Sidney A. Thayer

A New Tri-atomic Alcohol from the Urine of Pregnant Women.

A new alcohol differing from the pregnandiol of Marrian 1 and Butenandt 2 and also from the crystalline ovarian hormone isolated by Doisy, 3 Thayer and Veler, Butenandt, 4 Laquer 5 and Marrian 6 has been obtained from the urine of pregnant women. This alcohol has been isolated in the form of snow white crystals. The melting point by the open beaker method of 5 different preparations was 273°, 273°, 273°, 272.3°, 272° (uncorrected). The crystals melted sharply without decomposition. The molecular weight determination by Rasts micro procedure gave an average value of 294. The iodine numbers of 3 different preparations were 85.3, 86.2, and 88.5. The average of these values, 86.7, permits one to calculate a molecular weight of 292.8 if one double bond is assumed. Determination of the number of hydroxyls in 2 samples by the procedure of Peterson and West 7 indicates that 3 atoms of oxygen exist in this form. Found 124, 129; theory 129 gms. of CH3C = 0 per mole. The average molecular weight of the triacetyl derivative is 410. M. P. 126° uncorrected. The specific rotation of a 0.322% solution in 95% ethyl alcohol at 28° in a 2 dm. tube with a sodium flame was +68.3°. Another sample (0.148%) gave a value of +72.8°. Qualitative analysis did not detect sulfur, halogens or nitrogen. Quantitative micro analysis: carbon 75.15%; hydrogen 8.22%. Calculated for C18H24O3, carbon 74.95%; hydrogen 8.39%; mol. wt. 288.

Inactivation of vitamin K by light.

In an earlier note, 1 we reported the isolation of crystalline material from alfalfa which after 4 recrystallizations still had the activity ascribed to Vitamin K. Since our observations were still incomplete it was impossible to state with certainty that we had isolated Vitamin K. Some time after this work, it was found that although we could obtain crystals with approximately the same melting point, solutions of these crystals did not restore the coagulation time to nomal values. Frankly, we admit that we have been unable to duplicate the work reported last summer and, moreover, thus far do not have any explanation which appears to be satisfactory. In seeking an explanation of our unexpected results, we recalled that Almquist 2 , 3 had reported briefly on the loss of activity of preparations exposed to sunlight and on the stability of the vitamin exposed to artificial illumination. The effect of sunlight has been confirmed but in addition we have found that highly purified preparations dissolved in various solvents rapidly lose activity on exposure to the illumination from ordinary daylight bulbs. On the other hand, our crude extracts of alfalfa have been found to be quite stable, no special precautions regarding light being necessary. However, as the potency is increased to 500 or 1000 units (Thayer, et al. 4 ) per milligram, the lability is such that decomposition has frequently occurred in spite of our precautions. Experimental. Two preparations were used in our experiments on the effect of light: (a) a non-crystalline product obtained from alfalfa having a potency of approximately 1000 units per milligram and (b) a crystalline product (MP. 50.5–52.0°) obtained from putrefied fish meal. The latter had been recrystallized 10 times from various solvents without detectable alteration of its potency, which was approximately 600 units per milligram.

The Crystalline Ovarian Follicular Hormone

Due to the low concentration of estrus-producing material in ovaries, we abandoned their use several years ago in favor of the much cheaper and more concentrated source, namely, the urine of pregnant women and mares. However, with the isolation of so many different estrogenic compounds, it seemed desirable to determine the nature of the active substance in the ovary. Starting last year seriously to work on this problem we soon obtained the hormone in a crystalline condition but owing to the very low concentration in hog ovaries, have not secured enough to complete our work. Preliminary assays of the crystalline follicular hormone give the following data: (1) ovariectomized mice by the Marrian-Parkes procedure, 200,000 units and by the Butenandt procedure, 70,000 units per mg.; (2) ovariectomized rats by our usual procedure, 16,000 units per mg.; (3) immature rats by the Curtis-Doisy procedure, 5,000 units per mg. In our laboratory these results are from 4 to 8 times the values that we obtain for theelin, but with the immature rat the potency is equal to that of theelol. The assays for dihydro-theelin and for the follicular hormone by the respective methods give similar values. Although we have not yet accumulated sufficient material for complete analysis, our results indicate the identity of the hormone with dihydro-theelin. The m-bromobenzoate of the hormone was prepared, and after 3 crystallizations had a melting point of 154°–155°. After 4 crystallizations the m-bromobenzoate prepared from a sample of pure dihydro-theelin melted at 155°–156°, and the dihydro-theelin obtained from it by saponification with dilute alcoholic alkali melted at 171°–172° after one crystallization. By saponification of the m-bromobenzoate of the hormone in the same manner the crystalline hormone was recovered and found to melt at 170°–171°. All melting points are uncorrected but were taken with a Bureau of Standards long stem thermometer.

Assay of vitamin K concentrates.

Summary A curative method of assay of Vitamin K based on Trevans principles of bioassay has been found to give satisfactory results.

The Assay of Vitamins K1 and K2

Summary 1. The potency of Vitamin K1 is approximately 1000 of our units per milligram; K2 approximately 660. 2. The 18-hour assay procedure gives satisfactory results. 3. In a slightly modified Almquist 7-day curative method, 80 micrograms per kilo of diet of K1 and 160 micrograms per kilo of diet of K2 are adequate.

Recovery from the anemia caused by a diet deficient in vitamin K.

The purpose of this investigation was to ascertain whether the restoration to normal of the clotting time of chicks on a diet deficient in Vitamin K was accompanied by a cure of the existing anemia. This anemia has been observed by Dam, 1 and Hoist and Halbrook, 2 but thus far no extensive investigations on the regeneration of hemoglobin have been made. The changes toward normal produced by the administration of minute quantities of a Vitamin K concentrate were so rapid that it seemed desirable to report our results. The chicks used in our experiments were one-day-old white Leghorns hatched from eggs laid by hens which had been fed a diet relatively free from Vitamin K. They were raised in battery brooders and had free access to food and water. The basal diet used in these experiments was that of Almquist 3 with only minor modifications. Our active extract was prepared from alfalfa meal by an adaptation of the methods of Dam and Sch⊘Hnheyder 4 and of Almquist. 3 Blood was drawn from the brachial vein, samples being taken for the determination of clotting time, hemoglobin, and the erythrocyte count. The clotting time (Sch⊘nheyder 5 ) was determined by allowing the blood to flow directly into a small porcelain dish. Zero time was taken as the time when the first drop fell into the dish. When the blood no longer flowed upon tilting the dish it was considered to be clotted. The blood was observed for 300 minutes, if it failed to clot in a shorter time. The hemoglobin was determined by the method of Palmer 6 in which the hemoglobin is converted to carbon monoxide hemoglobin and compared with a standard solution of carbon monoxide hemoglobin in a colorimeter.

The Crystals of the Follicular Ovarian Hormone

The first preparation of crystalline ovarian hormone 1 was obtained from a combination of 2 acidic aqueous solutions containing approximately 7500 rat units of a potency exceeding 1000 units per mgm. The aqueous solution (volume 480 cc.) was extracted with six 150 cc. portions of ethyl ether, the ether distilled and the flask evacuated to remove the last of the solvents. The residue was leached with small volumes of anhydrous ethyl ether, this solution centrifuged, poured into a flask and distilled to dryness. Owing to the danger of ether peroxides 1 cc. of ethyl alcohol was added and distilled using a vacuum to complete the removal of the alcohol. The flask was put in the refrigerator (-10° C.) and crystals began to appear within a short time. The weight of the crystals, which possibly were not absolutely pure, was 2.07 mg. Upon an additional purification the weight diminished to 1.39 mg. Beginning with this initial crystallization, we have been able to convert all of our preparations into a crystalline form. Many individual preparations have been converted into the pure crystals. Several other products of a different crystalline form isolated during the course of preparation have proved to be inactive upon assay. One interesting observation regarding the crystalline structure has been made. The pure hormone crystallizes in at least 2 distinct forms of crystals, a phenomenon which is not exceptional but which is encountered quite frequently. For want of a more adequate description, we refer to the one form as clusters of needles (A) and to the other as rhombohedral plates (B). In one of our earlier preparations, A separated from a light yellow oil. Upon washing out the oil with cold ethyl alcohol, we obtained the A form apparently in a pure condition.

Potencies of Vitamin Ki and of 2-Methyl-1, 4-Naphthoquinone:

In a previous investigation 1 we have confirmed the report of the marked antihemorrhagic activity of 2-methyl-1,4-naphthoquinone. 2 A careful comparison of the potencies of this compound and of pure vitamin K1 by our 18-hour procedure 3 showed that the latter is approximately one-half as active as the former. In view of this observation and the report that by the 6-hour procedure vitamin Ki is only 1/30 as potent as 2-methyl-1,4-naphthoquinone, 4 it seems desirable to publish the results that we have obtained in a comparison of the potencies of the two compounds by the 6-hour observation period. Experimental. In our experiments, we have used for the evaluation of the response of the chicks: (1) the percentage of chicks showing a clotting time of less than 10 minutes; 3 (2) the mean clotting time; (3) the mean prothrombin time. 5 Following Ansbachers suggestion, a solution of the compound in cod liver oil was administered and the blood drawn 6 hours later for the evaluation of the reaction. Each assay included the response of the same lot of deficient chicks to the administration of one or 2 dosages of each compound and the mean clotting time of a control group. The data are summarized in the table. In Experiments 1, 2, 3 and 4 the volume of cod liver oil used for administration of the compounds was 0.10 cc; in Experiments 5 and 6 only 0.05 cc was used. From other reports and the data of this paper it appears likely that in experiments in which the response is restricted to a period of 6 hours or less the volume of oil used may play a role in the absorption of vitamin Ki and therefore in

Some Physiological Properties of a New Tri-Atomic Alcohol from the Urine of Pregnant Women.

A study of the responses of female mice, rats, guinea pigs and rabbits to the enteral and subcutaneous administration of the new triol 1 has given interesting data. Using rats 17-23 days of age it was found that minute quantities administered either subcutaneously or by stomach tube caused opening of the vagina in from 2 to 10 days. Vaginal smears made twice daily showed that cornification generally began on the third day following; the cornified cells persisted from 2 to 5 days. Animals sacrificed at the time leucocytes began to appear showed either large follicles or corpora lutea and sometimes both. That the new triol acts in the absence of the ovary is proved by the administration to ovariectomized rats 21-22 days of age. Opening of the vagina occurred in 2 to 7 days with the subsequent appearance of cornified cells in the smears. Although our experiments are still incomplete our evidence indicates that if the amount required to produce opening of the vagina of 21 day old animals be regarded as a unit, the rat unit weighs 0.10γ or less and the mouse unit 0.004γ (1γ = 0.001 mg.). Dr. W. D. Collier is making microscopic studies of our injected animals.

Bioassay of Water-Soluble Antihemorrhagic Compounds by Intravenous Administration.

Summary Using intravenous administration it has been found that on a molecular basis the potencies of all the compounds with the exception of the disulfate are approximately equal to that of the standard, Z-methyl-l,4-naphthoquinone. The disul fate is about onethird as potent on a niolecular basis, but owing to the much larger molecular weight its activity per milligram is somewhat less than one-sixth that of 2-methyl-1, 4-naphthoquinone.