Distribution of Ribofiavin
Riboflavin is one of the most widely distributed B vitamins, but there are very few common sources that contain high concentrations All cells of both plants and animals presumably contain the vitamin. Plant seeds synthesize the vitamin during germination. A few bacteria and the yeasts are rich sources. Muscle tissue is low in riboflavin, but certain internal organs, such as the liver and kidney, may contain appreciable amounts. . Note the high concentration in live, wheat germ, and yeast, and the very low content in celery, polished rice, and potatoes. It should be kept in mind that even though potatoes are low in the vitamin, the continued intake at one or even two meals a day by, the majority of many races makes the potato an important contributor to the total dietary intake It was pointed out previously that the thiamine oflive yeast is not available to the body. A similar situation exists with riboflavin.
Parsons and co-workers showed that the riboflavin in live bakers' yeast is only slightly available to humans but that the vitamin is completely available from samples of the same yeast after has been specially dried (dead cells). Other workers have reported similar findings. The methods used involve dearmnining the urinary and fecal riboflavin excretions after the oral administration of a known amount of pure riboflavin and comparing these with excretions after known amounts of the vitamin in yeast before and after special drying and heating treatments. Other foods do not yeld their total riboflavin content to the body. For instance, it was reported that in normal women the availability of riboflavin in ice cream was 90 per cent, whereas in green peas and almonds only 41 and 39 per cent, respectively, were available. The vitamin exists within many plant and animal cells, primarily in combination as one of :he two coenzyme forms. The retina and urine and milk contain principally the free vitamin. The nucieotides may in part be combined with specific proteins to form enzymes. In serum of 13 normal adults the sum of the free riboflavin and riboflavin phosphate was found to average 0.8 l!g per cent, and the riboflavin dinucleotide, 2.41 g per cent. The white cells plus platelets contained 252 fig per cent of total riboflavin (both derivatives plus the free vitamin) and the red cells only 22. In a later'study Bessey and others found somewhat similar values in plasma and cells, although the levels were lower in individuals on restricted vitamin intake.
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.
