Vitamin assay using rats

Biological, spectroscopic, and chromatographic methods have been used to assay vitamin A and the carotenoids. Biological methods have traditionally been based on the growth response of vitamin A—deficient rats. The utiUty and shortcomings of this test have been reviewed (52,53). This test has found apphcabiUty for analogues of retinol (54,55). Carotenoids that function as provitamin A precursors can also be assayed by this test (56). 

The methods now used to measure 25-OH-D are competitive protein-ligand binding assays that use either serum globulin (diluted rat serum) (27)28) or a vitamin D-deficient rat kidney... 

It is well recognized that in biological assays of vitamin A with rats, high variability in response is continually encountered. For this reason as many as 10 to 15 carefully selected animals, generally males, are used for each testing level, and the responses averaged. Certain stocks of animals then yield concordant results, whereas other stocks cannot be used. Through the courtesy of H. J. Deuel, Jr., the writer has been furnished the raw data on which several satisfactory vitamin A assays were based.34 When the level of intake was such that all the... 

The classical method for the deterrnination of vitamin K is based on the clotting time of a vitamin K-deficient chick. It is relatively easy to produce a hemorraghic state in chicks (17). Vitamin K-deficient rats have also been used for this assay (18). Owing to the development of modem chromatographic techniques, this method of analysis has been supplanted by other methodology. 

SA Tanumihardjo, HC Furr, JW Erdman Jr, JA Olson. Use of the modified relative dose response (MRDR) assay in rats and its application to humans for the measurement of vitamin A status. Eur J Clin Nutr 44 219-224, 1990. 

Food and feed samples, which contain vitamin D in very low concentrations, are not usually assayed by chemical methods. For these substances, bioassays are the only means available for the eissessment of vitamin D activity. Rats and chicks are used fis test animals rats respond equally well to 2 and D3, whereeis chicks respond only to D3. The assays measure the alleviation (curative test) or the development (prophylactic test) of vitamin D deficiency in terms of the degree of rickets produced. 

Vitamin D can be assayed using either rachitic rats or chicks. Vitamin Dg and vitamin D3 are equipotent on rats but vitamin Dg has little antirachitic effect in chicks. It could be assumed therefore that vitamin D preparations intended for chick use should be assayed on chicks but if the source of the vitamin is known the rat result is of course quite acceptable. 

Because the serum/plasma concentration of the active form of vitamin D3, l,25-(OH)2-D3, is extremely low (30-70 pg/ml), it has been conventionally measured by a radioreceptor assay technique using the vitamin D receptor in the chnical field. Although a method has been reported for the LC-MS assay of l,25-(OH)2-its applicabihty was proved only for the rat serum assay this method is of less practical use in the clinical field. Even if an up-to-date mass spectrometer model is employed, it may be difficult to measure the serum/plasma l,25-(OH)2-D3 levels with a clinically available sample volume (<1 ml). 

Cell fractionation studies of homogenates of the gastric mucosa have demonstrated that the intrinsic factor is found both in mitochondria and the supernatant fluid. The factor remains difficult to assay a variety of methods have been used, but none is entirely satisfactory. These methods include (1) administration of intrinsic factor preparation to gastrectomized animals (rats or hogs) (2) measurement of vitamin 6 2 binding capacity of the preparation (3) measurement of vitamin Bi2 uptake in tissue and (4) evaluation of the inhibition of cobamide coenzyme activity in the glutamate isomerase reaction. 

MEASUREMENT/ASSAY. The assay of vitamin A is accomplished by two basic methods biological, or chemical. The bioassay procedure is based on a biological response such as growth of rats or chicks deficient in vitamin A. It measures the total vitamin A, including provitamin A, present. But, because of the difficulties and time factor in bioassays, chemical assays are usually used. Until recently, dietary allowances of vitamin A were stated in terms of either International Units (lU) or United States Pharmacopeia (USP) units, which are equal. An International Unit (lU) of vitamin A is defined on the basis of rat studies as equal to 0.344 meg of crystalline retinylacetate (which is equivalent to 0.300 meg of retinol, or to 0.60 meg of beta-carotene). These standards were based on experiments that showed that in rats only about 50% of the beta-carotene is converted to vitamin A. In man, however, beta-carotene is not as available as in the rat, due to poorer absorption in the intestines and other factors, with the result that various factors have been used to compensate for this when vitamin A activity of foods and diets have been expressed in lU. 

The vitamin B-6 content in foods and tissues is determined by microbiological assay, chemical methods, and animal bioassays. The animal bioassays, using either the rat or chick, are time consuming, expensive, and variable therefore, they have been generally replaced by microbiological and chemical methods. 

High potency preparations of vitamin B-12 are usually assayed by spectrophotometry. Also, vitamin B-12 may be assayed colorimetrically or fluorometrically. Some assays involve measurement of cobalt. However, food sources are usually assayed for vitamin B-12 by either (1) the microbiological method, or (2) the biological method, using chicks or rats. 

According to the US Pharmacopoeia (USP), IIU of vitamin E equals Img all rac a-tocopheryl acetate, 0.67mg RRR-a-tocopherol, or 0.74 mg RRR-a-toco-pheryl acetate. These conversions were estimated on the relative biologic activities of the various forms when tested in the rat assay for vitamin E deficiency, the fetal resorption assay. These USP lUs are currently used in labeling vitamin E supplements and food for-tificants. It should be noted that the current RDA does not use vitamin E USP units but rather the recommendation for adults is set at 15 mg of RRR-a-tocopherol or IR-a-tocopherols. Most foods contain RRR-a-tocopherol naturally, but foods that have been fortified with vitamin E contain the synthetic form, e.g., fortified breakfast cereals. If the amount of vitamin E on the label is given in international units, then this must be multiplied by the factors given in Table 2 to obtain the amount of 2R-/3-tocopherol. 

Although there is little doubt that nicotinic acid synthesis takes place in the rat, sheep, cow, and other animals, the simple measurement of nicotinic acid excretion does not seem to be an adequate index. From ail available evidence the only index of nicotinic acid deficiency appears to be the urinary F2 fraction of Najjar and Wood (82-86). This was confirmed by workers using the Fj assay (87, 88) and the so-called trigonelline fraction (89, 90) which is mainly, if not all, F2 (91, 92). The F2 fraction, a derivative of V -methylnicotinamide (80, 91, 93-95) was found to be excreted in substantial quantities in rats and pigs while on a nicotinic-acid-deficient diet, a fact which indicates synthesis of the vitamin. 

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