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DIFFERENCE BETWEEN CHEMICAL ANALYSIS & BIOLOGICAL AVAILABILITY 539Vitamin AVitamin A is the generic descriptor for a variety of chemical substanceswhich possess a specified biological activity, including retinol and itsderivatives, a-, b - g- carotene, cryptoxanthin and some other minor carotenoids.Enormous effort has been devoted in a very few experiments in attempting todetermine human requirements for vitamin A. Fortunately, the average results ofthe few investigations show good agreement with extrapolations made from animalexperiments. But the work indicates that it would not be possible to use humansubjects to assay the vitamin content of a food. The best we can do is to use ananimal bioassay in which the biological potency of the food is compared with astandard amount of the vitamin and to assume, in the instance of vitamin A, withsome confidence, that the same comparison is valid in man.In the Sheffield experiment (Hume and Krebs, 1949) determination ofrequirements involved measuring the amount of vitamin A that was available in thefoodstuffs provided but later, with more sophisticated methods, the turnover oflabelled retinol was used and this does not provide information on availability.The advantages of physical/chemical methods listed above are usually considered tooutweigh the drawbacks when compared with the problems involved in bioassay, butthere are unrecognised problems exemplified by vitamin A. Until 1971, it wasconsidered that canning of fruits and vegetables had no effect on vitamin Apotency because colorimetric estimation of the extracted carotenoids showed nochange on processing. Sweeney and Marsh (1971), however, showed that heatisomerises the carotenoids and changes their biological potencies (Table 1) andconcluded that green vegetables, containing largely beta-carotene, lose 15-20% oftheir vitamin potency on processing, while red and yellow vegetables, containinglargely alpha-carotene, lose 30-35% of their potency. Bioassay would haverevealed this (but probably with less precision). Chemical work involvesseparation of the different chemical forms before measurement — also a long andlaborious procedure but presumably far more precise and reproducible.Table 1: Relative potencies of isomers of carotene (Sweeney and March, 1971)All -trans ß- carotene 100neo -ß- carotene-B 53all - trans - a - carotene 53neo -ß- carotene - U 38neo - a - carotene - B 16neo -a- carotene - U 13One of the more intractable problems in assaying the content of a nutrient,whether chemically or biologically, is that of extracting it (Bender andMacFarlane, 1965). There are reports in the literature of apparent increases inthe carotene content of foods because it was less completely extracted from theraw food than after the cell walls had been broken down by heat. Similarly, thereare considerable potential errors in the assay of folic acid (microbiological)because the food has first to be autoclaved to permit extraction of the vitamin.A measurement of bioavailability carried out by feeding the food itself wouldapparently yield the correct value, yet the findings of the Sheffield experimentbelie this. By measuring blood levels in depleted human subjects it was foundthat the proportion of carotene absorbed from carrots varied from 25% to 50%depending on the physical form of the food, namely on how finely it was minced.