INDUSTRIAL A N D ENGINEERING CHEMISTRY
January, 1924
As long ago as 1910, Biedl, in his work “Innere Secretion,” makes the following comment on the theory of pancreatic diabetes: All that can be said a t present t o be founded on reliable evidence is that normally an internal secretion is formed by the pancreas that plays a deciding role in the metabolism of the carbohydrates and whose absence is the sole cause of the deviations forming the diabetes. The Eact that only the fewestobservers (capare1li, zuelzer, Dohrn and Mayer) saw any definite improvement following the ingestion of fresh or dried pancreas, while most of the leading men give statements to the contrary, is no proof against this assumption. It proves merely that the generally accepted methods of organo-therapy are not applicable in the treatment of diabetes. All the experience given by transplantation of the organ as well as by the establishment of “parabiosis” (connecting the blood circulation of two animals) gives sufficient support to the said theory.
I n striking confirmation Of Biedl’s critical comment, we see that the very first experiments “asking the question Of Nature”-will a heterologous pancreas hormone extract, by Parenteral administration, give antidiabetic action in experimental and in human diabetes?-were answered in the affirmative.
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I n that development of the plan which rested upon the essential feature of highly specialized clinical trial, individual and institutional, the difficulties and the hazards which beset the problem of establishing the therapeutic properties of insulin injection and usage become more and more evident, and with recognition of the wisdom of this plan. Whatever may be the method of manufacture finally shall be perfected, and regardless of the matter of inventions in chemical methods, the product itself should be of a uniform standard everywhere, and as it has been worked out in consequence of the program of the Toronto University. The method of preparation, standardization, is one which is widely available, and of a more practicable nature than a t first appeared. This also is likely to be a matter of progress. The \videst participation of the manufacturing chemist is obviously desirable in order that the sources of supply shall be utilized to the best economical and technical advantage and with that highest possible attainment which is inevitably to be anticipated under such circumstances; and, lastly, that the preparation shall be correspondingly available in medicine.
Observations on t h e Baudouins Test‘ By C. 0. Gravenhorst A A ~ H UOLIEFABRIK, S AARHUS.DENMARK
R O M time to time the addition of sesame oil to margarine has been required by law for the purpose of preventing its fraudulent sale. This requirement is found in the laws of several nations, Belgium requiring the addition of 5 per cent sesame oil, and Austria, Denmark, Finland, Germany, Portugal, Sweden, and Switzerland 10 per cent. While a number of color reactions are found to be applicable to the detection of sesame oil, the Baudouins reaction is the one most generally used and regularly stipulated by law. The laws of some nations require only that a definitely red acid layer be produced in this test, while others--for instance, Denmark and Sweden-require that the color shall be up to a certain definite standard, and have issued tables showing the required color. Since the color given by genuine sesame oil is variable, compliance with such a requirement is secured by using an amouni, of oil sufficient to reach the standard shown by the color table. It is often important to determine the exact amouni, of oil required to give the standard reaction. I n testing oils for this purpose, it is necessary to carry out the analytical procedure in conformity with legal requirements and to take care that all chemicals used are standardized. The test is best carried out in accordance with the procedure described by Villavecchia and Fabris, mixing 0.5 cc. of the melted and filtered margarine fat with 9.5 cc. peanut or cottonseed oil and adding 0.1 cc. (4drops) of a 2 per cent solution of furfural in alcohol. This is next mixed with 10 cc. hydrochloric acid (specific gravity 1.19) in an 18-mm. test tube and shaken vigorously. After standing 15 minutes the color of the acid Iayer is compared with the color table. The color shown by the Swedish table corresponds to 0.75 red and 0.30 yellow by the Lovibond tintometer. The color c,hown by the Danish table corresponds to 1.0 red and. 0.20 yellow, and this color will be taken as standard, as it is identical with that obtained in the reaction. As has been previously pointed out (Soltsien, Kerp), a violet color may be obtained from the reaction of hydro-
F
1
Received September 13, 1923.
chloric acid and furfural, but this color does not appear with the strength of furfural solution specified. A slight color of the acid layer may be sometimes shown with peanut or cottonseed oil. Care must be taken to avoid an excess of furfural, not more than six or seven drops being used in any case. It has been stated that the hydrochloric acid must be free from chlorine, but the writer has found that a comparatively large amount does not affect the reaction. An amount of free chlorine much larger than is usually present in hydrochloric acid is required to affect the reaction, as shown by the following tests: No.
Free Chlorine Per cent
I@’,
i ::E34]
Hydrochloric acid with
i
5 6
2.89 5.52
COLOR Standard weaker than (1)
358
It is worth noting in this connection that peanut oil will absorb the free chlorine when shaken with hydrochloric acid containing it, thereby rendering the acid satisfactory. The exact strength of the hydrochloric acid is of much more importance. The writer obtained the following results: HYDROCHLORIC ACID ’
No. 1 2 3 4 5 6 7
Specific Gravitv
Per cent HC1
COLOR %$ltronger Standard than (1)
1,190
37.35 38.52
1.187 1.176 1.166
36.56 34.63 32.75
60%
1.137
30.78 27.03
75% 9370
1.197
1 . I56
weaker
than
(1)
All measurements were made by adding sufficient amount of sesame oil to give the standard color. The statement that No. 2 showed a 25 per cent strength reaction means that four-fifths of the amount of sesame oil used in KO. 1 was sufficient to produce the standard color. Several authors (Uta. Soltsien. Lauffs and Huismann. Kreis) have stated that the reaction is influenced by rancidity:
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
These statements have been confirmed. A mixture of sesame and peanut oil containing 0.25 per cent sesame oil giving the standard reaction shown by the Danish table was exposed in thin layer in a Petri dish. After 2 days it was observed that the reaction had become weakened, and after 10 days no reaction a t all was obtained. A similar result was obtained with a mixture of butter and sesame oil. The reaction was not weakened, however, when pure sesame oil was exposed to light and air for 4 weeks. Despite the fact that the oil became badly rancid, a mixture of it with fresh peanut oil gave a reaction equal to that given by the original oil. When peanut oil was exposed to air and light in a thin layer, a distinct rancidity was developed in 2 days. After 4 days' exposure, the reaction obtained when mixed with sesame oil was observed to be weaker. After 6 days the weakening of the reaction mounted to approximately 25 per cent, after 12 days to approximately 50 per cent, after 16 days to approximately 75 per cent, and after 20 days no reaction a t all was obtained with an amount of sesame oil sufficient to give the standard color when mixed with fresh peanut oil. A similar experiment was made with cottonseed oil and with soy bean oil, and identical results were obtained. Since rancid sesame oil when diluted with sweet peanut oil gives a reaction of normal strength, these results show that the reactive substances in sesame oil are not destroyed by the development of rancidity, but that the reaction is interfered with by substances contained in the rancid oil. It has been found possible to remove interfering substances by treating the rancid oil with lye. Butter fat to which sesame oil had been added, but which had become rancid so that it no longer reacted to the Baudouins test, was agitated with a 5 per cent solution of sodium hydroxide at 35" C. The temperature was raised to 70" C. with continuous agitation, the oil separated from the soap and alkali, dried by agitating with a small amount of dry sodium sulfate, and filtered. After tliis treatment the fat gave a Baudouins reaction showing the same intensity of color as had been obtained before rancidity developed. The same experiment was made with peanut oil to which sesame oil had been added, with identical results. Rancid peanut, cottonseed, and soy bean oils were all treated in the same way, and after treatment were found to show a satisfactory reaction when mixed with sesame oil. Commercial sesame oils are often found to give a reaction of less than normal intensity. This may be due to the sesame oil being impure or adulterated, but may also be due to the use of rancid oil for diluting it. To overcome the latter objection, tests of a number of possible diluting agents were made. Ether, chloroform, benzene, toluene, xylene, petroleum ether, and yellow paraffin oil were tried and found unsatisfactory. White paraffin oil (liquid petrolatum U. S. P,) was found satisfactory, the reaction obtained being approximately four times as strong as when peanut oil was used for diluting. This agent is not affected by exposure to light and air, and is therefore a n excellent medium for dilution. The amount of crude sesame oil required to give the standard color varies with the origin of the seed. While there are seeds that yield oil which when mixed with two hundred parts of peanut or cottonseed oil will show a reaction equivalent to that required by the standard color table, there are other seeds that yield oil giving a stronger reaction. Seed from India and the Levant usually yield an oil giving the standard reaction, Many Indian, African, American, and Chinese seeds yield oil giving a reaction more than twice as strong as the standard. The strongest reaction observed was 3.8 times the standard color and was found in the oil from a sample of white Chinese seed.
VoI. 16, No. 1
These figures refer to the crude oil. Refining with fuller's earth causes a loss of reactive power so that refined oil giving a reaction more than two times the standard is seldom found. Some fuller's earths are more active than others in removing the reactive substances, so that in case the reaction of the finished oil is important it is advisable to select the earth with caution. The effect of fuller's earth is not always evident until after deodorizing. As an example, a certain Frankonit earth reduced the intensity of the reaction 20 per cent, but subsequent deodorizing further reduced the reaction, so that the reaction of the finished oil was 50 per cent less than that of the original. Another Frankonit earth gave a scarcely perceptible reduction. Floridin earth seldom causes a reduction of more than 15 per cent, and this suffers no further reduction when the oil is deodorized. If the sesame oil used in the margarine or in a fat mixture is unknown, it is not possible, therefore, to do more than determine the minimum content of sesame oil. This can be done by calculating that an 18-mm. acid layer and Lovibond glasses 1.0 red and 0.20 yellow correspond to each other when 0.25 per cent of sesame oil is present when peanut or cottonseed oil is used for dilution. If liquid petrolatum is used for dilution, a mixture containing 0.0625 per cent sesame oil will give the standard color. If, for example, it is found that a margarine fat gives the standard color when 0.2 cc. is made up to 10 cc. with peanut oil, the amount of sesame oil is not less than 12.5 per cent; and if the same reaction is obtained when 0.2 cc. is made up to 10 cc. with liquid petrolatum, the amount of sesame oil present is not less than 3.125 per cent. These figures are, of course, only approximate. 1
COLORIMETER FOR USE WITHBAUDOWINS TEST
The colorimeter described in the accompanying sketch has been found very satisfactory for comparing the reaction of oils with the standard color tables. It consists of a small box 45 x 80 x 250 mm. The color table, which is printed on a white sheet of paper, is fixed in a slide placed a t the back wall of the colorimeter. The remainder of the inside surface is lined with white porcelain plates. A round hole 25 mm. in diameter is cut through the front so that a part of the color table is seen through it as a half circle. The test tube is inserted through a hole in the top of the colorimeter so that the acid layer is placed exactly over the other half of the circle seen through the hole in the cover. A tube 200 mm. long with an inside diameter of 20 mm. is placed in the opening in the cover. I n using the colorimeter the operator sits with his back to the light so that the color in the tube is seen by the light reflected from the white porcelain background. A good comparison of the colored acid layer in the test tube with the standard table can be obtained by this method.
