D. W. MacCorquodale

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.