INDUSTRIAL S N D ENGINEERILVG CHEMISTRY
840
of flow of variations in d, K , and y is brought out in Table 11, giving calculated times of flow for liquids of suitable viscosity, taking for d the values 0.025 and 0.030 cm., and for K the values 1/29 and ‘/a2. It was assumed that b, h, and a had the same values as for the Bureau of Standards instrument. Figure 3, which has been compiled from the data available, may be used for estimating surface tensions of petroleum products with sufficient accuracy.I2 If the gravity of kerosene is assumed to lie between 50” and 40” A. P. I. (sp. gr. 0.780 to 0.825),13the surface tension will lie between 29 and 33 dynes per centimeter. The last two lines of Table I1 12 Francis and Bennett, Egloff, I b z d , 7, 481 (1915) 1s
THISJOURNAL,
14, 626 (1922), Rittman and
Buv Scandavds C Z Y C164 (1924)
1-01, 19, No. 7
were calculated by assuming these values, and taking a viscosity of 0.022 poise in both cases. Then, as shown by the table, an increase in surface tension of this amount will decrease the calculated time of flow about 9.5 per cent, which is substantially the same decrease as that due to a change in the value of K from 1 / / 3 2 to 23. Conclusion
If the experirrientally determined coefficient, I//?g, is used with the theoretical equation for the flow into the capillary tube of a Saybolt thermo-viscometer, the calculated times of flow will agree within the experimental error with the results of tests, over the range of conditions examined.
Cod-Liver Oil as Food’ Observations on the Existence of Vitamin E By V. E. Nelson, R. L. Jones, Georgian Adams, and L. T. Anderegg C H E X I C A L LABORATORY,
O S S I D E R A B L E interest has been manifested during the past few years concerning the existence of a substance designated as vitamin E. Supposedly this unknown dietary factor is required for normal reproduction, and in its absence animals become sterile, although they may grow to full adult size a t the normal rate. Previous Investigations
C
Iowa S T A T E COLLEGE,
4MES, IA.
clusively to the existence of this unknown factor. Several articles have been published from this laboratory on the role of diet in rep r o d u c t i o n and rearing of young. It has been found that reproduction is possible but not normal on synthetic diets containing all the known factors necessary for normal growth and general well-being. These synthetic diets had the following coniposition: casein 18, salt mixture 185,63.7, filtered butter fat 5, Fleischmann and Harris yeast at various levels from 1 to 24. and dextrin to 100 per cent. -1large number of young were born on these rations and animals have been continued for six generations on this synthetic food supply. Reproduction was not normal, however, since the vast majority of the females had only one litter of young, although a few had two and even as many as three or four litters. The number of litters and total number of young born were far below that produced on the standard growing ration employed in this laboratory. Evidently, these results are associated with diet, since on our growing ration large numbers of litters are produced and the mortality is very low. Evans and Bishop’ explain the birth of young on the above synthetic rations by assuming that butter fat a t times contains small but demonstrable amounts of vitamin E. XIthough butter fat is supposed to contain only a small quantity of this vitamin, other foods, especially green leafy vegetables and certain oils obtained from seeds, are potent sources of this dietary factor. Wheat oil and corn oil are unusually rich sources of this principle. On the other hand, Evan? and Burr8 and Sureg claim that cod-liver oil is very deficient in vitamin E. Evans and Burr say:
The food value of cod-liver oil has been tested by the biological method. It has been shown that a synthetic diet containing casein, salts, yeast, dextrin, and codliver oil as the sole source of fat-soluble vitamins suffices for normal growth and normal reproduction. The results obtained with cod-liver oil depend on the manner in which the oil is fed. When fed separately abnormal reproduction records were obtained. Results were not nearly so favorable when cod-liver oil was incorporated in kilogram quantities of the ration as when the oil was mixed in the ration daily. This is no doubt due to decomposition of the oil, either with formation of toxic products or the destruction of the vitamins or both. The odor of the decomposed oil is similar to acrolein. Reproduction is much less favorable with filtered butter fat than with cod-liver oil.
The leading investigators in this field-namely, Evans and associates, Mattill and co-workers, and Sure-have presented a large amount of experimental data which, according to them, point conclusively to the existence of an unknown dietary factor, a t first called vitamin X, but now more widely and logically known as vitamin E, the latter designation being adopted by Sure to be in harmony with the nomenclature of the other vitamins. Evans and Bishop2 were the first to announce the existence of this unknown dietary factor. They reported that the function of substance X is to cure sterility by preventing resorption of the fetus during gestation. Sterility is not confined to the female but, as shown by Mattill and coworkers3 and subsequently by Evans,4 males also become sterile when deprived of a sufficient quantity of vitamin E. Histological examinations of the tissues show that the sterility disease affects males and females differently. Sure5 has studied the distribution of this vitamin in many naturally occurring foods and claims that his experiments point con1 Presented by V. E. Nelson and R. I ,. Jones under the title “Synthetic Diets for Growth and Reproduction” before the Division of Biological Chemistry a t the 72nd Meeting of the American Chemical Society, Philadelphia, Pa., September 5 t o 11, 1926. 2 Science, 66, 650 (1922); J. Metabolic Research, 3, 233 (1923). Mattill, Carman, and Clayton, J. Biol. Chem., 61, 729 (1924). 4 Proc. N a t . A c a d . Sci., 11, 373 (1925). 6 J . B i d . Chem., 69, 29 (1926).
8
7 8
9
McCollum and Davis, J . Biol. Cham., 19, 246 (1914). J . Metabolic Research, 3, 233 (1923). Proc. N a t . Acad. Sed,, 11, 334 (1925). J. B i d . Chem., 69, 53 (1926).
July, 1 9 2 i
INDUSTRIAL A-VD EATGIXEERIiVG CHEMISTRY
Cod-liver oil, though high in vitamins A and D, is notably lacking in E. Throughout the life of animals, 9 per cent by weight of the ration may be constituted by cod-lilrer oil, a single drop of which daily is adequate for A requirements, and yet sterility result.
The ration employed by Evans and Burrs had the following composition: casein 18 per cent, cornstarch 54 per cent, lard 15 per cent, milk fat 9 per cent, salts 4 per cent, and Harris yeast tested for its vitamin B content 0.4 t o 0.6 gram daily per rat. They substihted 9 per cent of cod-liver oil for the 9 per cent of but.ter fat in the diet of fire rats and obtained on the basic ration nine resorptions and no litters, and on the modified basic ration containing cod-liver oil thirteen resorptions and no 1itt.ers were born. They found that the vitamin h content of the particular lot of cod-liver oil d i i c h they used was a t least eleven times as rich as milk fat. These same investigators found no effect on fertility when 24 per cent Xorwegian cod-liver oil was present in the basic ration. Hogan and Harshaw'O have recently in part confirnied data from this laboratory. They have also shown that an increase of \-itamin B in the form of yeast and the addition of cod-liver oil to the basic ration containing butter fat as the only source of fat-soluble vitamins enhance the value of the diet for reproduction. The fact that certain vegetable oils, such as those obtained froin wheat and corn, markedly increase the reproduct'ive value of the diet has been accepted as proof of the existence of vitamin E. Experiments along this line in this laboratory are not yet complete. Anderegg,*l of this laboratory. and Anderegg and Selson12 have made an extended investigation concerning the nutritive value of whole and skimmed milk powders and have found that there is a difference in their nutritive value even when butter fat is added to the skimmed milk powder t o make it comparable in composition with the whole mi;k powder. They found that if natural u-heat embryo were added to the skimmed milk powder dirt (skimmed milk powder 60, butter fat 5 , iron citrate 0.2, wheat embryo 8, and dextrin to 100 per cent), young were born to the first generation females, but no third generation animals were born even though the second generation animals were carefully observed for 5 months. Another group of rats was placed on a similar diet except that yeast replaced the wheat embryo. I n this case also young were born, but no advanced pregnancy resulted from the second generation at the end of 5 months. This is an important observation since a yery small amount of wheat embryo is supposed to confer fertility on females deprived of vitamin E. Anderegg and Selson'? conclude with the following statement :
84 1
ther light on the existence of vitamin E and the relation between the dietary factors essential for successful reproduction and t'hose necessary for growth. Vigorous rats weighing from 50 to 60 grams were employed in these experiments. These animals had been reared on the growing ration employed in this laboratory, which has been shown to be adequate for the needs of the animal body from the standpoints of both growth and reproduction. The animals were weighed once a week, and their general condition and behavior mere noted several times daily. The casein used in the diets was a high-grade commercial product purified by washing with water acidified with acetic acid until vitamin-free and containing approximately 0.2 per cent of ash. The cod-liver oil was a widely distributed brand and was purchased on the open market. The salt mixture was that devised by McColluin and Davis6 and called by them salt mixture number 185. Yeast was obtained from the Fleischiiiann Company. Dext.rin was prepared by autoclaving starch acidified with citric acid solution at 15 pounds ( I kg. per sq. em.) pressure for 3 hours. All the diets contained the same percentages of casein and salt mixture. Vitamin B was supplied by yeast varying in quantity in the different experiments. -211 the animals received cod-liver oil either fed separately or with 5 per cent in the diet. The sum of casein, salts, yeast, and cod-liver oil subtracted from 100 represents the amount of dextrin. The composition of the different diets is recorded in Table I. Table I-Composition
of Diets (Figures in per cent;i SALT COD-LIVER METHODOF FEEDING DIET CASEIN hfIXTURE OIL YEAST DEXTRIX COD-LIVER OIL 1 18 3.7 3.9 3 70.3 Separately 2 18 3.2 4.8 5 68 3 Separately 3 18 3.i 2.9 8 65 3 Separately 4 18 3.7 3.4 12 61 3 Separately 5 18 3.2 8 65 3 Mixed in ration daily 6 18 3.t 12 61 3 Mixed in ration daily 7 18 3.7 J 8 65 3 Mixed in kilogram quantity of ration 8 18 3,i 5 12 61.3 Mixed in kilogram quantity of ration
;
The results of the experiments are recorded in Table 11. Growth curves (not shown) were made for each animal, and these curves compared with the normal. The results are for the most part self-explanatory, but some of the most important points should be emphasized. Codliver oil was administered to the animals in three different ways. Some animals received the oil separately, others reccived it mixed in the ration daily, while another group obtained cod-liver oil mixed in considerable quantities of the ration (kilogram lots). It had already been shown in Clearly wheat embryo and yeast supplement the skimmed this laboratory by Anderegg and NelsonL2that some codmilk powder, making it resemble somewhat nearer the whole liver oils, when incorporated in certain rations and allowed milk powder as concerns its nutritive value for reproduction. to stand, undergo decomposition with the production of The assumption of a vitamin having t o do specifically with repro- substances very suggestive of acrolein. It was deemed diiction makes easy the explanation of the observations. If it is important, therefore, to administer the cod-liver oil in such a vitamin t h a t has t o do specifically with reproduction, then it appears t o be present in the air-dried yeast to an extent com- a manner as to obviate as much as possible the formation of parable t o t h a t present in wheat embryo. decomposition products which appear toxic, and a t the same In a previous publication Nelson, Heller, and F ~ i l m e r ' ~ t,ime to ascertain if this particular brand of oil acts like others showed that modification of the basic ration of Evans and which have been tried in somewhat different rations. OIL FEDS E P A R - ~ T E L Y - oil T ~ was ~ placed in an inverted Bishop by substituting for 15 per cent of lard 15 per cent of a cork through which passed t'est tube in which was inserted dextrin made the diet so that although reproduction was not normal, nevertheless animals were carried through six a glass tube bent at' right angles, closed a t one end, and having generations, whereas no young were born on the high-fat diet. a hole blown out fairly close t o the elbow. Accurate consumption of cod-liver oil could be assured by this procedure, Potency of Cod-Liver Oil in Synthetic Diets and the device also prevented loss of the material. Results for animals receiving 3 per cent of yeast in the It was deemed necessary to ascertain the potency of codliver oil incorporated in synthetic diets in order to shed fur- ration and cod-liver oil separately are given by lot 61. The average amount of cod-liver oil consumed during a period '"J . Metabolic Resenrch, 5, 111 (1924). of 6 months was 3.9 per cent. One female of the three had 1 l J . Bioi Chem., 69, 587 (1924). one litter born dead and then ceased to have more young THISJ O U R N A L , 17, 451 (1925); 18, 620 (1926). '3 J . Biol. Chem., 67, 415 (1923). even though the animals were on the experimental diet for
IiYD USTRIAL A S D ENGIAVEERINGCHEMISTRY
842
Vol. 19, No. 7
Reproduction, a n d Rearing of Y o u n g on S y n t h e t i c D i e t s C o n t a i n i n g Cod-Liver Oil FEMALESMALES TOTAL YOUNG YOUNG RATEOF MONTHS GENERATION FEMALESMALES DIED DIED LITTERS YOUAG WEANED DIED GROWTH=ON RATIONS
Table 11-Growth,
LOT
DIET
OIL F E D S E P A R A T E L Y
61 68
-1
69
2
72 84 S5
3 4
1 1
4
>
89 90
107 108 109
7
2 6 6 6
1 1 2
3 6 1 4 6 5
2 2 2 7
2
I
0 3
0 1
1 6
0
0
0
1 3
0 0 1
2 8
2
0
3 31
0 4
0
0
18 54 0
4 13 0
46 34 146 66 69
31 9 106 46 35
3 27 0 14 41
N
x
N
11 11 71/.
N
9 li;
O
N-
82/3
15
N N
A-
8’/2
OIL M I X E D I N RATIONS DAILY
1 2 1 2 3
3 6 3 5 4
2 3 2 3 2
1 1 0 0 0
0
1 1 1 0
8 7 18 11 11
25 29 20 34
K+
N+
N+
14 11 13’/2 6 b
OIL M I X E D I N KILOGR.4M O U A N T I T I E S O F RATION
N = normal growth; N *
= growth better than normal, S - = growth below normal
11 months. The remaining females did not reproduce. More young were born on higher levels of yeast intake and with cod-liver oil fed separately. For instance, on the 5 per cent yeast level (lots 68 and 69) 6 females had 6 litters or a total of 31 young. Only 4 of the young were reared. Two of the females had 2 litters each. The mortality of the mothers on this diet was high. Three females died shortly after birth of young. Of the 4 females on the 8 per cent yeast level (lot 72) 2 were sterile, for when placed with normal males no evidence of pregnancy was observed. One of the females died in parturition and had 9 young in the uterus. The remaining female had 2 litters of 18 young, of which 14 were consumed by the mother and 4 reared. This female had no more young for 4 months. She was then bred with a normal male taken off the growing ration. A litter of 5 young were born, all of which were reared. These animals on the 8 per cent yeast level consumed on an average 2.7 per cent of cod-liver oil over a 6 months’ period. Six females on the diet containing 12 per cent of yeast and cod-liver oil fed separately (lot 84) had 8 litters or a total of 54 young, of which 34 died before they were weaned, 7 were consumed by the mothers, and 13 were reared. Three females died during the reproduction period, one died in parturition with 8 young in the uterus, and the two remaining females died immediately after the birth of the young. This lot of animals consumed on an average over a 6 months’ period 3.4 per cent of cod-liver oil. The second generation on 5 and 12 per cent of yeast (lots 69 and 85) were continued on the ration of the mothers. Those on 5 per cent (1 female and 2 males) have been on the diet 71/2months, but no sign of pregnancy has been observed, although growth has been a t the normal rate. The animals on 12 per cent of yeast (5 females and 2 males) have been on the diet 82/3 months with no evidence of pregnancy. The growth curves of these rats average slightly below normal. Two of the second generation feinales on the 12 per cent level of yeast died, one from infection of the middle ear and the other from an unknown cause. One male also died from ear infection. The results obtained by administering the cod-liver oil separately show that young are born but that reproduction is far from normal. At least two factors appear responsible for the abnormal results on reproduction on the above rations. Three and five per cent of yeast apparently do not supply a sufficient fact quantity of vitamin B for normal reproduction-a substantiated by data that follow. The consumption of cod-liver oil by the animals is erratic-some weeks being fairly high and a t other times low-which may have a decided effect on such a delicate process as reproduction. OIL MIXEDIN RATIONS DAILY-In these experiments 8 and 12 per cent of yeast were used and 5 per cent of cod-liver oil was mixed in the rations each day. There were 3 females and 2 males in the first generation on 8 per cent of yeast and 5
per cent of cod-liver oil. One of the females was apparently sterile. A second female had 6 litters or a total of 33 young; 6 young died, 2 were consumed by the mother, and 25 were weaned. The remaining female had 2 litters or a total of 13 young, 7 died, and 6 were weaned. Six females and 3 males (lot 90) of the second generation have been on this ration for 11 months. One female died in parturition with 4 young in the uterus. Another female had 5 litters or a total of 20 young, but only 3 were weaned. Another female had a litter of 8 young. Two females have had no young and one other 1 litter of 6 young, 3 of which were weaned. The first-generation animals on the ration containing 12 per cent of yeast with 5 per cent of cod-liver oil mixed in the ration daily (lot 107) have performed exceptionally well, in fact better than any other group. Three females and 2 males were placed on this diet and have been on this food supply for 131/2months. Three females have had 6 litters each, or a total of 18 litters consisting of 146 young (11 recently born), 106 of which were weaned. One male on this diet died from infection of the middle ear. Five secondgeneration females (lot 108) have had 11 litters or a total of 66 young and 4 third generation females (lot 109) have also had 11 litters consisting of a total of 69 young. Two of the fourth generation have very recently had young. The young from the mothers on this ration are exceptionally vigorous and look normal in every way. The total number of litters given for the females does not represent the maximum they were capable of producing. Soon after thirdgeneration animals were weaned the second generation was discarded; and likewise the third generation was discontinued when the fourth generation was fairly well started. The first generation, however, has been kept on the diet to the present time. It is interesting to note that the females of the first generation are still reproducing, although they are 15 months old and 13 months is supposed t o mark the end of the fertility period in the rat. OIL MIXEDIN KILOGRAM &UANTITIES-Five per cent of cod-liver oil was mixed in kilogram quantities of ration. Five females and 2 males received ration number 7 containing 8 per cent of yeast (lot 123). Only one litter (consisting of 2 young) was born; one of which died, the other being consumed by the mother. Lot 127 received diet 8 containing 12 per cent of yeast. Five litters or a total of 36 young were born, 18 were weaned, 5 died, and 13 were consumed by the mothers. The third generation on diet 8 (lot 133) have not reproduced, although they have been on the experiment for 6 months. The results with 5 per cent of cod-liver oil mixed in kilogram quantities of ration are not so good as those obtained when the oil is added to the ration daily. Decomposition of cod-liver oil was very noticeable in the diets in which the oil was mixed in kilogram quantities of the ration. A charac-
July, 1927
ILtTDCSTRIALd S D ESGI,YEERISG CHEMISTRY
teristic odor suggestive of acrolein is produced. S o doubt this is the result of oxidation and the abnormal results obtained on rations where decomposition is evident may be due to destruction of vitamin A, the toxicity of the decomposition products, or both. Hart, Steenbock, and Lepkovskyi4 have shown that vitamin D is not destroyed when cod-liver oil is mixed with ground grains and the mixture stored for 6 months. Effect of Substituting Butter Fat for Cod-Liver Oil
One of the most interesting facts brought out by the studies in this laboratory is the difference in results obtained with filtered butter fat and cod-liver oil. Reproduction is much superior on rations containing cod-liver oil (added daily) than on the same diets with filtered butter fat in place of cod-liver oil. Hart, Steenbock, Elvehjein, arid Waddellj say : I t is unfortunate that in several recent investigations with the rat the original fact observed by Abderhalden that inorganic iron added to a milk diet of some animals will not improve the ration in respect to its capacity to build hemoglobin has not been given the consideration that it deserves. In these investigations by hlattill and associates, Anderegg and Nelson, and Sure, no data have been presented in reference to the normality or abnormality of the blood, especially in respect t o its content of erythrocytes and hemoglobin.
It appears to the authors that such determinations might reasonably have been overlooked or deemed unimportant to a solution of the problem a t the time. Little if any consideration has been given to hemoglobin determinations in the dietary analysis of milk diets since the discovery of the vitamins. Evidently such determinations were not considered of major importance in the solution of nutrition problems, indicating that the work of dbderhalden did not impress investigators so strongly as it should. The positive results obtained on synthetic diets for growth and general well-being were partly responsible for this attitude. The work of Hart, Steenbock, and co-workers is to be highly commended since it opens up a new avenue for nutrition investigations, but there is ample justification from work already done for failure on the part of the present writers to deal with blood determinations. I n studies attempting to prove the existence of vitamins A, B, C, or I), no henioglobin determinations were made, and apparently long after their discovery it did not seem to be of immediate concern to run blood counts. The diets employed in proving the existence of vitamins A and B were largely or entirely of milk components, such as casein, butter fat, lactose, and salts in imitation of milk ash. And yet during all this time the work of Abderhalden was known. It appeared in 1900, twelve years before the discovery of vitamins; and yet, as far as the authors are aware, work on all the vitamins has progressed nicely without consideration of his data. I n practically all textbooks on nutrition during all these years the one outstanding example of a perfect food except for its iron content has been milk. Only in the past few years, as a result of the work in this laboratory and elsewhere, has it been shown that certain milk diets do not give the results that might be expected. The statement of Hart, Steenbock, and co-workers is possible-so it appears to the writersonly after the specific problem of iron metabolism has been undertaken for solution. The fact that normal growth could be obtained on synthetic diets containing casein as the sole source of protein (except for the small amount in the material carrying vitamin B) led investigators to believe that inorganic iron could be utilized by the animal body and that milk contained everything necessary for normal well14 J Brol Chem , 66, 571 (1925). 15
Ibrd
, 66, 67 (1925).
843
being except sufficient iron. It is not surprising, therefore, that work on reproduction did not' involve iron metabolism. The writers feel that it is as logical to assume that uric acid, purine bodies, etc., should have been determined since milk is deficient in purines. Hart, Steenbock, and eo-workers go on to say: In the work of the afore-mentioned investigators divers natural materials have been added to the diet with great improvement in the condition of the rat, especially in respect to its capacity to reproduce. The conclusion that they reached was that vitamin E was deficient in the milk and was introduced by the inclusion of various materials, such as wheat embryo and green lettuce leaves. T t is, however, true that in most of the recent investigations on the nutritive properties of milk an exclusive milk diet has not been used, but generally one in which a whole milk powder constituted 50 per cent or more of the ration, and of which the remaining portion consisted of lard, cornstarch, and a salt mixture.
The authors are of the opinion that the methods employed in studies concerning the existence of vitamin E do not differ in any essential way with respect to the complexity of the materials used from those employed in the investigations which led to discoveries of vitamins A, B, C, or D. The proof of the existence of these vitamins resulted from experiments containing materials just as complex as those employed for the solution of the vitamin E problem. The statement that' t'he present writers concluded that vitamin E is deficient in milk is not in accord with their published results. Anderegg and Nelson12say: Whole and skimmed milk powders have been tested by the biological method concerning their food value. I t is definitely shown that whole milk powder properly supplemented with iron salts and carbohydrate, or iron salts alone, furnishes everything necessary for growth, reproduction, and rearing of young. The relative amounts of protein, fat, and salts have a decided influence upon the nutritive value of the ration. The amount of iron salts added to whole milk powder has a pronounced effect on reproduction. Skimmed milk powder has an entirely different food value than whole milk powder, etc.
They placed two lots of animals on 99.8 per cent of whole milk powder plus 0.2 per cent of iron citrate. One lot did not reproduce but the other lot gave birth to 3 litters of a t least 15 young from 3 females. Another lot received whole milk powder 99 per cent and 1 per cent of iron citrate. Three females of this lot had 4 litters of 31 young. One lot of animals received whole milk powder 60, casein 6.0, salt mixture 2.4, iron citrate 0.2, agar-agar 4.0, and dextrin 27.4 per cent. Three females on this ration in 29 weeks had 11 litters consisting of a total of 94 young, 87 of which were weaned, or a mortality of 7.44 per cent. Analysis of this diet shows that milk constituents form the entire ration with the exception of starch, iron citrate, and agar-agar. Starch, in so far as we know, serves the same purposes as other carbohydrat'es in the diet, and agar-agar is conceded by investigators not to furnish any nutrients to the mammal. Furthermore, on a diet consisting of 85 per cent of whole milk powder, 0.5 per cent of iron cit'rate, 4.0 per cent of agar-agar, and 10.5 per cent of dextrin, 3 females after being on the diet 13 weeks had 3 litters of 30 young and all but 2 of the young were weaned. It is perhaps too much t,o expect' any single food to be perfect in all dietary respects. I t is also possible that a food may contain all the essential dietary factors and yet fail to yield optimum results, because these dietary coniponents are not present in optimum proportions. RobertsonI6 states: There is every reason for supposing that the pyrrole group cannot be synthesized by animals, but must be obtained by them preformed; that is to say, from the tissues of plants or from the tissues of animals, which acquired it from plants. This
-16
"Principles of Biochemistry," p. 47.
844
I N D USTRIA4LA N D ENGISEERING CHEMISTRY
pyrrole grouping is contained in small amounts in the majority of proteins, and i t forms a very important component of chlorophyl, the green coloring matter of plants which, as we shall see, is very closely related, chemically, to hemoglobin. I t is not improbable, therefore, that inorganic iron salts added to an exclusive milk diet are not utilized for building up hemoglobin simply for the reason that other component parts of the hemoglobin molecule as essential as iron itself are either lacking altogether in the milk diet or present therein in insufficient amount to subserve the needs of the blood-forming tissues and those of the other tissues as well.
The authors' work on milk powders and synthetic diets containing butter fat and cod-liver oil indicates that this statement must now be modified in the light of newer disroveries. Conclusion The following results of this investigation should be emphasized : I-Reproduction results obtained with cod-liver oil depend upon the manner in which the oil is administered.
Vol. 19, No. 7
2-Reproduction is much better on synthetic diets containing cod-liver oil than on synthetic diets with filtered butter fat. If the animals on the synthetic diet containing butter fat have a low hemoglobin content (and such may be the case) whereas those on cod-liver oil have a normal erythrocyte count and hemoglobin content, then there is present in cod-liver oil a specific vitamin which is required for normal iron metabolism. This conclusion is based on the supposition that the results of Hart, Steenbock, Elvehjem, and Waddell are correct-namely, that their animals suffering from anemia received a sufficient quantity of vitamins A, B, and C ; and that ultra-violet light did not remedy the condition. This problem is now under investigation in this laboratory and results d l be published when they are complete. Acknowledgment The writers desire to express their appreciation t o the Fleischmann Company for kindly supplying the yeast.
Estimation of Methanol in Alcohol and Alcoholic Beverages, Using the Immersion Refractometer' By John F. Williams
u. s. INTERNAL
REVENUE B U R E A UBRANCH , LABORlTORY, BUFFALO, N. Y .
This method is based upon the refractive indices of the alcohols, and the apparent total percentage of alcohol as ethyl alcoholfrom the speczJic graoity, using a 20 per cent solution. It is simple, rapid, accurate, and eliminates caZculations.
N
CMEROUS methods have been proposed and used
reading oi 41 35, and specific gravity 0 97513 (20°/4' C )
By the applica-
of these figures to the method given one will obtain a minus quantity for t'he estimation of methanol in alcohol, depending tion of 4 9 per cent of methanol generally upon chemical reactions. Among these Method are the Riche and Bardy method,2 which converts the alcohols into methyl and ethyl aniline dyes, comparing strips of wool The method given here depends upon finding the numeridyed by these products; the method of Thorpe and Holmesj3 cal difference between the immersion refractometer reading, which uses potassium bichromate and sulfuric acid t o oxidize R , and the exact percentage of total alcohol, P , calculated the methanol into water and carbon dioxide, and the esti- as ethyl alcohol from the specific gravity, using a 20 per cent mation of the latter; the method of Deniges-Simm~nds,~solution. Both the refractometer reading and the percentage which depends upon the colorimetric est'imation of the form- of total alcohol are taken on a 20 per cent alcoholic solution aldehyde oxidized from methanol; method based upon the of the original sample. That is, the original sample is estimation of formic acid oxidized from methanol; and diluted to 20 per cent total alcohol by volume. This is method depending upon combustion analysis. done by taking the specific gravity, as usual, on the distillate The method most universally used in this country is of the original sample, and assuming that all the alcohol that given by Leach and Lythgoe in 1905) based upon the present is ethyl alcohol, diluting a measured amount, repredifference in the indices of refraction between the two alcohols. senting 20 volumes of absolute alcohol, to 100 cc. On this The method is intended to be used with the Hehner Alcohol 20 per cent solution is then obtained the immersion refracTables 15.6"/15.6" C., but this fact was not' stated by the tometer reading, R, and also, from the specific gravity, the authors in their original article,fl and the method was un- exact percentage, P , of total alcohol by volume, calculated fortunately inserted, without revision, in the Methods of as ethyl alcohol. This last value may be slightly above Analysis, Association of Official Agricultural Chemists, 20 per cent, depending upon the amount of methanol present, 1919 and 1924, which uses the Bureau of Standards Alcohol as explained later. The percentage of methanol is then read Tables 2 O o , / 4 O C. This method will give an error of the off on a graph or table from the difference, R - P. A more methanol value of 4 to 5 per cent in some cases, mainly detailed description is given under Procedure. because of the difference in the two temperature standards. The method is designed to eliminate the inaccuracy, as well as the calculations necessary, in the method of Leach Noh-This fact may be shown abstractly b y assuming a pure ethyl alcohol solution, 15 per cent by weight, which will have a true refractometer and Lythgoe as printed in the h1ethods of Analysis, A. 0. A. C. The new method also takes into account the fact Printed b y permission of the Prohibi1 Received February 28, 1927. that when ethyl alcohol and methanol are diluted the specific tion Unit, U. S. Internal Revenue Bureau. 2 Compl. rend., 80, 1076 (1875). gravities do not run parallel. The difference between the 3 J . Chem. SOC.(London), 85, 1 (1904). specific gravities of the two are greater for dilute solutions 4 Simmonds, "Alcohol," p. 183 (1919). than for strong solutions. When a 20 per cent by volume 6 2. X a h r . Genussm., 24, 7 (1912). solution of pure methanol is calculated as ethyl alcohol, 6 J . A m . Chem. Soc., 27, 964 (1905).
