Determination of Riboflavin
Rats and chicks have served as test animals in the biological methods. In the rat growth method, young animals are fed a special riboflavtin-deficient diet for two or three weeks, after which time growth ceases. Then groups ofthe depleted rats arc given supplements of known amounts of the vitamin, and other groups are supplemented with graded quantities of the test material. Comparisons of growth response over four weeks or more allow the calculation of approximate vitamin content of the substance under examination. Such methods have largely been replaced by shorter and less expensive procedures. The original microbiological method of Strung and Snell using L. casei has been modified many times and is the basis of the official USP method. This organism requires riboflavin for growth and lactic acid production.
The culture medium can be prepared so that riboflavin is the limiting factor. With such a culture medium the organism responds to the addition of graded doses of the vitamin by producing corresponding increments of acid. In the test the acid production in response to supplements of known amounts of pure vitamin is compared to the acid production produced by adding graded amounts of extracts of tissues or other substance under examination. From such data an estimate of the riboflavin content of the unknown can be made. Marked variations in technique are required for the preparation of different types of materials for assay. This is especially true in the fluorometric methods, since substances other than riboflavin present in plant and animal tissues are known to fluoresce under the conditions of the test.
The intensity of fluorescence of a solution of riboflavin (under specific conditions) is proportional to the concentration of the vitamin. Light of wavelengths in the region 400 to 500 mm induce the fluorescence, and the intensity of this fluorescence can be measured by one of the various fluorophotometers on the market. In practice riboflavin is determined in an extract of the test substance in terms of the intensity of fluorescence before and after destruction of the vitamin. This is compared with the fluorescence of a known sample of riboflavin or with a solution of a substance such as fluorescein that has been standardized against the vitamin. Yagi described separation procedures for the various forms of riboflavin employing paper chromatography and paper electrophoresis, and a simplified technique for their determination. Murphy and others described procedures for liberating riboflavin from natural sources. An enzymatic method for FAD was developed by DeLuca and co-workers.
Riboflavin Requirement
There is no USP unit or IU for riboflavin. It is becoming common, and logically so, to refer to quantities of all vitamins of known structure on a weight basis. As is the case with most, of the vitamins, the human requirement of riboflavin is not known with any certainty. There are too many complicating factors, and the experimental clinical approach is impractical. Various estimates have been made for average requirements of a large group of/people, but these figures cannot be translated to represent the required intake of anyone in-dividual. Horwitt and co-workers set the need of an adult on 2200 Cal at between 1.1 and 1.6 mg per day. A riboflavin-depleted individual was able to assimilate at least 6 mg of the vitamin at one time.
The Food and Nutrition Board, National Research Council, 1963 revision, gives as the recommended allowance from 1.0 to 2.0 mg of riboflavin daily depending upon body weight. During pregnancy and lactation, the values given are higher. Bro-Rasmussen has published a comprehensive treatment of available work on riboflavin requirements of man and animals. In animals more exact data concerning requirements are known. The practical value in agriculture of such information is at once obvious. The establishment of required intakes for optimum growth rate, etc., in various species has been of great value in animal industry.
There are reviews on various aspects of ribofiavin.
