Methods of Analysis and Units of Activity

Historically, the vitamins, like hormones, presented chemists with a considerable challenge. They are present in foods, tissues, and body fluids in very small amounts, of the order of ^moles, nmoles, or even pmoles per kilogram, and cannot readily be extracted from the multiplicity of other compounds that might interfere in chemical analyses. Being organic, they are not susceptible to determination by elemental analysis as are the minerals. In addition, for several vitamins, there are multiple vitamers that may have the same biological activity on a molar basis (e.g., the vitamin B6 vitamers, Section 9.1), or may have very different biological activity (e.g., the vitamin E vitamers, Section 4.1).

The original methods of determining vitamins were biological assays, initially requiring long-term depletion experiments in animals, and later using a

Table 1.4 Compounds that Are Not Dietary Essentials, But May Have Useful Protective Actions

Anthocyanins

Plant (flower) pigments, antioxidants

Bioflavonoids

Polyphenolic compounds with antioxidant action, at one time known as

vitamin P

Glucosinolinates

Modify metabolism of foreign compounds and reduce yield of active

carcinogens from procarcinogens

Glycosides

Modify metabolism of foreign compounds and reduce yield of active

carcinogens from procarcinogens

Polyterpenes

Inhibit cholesterol synthesis

Squalene

Final acyclic intermediate in cholesterol synthesis, acts as feedback

inhibitor of cholesterol synthesis

Phytoestrogens

Weak estrogenic and antiestrogenic actions, potentially protective

against estrogen- and androgen-dependent tumors and osteoporosis

Polyphenols

Antioxidants

Ubiquinone

Redox coenzyme in mitochondrial electron transport chain,

(coenzyme Q)

coantioxidant with vitamin E

Vitamin A

Antioxidants

inactive

carotenoids

variety of microorganisms with more or less defined requirements. Microbiological assays are still commonly used for many of the vitamins; problems of both overestimation and underestimation may occur:

1. Overestimation of the vitamin content of foods will occur if the test organism can use chemical forms and derivatives of the vitamin that are not biologically active in, or available to, human beings.

2. Underestimation will occur if the test organism is unable to use some vitamers, although human beings have appropriate enzymes for interconversion.

Before some of the vitamins had been purified, they were determined in terms of units of biological activity. All should now be expressed in mass or, preferably, molar terms, although occasionally the (now obsolete) international units (iu) are still used for vitamins A (Section 2.1.3), D (Section 3.1), and E (Section 4.1). Where different vitamers differ greatly in biological activity (e.g., the eight tocopherol and tocotrienol vitamers of vitamin E, Section 4.1), it is usual to express total vitamin activity in terms of milligram equivalents of the major vitamer or that with the highest biological activity.

Many of the methods that have been devised for vitamin analysis are now of little more than historical interest, and, in general, unless there is some reason, no analytical methods are listed in this book. A number of recommended methods for vitamin analysis in foods were published as the outcome of a European Union (EU) COST-91 project (Brubacher et al., 1985); since then, the development of ligand binding assays (radioimmunoassays) and highperformance liquid chromatography techniques has meant that individual chemical forms of most of the vitamins can now be determined with great precision and specificity, often with only a minimal requirement for extraction from complex biological materials. Nevertheless, microbiological assays are still sometimes the method of choice, and biological assay is still essential to determine the relative biological activity of different vitamers.

Although modern analytical techniques have considerable precision and sensitivity, food composition tables cannot be considered to give more than an approximation to vitamin intake. Apart from the problems of biological availability (Section 1.1.2), there is considerable variation in the vitamin content of different samples of the same food, depending on differences between varieties, differences in growing conditions (even of the same variety), losses in storage, and losses in food preparation.

When foods have been enriched with vitamins, because of the requirement for the food to contain the stated amount of vitamin after normal storage, manufacturers commonly add more than the stated amount - so-called overage. One of the problems in the debate concerning folate enrichment of flour (Section 10.12) is the relatively small difference between the amount that is considered desirable and the amount that may pose a hazard to vulnerable population groups, and the precision to which manufacturers can control the amount in the final products. In pharmaceutical preparations, considerable latitude is allowed; the U.S. Pharmacopeia permits preparations to contain from 90% to 150% of the declared amount of water-soluble vitamins and from 90% to 165% of the fat-soluble vitamins.

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