Biological Assay of Vitamin A Liver Storage Test of Guggenheim and Koch JOHN R. FOY AND KENNETH MORGAREIDGE S o p c o Chemical Company, Harrison, N. J .
A biological assay procedure for vitamin A is presented w-hich depends on the colorimetric estimation of the amount of this factor deposited in the livers of previously depleted weanling rats follow-ing administration of standard and sample doses. The method is recommended for its specificity, precision, and economy over the U. S.P. curative-growsth method, with which it shows substantial agreement. Application of the method to the assay pf a series of
T
HE need for an improved bioassay for vitamin d has not
vitamin A oils shows, in confirmation of previously reported w-orlr, that nonbiological assay methods, with the possible exception of the colorimetric estimation on the unsaponifiable fraction, overestimate the sample potency in direct proportion to the divergence of the ultra\ iolet absorption curl e from that of pure vitamin A. The nature of the feeding oil diluent influences the biological response, in relation to its composition and antioxidant content.
tocopherol to the diet as well as other protective factors advantageous in the economy of vitamin A in vivo. The depletion period was purposely made as short as possible, since it is desired to avoid any reduction of the vitamin h in the tissues or in circulation. Si.; days nere found sufficient t o reduce the liver vitamin to about the limiting level of the analytical method. The number and size of the doses necessary to give an optimum storage response in the shortest time were determined experimentally. The idea of a single dose was discarded when it was found to result in erratic values, whereas two doses on successive days resulted in sufficiently uniform data for the purpose of the test. Throughout the work, corn oil (Nazola) was used for the routine dilution of the standard and test samples. This is an important factor, since the diluent oil has been found to exert a marked influence on the absorption and storage of the vitamin. The relationship between the potency of the original sample and the percentage of diluent oil in the solution “as dosed” is demonstrated in Table 11. This txyo-dose procedure is limited t o samples having potencies greater than 3000 units per gram. While this limit may be lowered by altering the number or size of the doses, special consideration must be given to the assay of materials nhose potencies fall below the range of 10,000 to 20,000 units per gram, because of the disproportionate amounts of diluent oil in the two dosing dilutions. From the beginning of work with this method, it was realized that the confused state of affairs with regard t o the vitamin A standards would have to be side-stepped. Following Gridgeman’s suggestion ( 8 ) , a “spectroscopic unit” \vas adopted and defined as the quantity of vitamin A necessary to give a value for E;?&, of 0.0005 at 3280 A. Since this corresponds t o a con-
been eliminated by the recent advances in our knowledge of this factor. From a purely analytical point of view, the more precise nonbiological methods are still based upon and derive what accuracy they possess from conversion factors obtained from bioassay data. At the same time, it is obvious that a study of the physiological properties of vitamin A cannot he dependent on nonbiological methods of assay. The widespread use of these latter methods a t the present time is not due to a lack of appreciation of these facts, but rather to the lack of a bioassay method that is accurate and convenient in regard to cost, time, and labor. The curative-growth (U.S.P.) method now employed discourages extensive investigation of the problems involved in the physiology, utilization, and assay of vitamin A because it is laborious, costly, and inaccurate. Guggenheim and Koch ( 3 ) have recently proposed a method which offers a t least a partial solution to the bioassay problem. Their method depends on the well recognized fact that the amount of vitamin -4stored in the liver varies directly with the dose fed. ASSAY PROCEDURE
I n repeating this work and applying it to a routine bioassay procedure, the authors have established the following scheme, the details of which, naturally, will vary from laboratory to laboratory depending on local conditions. Weanling rats {IS to 22 days of age and 40 to 45 grams in weight) from a piebald strain reared in the authors’ laboratory are arranged into groups of 12 for each test level and given the usual U.S.P. vitamin A-free ration for a depletion period of 6 days. The animals are then dosed on 2 successive days with 0.1 ml. of oil containing from 150 to 300 units of vitamin B diluted from either the standard or a test sample. On the fourth day after the first dose the livers are removed and composited in groups of 4 (2 males and 2 females) for analysis by a modified Carr-Price procedure according to the method of Gallup and Hoefer ( 1 ) .
Table I. Typical Data from a Liver Storage Assay
I n practically all cases, multiple-level assays have been employed. -1typical protocol (Table I) illustrates the arrangement of data. It is seen that each assay group yields three analytical values from which separate calculations of the average number of units per liver are made. The potency of each test dilution is obtained graphically from the plot of the standard data. The known dilution factor then allows the potency of the original sample to be determined
Standard
Sample
Dose Level“ per R a t per Day 145 units
2 . 6 6 mg.
DISCUSSION OF METHOD 5 . 3 0 mg.
For the sake of clarity, certain points involved in the development of the method require mention, First, the U.S.P. vitamin A-free ration (6) as used by the authors contains corn oil (Mazola) as the fat component. This oil imparts appreciable quantities of
@
304
Liver Response, Units per Liver Group .iv. 74 78 75 76
Equivalent Vitamin , Potency, A , Units Units per Dose per G r a m
...
...
57 60 68
62
129
48,400
189 183 200
191
283
53,300 Ax., 50,900
Each dosage level made u p of 3 groups of 4 rats each.
305
V O L U M E 20, NO. 4, A P R I L 1 9 4 8 version factor of 2000, the unit has the same spectrophotometric value as that which has been widely used by the industry. -kt the same time, the authors employed crystalline vitamin -4 acetate (Distillation Products, Inc.) as the reference standard for the bioassay. This combination of unit and standard permit-., the assay results to be calculated in terms of either the common (U.S.P.) unit or a definite weight of a pure, active vitamin .1 derivative.
‘Iul~le11.
Percentage of Corn Oil in Feeding Dilutions with Samples of Decreasing Potency Percentage of Corn Oil 150-unit level 300-unit lei-el
Hainpie I’otency. r n i t s per G r a m 1,000,000 62,200 32,600 16,300 8,150 5,400 4,100 3,260
55.85 57.5 55 90 80 70 60 50
YH . 7 93 90 80 60 40 20
0
Table 111. Range in Liver Response to Reference Standard
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