE calciferol is considered to be a t 147" C. and that of vitamin D3 a t about 150" C.,,then on the same scale the lowest boiling vitamin evaporates a t 105" and the highest a t 185" C. This suggests that the most volatile vitamin contains nine carbon atoms less than calciferol and is thus devoid of a side chain. The heaviest vitamin yet detected would have a chain containing eight or nine carbon atoms more than calciferol. The antirachitic materials were distilled from many more fish liver oils than are mentioned in this paper. The peaks of the curves are all situated between 140" and 160" C. It would seem that a side chain with eight to ten carbon atoms is preferred in nature and that compounds with shorter or longer appendages occur in lesser quantities, determined by a fairly simple probability relation. The probability that a vitamin with "less than zero" atoms in the side chain will be found is, perhaps, zero, because this would involve depletion of the nucleus. That vitamins of very much higher molecular weight than any yet detected exist in small quantities is not unlikely, in view of the high potencies occasionally associated with fractions coming over about 200" C. Generally, however, the heaviest vitamins would be destroyed during distillation or would be broken down into simpler forms which would appear in the distillate a t lower temperatures.
condition because it will probably be some time before the final stage can be reached. For the final stage a larger scale of distillation and a greater degree of separation will be required than can now be obtained in one operation. There is some hope that the lowest boiling vitamin D can soon be isolated. In the first fraction of a commercial first fraction of cod liver oil (sample CLO-1-1) are available about 4.5 grams of low-boiling vitamin D, which perhaps can be separated from the kilograms of nonsaponifiable matter in which it is dissolved. A portion of the crude distillate was submitted to C. E. Bills for an estimate of the relative rat-chicken potency He reported that the potency is abnormally low to chickens, on a rat basis, so that the lowboiling vitamin differs greatly from the main bulk of the cod liver vitamin D. His experiments are to be published elsewhere. The relative behavior to rats and chickens is only one of the distinguishing tests for the various vitamins. Evidence shows that the thermal stabilities vary markedly. Also the change in degree of healing with change of dosage differs from one vitamin to another. While the distillates coming over below 145" C. produce 4+ healing in the knee joint of the rat
TABLEI. DATAON CODLIVEROIL Temp. of Distillates, ' C. 100 105 110 115
Degree of Healinga a t Following Assay Level in U. S. P. Units per Gram
2 0 2 2 0 80 160 240 2 . 5 1.5 0 . . . . . 3 3 2 0 3 3 1 0 0" . . . 3.5 2 { T . ' f 1 . 5
320
420
a
550
700
900 1150
. . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . 1.5 0 . . . . . .
0
120
. . . . . .
125
. . . . . . . . 3
2.5
130
. . . . . . . . . . .
3
135
. . . . . . . . . . . . . 3
0
...
140
. . . . . . . . . . . . . . .
1.5
0
145
. . . . . .
i
0.5
150
(: . . . . . . . . . . . . . . . . . . .! 2
3
2
{;I
0
0
. . . . . . . . . . . 12.5
42.5
160
2.5 . . . . . . . . . . . . . . . . . 2.5
165 170
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0 1 0
175
. . . . . . . . . . 1
0
180
. . . . . . . . 2.5
0
0
190
. . . . . . 2 0.5
0.5
4+
{E]
... ...
1
155
Status of the Problem Although evidence presented here indicates that many vitamins exist in fish oils, they have not been isolated, nor have distillates of any one been obtained entirely free from the others. The investigation is reported in this unfinished
801
/
0.5 2
0.5 0
{g}
{g] 0
2
2 0 ..255 1 1 0.5 0.5
B
1 0
2
0.5 2 1.5 0
{A]
1 0.5 0
0 0
0 0.5
... ...
0
0
0
0
. . . . . .
0
.........
= complete healing.
TABLE 11. DATAON SPEARFISH Temp. of Fraotion, r C. 100 110 115 120 125 130 135 140 145 150 155 160 165 170 175 1SO a
4+
0
... ... ... ... ... ... ... ... ... =
Degree of Healingo a t Following Assay Level in U. S. P. Units per Gram:
125
150
180
210
0
0
0 0
... 0
...
...
... ... ... ... ... ...
... ... ... , . .
...
... ... ...
oomplete healing.
... ... ... ...
... ... 3
260
310
... 0
... 0
... 0
...
1.5
... ... ... ...
0.5 2
... ...
.,. ,..
... ... ... ... ... ...
2.5
3 3
... ...
... ...
...
370
450
540
650
...
...
...
0 1 1.5
0 0
...
... ...
...
... ... ... ...
3 2
... ...
3.25 3 1
...
2.5 2
...
... 3 3 1
...
...
0 0 1
...
2.5 3.5 2.5
... ...
780
940
1130
0 0 2 3 3 3 3.25 2.5 2.5 1.5
0 '. 1.5 2 2.25 3 3 2 2
n
...
...
...
...
...
1360
1630
0'. 0 1.5 2 2 1.5 2
0 0 0 1.5 1.5
...
6.5 0.5 1.5 2.5 2.5 2 2
...
,
.
...
I
802
INDUSTRIAL AND ENGINEERING CHEMISTRY
when fed in doses double that required for 2+ healing, the fractions distilling a t about 160" C. often fail to produce more than 2.5f healing and in large doses may even show diminished healing. Stated differently, the biological response curve of the lower boiling fractions is steep, whereas the curve for the distillate a t 160" C. is flat. This makes assay of the later fractions uncertain, an uncertainty which is reflected in the in broader limits assigned to the curves a t 155" to 165" most of the figures. The given in this paper are considered an adequate substitute for tables of distillation data and biological assay levels. However, for the sake of example, and to lend point to the subject of the last paragraph, the data for whole cod liver and spearfish liver oils are given in Tables I and 11.
c.
*
Acknowledgment The rat assays in this paper were done by H. J. Cannon, of the Laboratory of Vitamin Technology, Chicago, to whom the writers are indebted for criticism and advice.
Literature Cited (1) Baxter, J. G., Gray, E. LeB., and Tischer, A. O., IND.ENG. CHEM.,29, 1112 (1937). (2) Bills, C. E., Cold Spring Harbor Symposia Quunt. Biol., 3, 328 (1935). (3) Bills, C. E., J . Am. M e d . Assoc., 108, 13 (1937). (4) Bills, C. E., Massengale, 0. N., Imboden, M., and Hall, H., J . Nutrition, 13, 435 (1937).
VOL. 30, NO. 7
(5) B r o c h a n n , H., 2. phusiol. Chem., 241,104 (1936), 2 4 5 , 9 6 (1937); Brockmann, H., and Busse, A., Ibid., 249, 176 (1937), Nuturwissenschuften, 26, 122 (1938); Haslewood, G. A. D., and Drummond, J. C., Chemistry and Industry, 55, 598 (1936);
Zucker, T. F., Simons, E. J., Coleman, H. C., and Demarest, B., Nuturwissenschuften, 26, 11 (1938).
:; EzF; 2;~ 609 (1930).
; ~ ' s " U " d ~ ~ ~ Med., c ~ 27, ~ ~
(8) Hickman, K. C. D., IND. ENG.CHEM.,29, 968 (1937). (9) Ibid., 29, 1107 (1937). (10) Massengale, 0 . N., and Nussmeier, M., J. B i d . Chem., 87, 423 (1930). (11) Muss&], F. E., and ~ ~ kpoultrysei., ~ 9, 334 ~ (1930). (12) W i n d a w A., Lett& H., and Schenck, F., Ann., 520, 98 (1935) ; Windaus, A., Sohenck, F., and Weider, F.V., 2. physiol. Chem., 241, 100 (1936) ; Schenok, F., Nuturwissenschuften, 25, 159 (1937). (13) Windaus, A., Linsert, O., Luttringhaus, A., and Weidlich, G., Ann., 492, 226 (1932); Kuhn, R., and Moller, E. F., Angew. Chem., 47, 145 (1934); LettrB, H., Ann., 511, 280 ( 1 9 3 4 ) ; Muller, M., 2. physiol. Chem., 233, 223 (1935); Heilbron, I. M., Samant, K. M., and Spring, F. S.,;Vature, 135, 1072 (1935); Windaus, A., and Theile, W., Ann., 521, 160 ( 1 9 3 5 ) ;
c. w,,
Heilbron, I. M., Jones, R. N., Samant, K. M., and Spring, F. S., J . Chem. SOC.,1936, 905; Auwers, K. V., Ann., 533, 255
(1938). (14) Wunderlich, W., 2. physiol. Chem., 241, 116 (1936); Linsert, 0.. Ibid., 241, 125 (1936). RECEIIVED March 24, 1938. Communication 664 from the Kodak Research Laboratories.
Factors Affecting the.Vitamin B, Content of Yeast c. P. L. PAVCEK, W. H. PETERSON, AND A. ELVEHJEM University of Wisconsin, Madison, Wis.
M
OST of the work on the vitamin B1 content of yeast has been done on bakers' or brewers' yeast and hence is limited to the genus Saccharomyces. Recently an entirely different type of yeast, Torula utilis, grown on wood sugar medium (7), was reported (11) to contain about the same amount of vitamin B1 as bakers' yeast grown on a molasses medium. I n a previous paper from this laboratory (10) it was shown that a strain of bakers' yeast, when grown on different media, varied greatly in vitamin B1 content, depending on the richness of the medium in the vitamin itself or possible precursors of the vitamin. Since yeasts differ in physiology, it was thought that they might differ in their ability to produce vitamin B1. Accordingly, several morphologically or physiologically distinct yeasts were grown on the three types of media used previously. The yeasts studied included Saccharomyces logos, reported by Drummond and Whitmarsh (5') to be superior to S. cerevisiae in ability to synthesize vitamin B1; Willia anomala, chosen because of its distinctive fermentation products as well as for its peculiar spore morphology; Endomyces vernalis, chosen because of its high fat-forming ability; a strain of bakers' yeast different from the one previously studied; and a brewers' yeast. The last two were selected because they represented extremes of the X. cerevisiae group. All the yeasts were grown in the same media under conditions of aeration, pH, and temperature approximating as nearly as possible those used in commercial practice. The biological assays were all made in the same manner, so that the results are on a strictly comparable basis.
Certain other questions raised in the previous paper regarding the ability of yeast to assimilate B1 from the medium and to regenerate the inactivated vitamin were also investigated.
Growth of Yeast T o obtain enough of the noncommercial yeasts for inoculation of several subsequent runs, a batch of yeast was grown in a 500-liter pilot plant on grain medium. The procedure employed was as follows: The culture on a glucose yeast-water slant was transferred t o 250 cc. of grain medium (5" Balling) and incubated 20 hours at 28" C.; 10 cc. were used to inoculate a second 250 cc. After 20-hour incubation, the entire 250 cc. was poured into 1000 cc. of the wort. This was in turn transferred into 15 liters of grain medium which, after 12 to 15 hours of incubation, served as inoculum for 130 liters of medium (the medium was slightly aerated at this point). After 12 hours of growth, the 130 liters were run into 350 liters of strong wort (5" t o 6" Balling) and moderately aerated during the next 8 hours of fermentation. From the pilot run about 20 to 2 5 pounds of yeast were obtained, which was stored at 4' C. until used. Some of this yeast was dried and assayed, and other portions were used as inoculum for subsequent laboratory runs on various media. In the case of bakers' and brewers' yeast the commercial product was used as inoculum. The laboratory runs were made with 30 to 60 liters of medium, in the manner described in the earlier paper (IO)..
