Introduction
Vitamin status may be assessed using a variety of direct and indirect measurements. Concentrations of vitamins can be measured in serum or blood cells. Vitamin metabolites may be measured in blood or urine. Changes in response to added vitamins may be measured as specific enzyme activities in blood or growth of leukocytes in cell culture. Finally, functional adequacy of a particular vitamin can be revealed by the urinary levels of specific metabolic intermediates controlled by the action of the vitamin.
Urinary organic acid analysis has traditionally been used for the detection of genetic diseases leading to frank inborn errors of metabolism.1 Although up to 30% of genetic polymorphisms are associated with decreased coenzyme binding affinifty,2 it is now widely accepted that genetic polymorphisms have a range of severity. One example is maple syrup urine disease (MSUD). The condition is primarily known to result from a faulty enzyme complex (branched-chain alpha-keto acid dehydrogenase). However there are numerous subunits and configurations that can be affected, so there is a range of possible downstream consequences. Research has identified patients diagnosed with MSUD who were found to be responsive to moderate to large doses of thiamin.2 Similarly functional deficits of coenzyme vitamins are revealed in organic acid testing. It follows logically that there may be many yet undiagnosed milder forms of these genetic polymorphisms with symptoms not generally associated with the frank disease. These milder genetic variants may not appear until a patient encounters a specific stress, or life stage such as adolescence, or a nutrient deficiency that slows the enzyme reaction. For routine clinical purposes, the most useful assay gives a clear answer to the question of whether body pools are adequate to meet current demands. Table 2.1 shows a summary of the laboratory evaluations of vitamin status that are discussed in this chapter.
Methods of Vitamin Assessments
Vitamins encompass several classes of compounds. The challenge of determining adequacy of body stores is complicated by the diversity of structures, the presence of multiple forms and the availability of specimens reflecting the whole body vitamin stores. No single assay can give adequate information about all vitamins. Technological advances for detecting metabolic markers of vitamin deficiencies have made functional testing available. Concurrently, the knowledge of metabolic intermediates that can serve as markers of functional deficiencies has also grown.3
For various reasons specific to each vitamin, it is possible for an individual to have normal serum levels of a vitamin while exhibiting signs of insufficiency for that vitamin owing to a lack of adequate intracellular concentration to meet the metabolic demands of the cells. For example, serum vitamin B12 can be normal while the metabolic intermediate methylmalonate is elevated owing to inadequate intracellular vitamin B12 to sustain the function of the enzyme methylmalonyl-CoA mutase.
An intermediate in a pathway that normally has high flux owing to macronutrient processing provides the greatest sensitivity of a metabolic marker for revealing early or mild vitamin insufficiency. The amino acid catabolic pathways offer this advantage. One example of this is the degradation of excess histidine to glutamic acid through the intermediate formiminoglutamic acid (FIGLU). The conversion of FIGLU to glutamic acid requires the participation of folic acid and mild insufficiencies of the vitamin produce elevations of FIGLU. The severity of FIGLU elevation may be related to the severity of folic acid deficiency, under standard dietary protein intake conditions. Using this type of relationship, it is therefore possible to specifically tailor nutrient supplementation according to the need for repletion to restore the metabolic capacity. Since many of the metabolic markers we will discuss are amino acid pathway intermediates, a high protein meal or challenge dose of amino acids prior to specimen collection may be employed to maximize the sensitivity of the test.
The first widely available tests of vitamin concentrations in serum were microbiological growth assays. Protozoan organisms whose growth is dependent on the presence of vitamins are grown in media that supply all but the vitamin being tested. The assays are very labor intensive and, because of the large number of variables affecting growth of whole cells, the reliability of results is frequently poor. These methods have been replaced by immunoassays with greater sensitivity and reproducibility and lower cost.
Vitamins A, E, beta-carotene and coenzyme Q10 are typically measured in serum using high performance liquid chromatography with various detectors. The fat-soluble components are extracted from the specimen and need no further preparation prior to injection into the instrument for chromatographic separation. Vitamin D, also in serum, can be measured by immunological assay or HPLC. The urinary organic acid markers of vitamin status, however, are more accurately measured by chromatographic separation (either gas or liquid) coupled with mass spectrometric detection. The newer technology of liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) requires less sample preparation and therefore is more accurate than the older methods using gas chromatography (GC/MS). For further discussion of instrumentation, see Chapter 1, "Basic Concepts."
An enzyme stimulation assay works by adding saturating amounts of a vitamin to an enzyme whose activity is dependent on that vitamin to cause an increase in activity. The increase in enzyme activity corresponds to how poorly the enzyme was initially supplied with the vitamin (called cofactor saturation). Erythrocyte transketolase activity, for example, is dependent on thiamin pyrophosphate (TPP). The activity is measured without any added TPP and again after adding excess TPP. The increase in activity may be expressed as a ratio or percentage.4 Abnormally high increases in activity indicate the degree of deficiency. An inherent limitation of the assay is that only one enzyme in erythrocytes is examined so total body functional status of the vitamin may not be revealed.
Another type of stimulation assay involves whole cell responses. Leukocytes are removed from the patient sample and grown in media with increasing concentrations of the vitamin to be assayed. Vitamins must be added in their active cofactor forms. Changes in growth rate at increasing levels of added nutrients provide a measure of the initial state of nutrient adequacy. Cells from a vitamin-deficient patient will require more added vitamin to achieve maximum growth than those from a well-nourished patient. This approach has great potential because it measures the total cellular response to the nutrient. The limitation is that, as with protozoan growth assays, there are many variables controlling the growth of whole leukocytes. Current techniques present extreme challenges to control all possible variables in the growth media. Because the technique depends on the initial viability of the cells, specimen transport also adds a significant variable. It is available for most vitamins and other classes of essential nutrients.5
