Measurement of Vitamin C Status Biochemical Tests for Adequacy and Deficiency

In species (such as humans) that cannot synthesize vitamin C in their bodies, the vitamin concentration in tissues and blood compartments (plasma, erythro-cytes, and white blood cells) varies characteristically with the dietary intake of the vitamin. Since the blood-compartment concentrations mirror the concentrations in most other cells and tissue compartments, tissue vitamin C status can be monitored by measuring the concentration in plasma or blood, even though the blood concentrations are generally lower than those in most tissues. The concentration ratios between extracellular and various intracellular compartments are determined by active transport systems that concentrate the vitamin inside many cell types. At high intakes of the vitamin, the intestinal absorption process is overwhelmed, so that some of the ingested vitamin remains unabsorbed and is destroyed in the lower intestine by intestinal bacteria. The maximum steady-state level in plasma can be temporarily exceeded following a high bolus intake, but the excess vitamin is rapidly excreted in the urine once the renal threshold for filtration and reabsorption is exceeded. These safety mechanisms limit the maximum concentration of the vitamin to which the tissues are exposed.

For many years, the best biochemical measure of vitamin C status was considered to be the buffy coat, or total white-cell concentration of the vitamin (Table 2), expressed as micrograms per 108 white cells, the cell count in the assay sample usually being estimated by an electronic cell counter. This status index varied predictably with the magnitude of the total body vitamin C stores during controlled (animal) depletion studies. However, in practice it has proved to be a difficult test to use in human studies and especially in surveys, as it requires complex laboratory operations to be performed immediately after collecting the blood. It is also difficult to harmonize between laboratories, and, since it measures the average vitamin C content across several different white-cell types, whose individual proportions and relative vitamin C contents may vary considerably, its interpretation was not always straightforward. In addition, infection has rather unpredictable effects on the values obtained. For all of these reasons, this assay has fallen out of favor and is now rarely used. The concentration of the vitamin in erythrocytes or whole blood is not an ideal alternative, partly because hemoglobin can catalyze the oxidative destruction of the vitamin in vitro and partly because erythrocyte concentrations do not mirror other body compartments in a simple manner.

Serum or plasma vitamin C has therefore become the most commonly used status assay. In order to avoid short-term fluctuations caused by recent bolus intakes from food or supplements, it is preferable to collect an overnight-fasting blood sample. Since the vitamin is extremely easily oxidized, the sample must be carefully preserved unless the assay is to be performed immediately. The usual approach is to add freshly prepared metaphosphoric acid, usually at between 2 and 5% w/v, which precipitates plasma proteins, chelates transition-metal ions, and provides a protective acidic environment of a suitable pH. If stored, the samples must be kept at a low temperature, e.g., at —25 °C for not more than a week or two or at —80 °C for up to 1-2 years. There are many alternative physicochemical and chemical assay methods for measuring vitamin C in extracts of plasma or serum. These include (a) the measurement of its chemical reducing action on reducible dyes such as dichlorophenol indophenol and (b) the formation of either a colored osazone or a fluorescent derivative with orthophenylene diamine, after conversion to dehydroascorbate. Quantitation by absorbance or by electrochemical detection after separation by high-performance liquid chromatogra-phy is favored by many workers. This procedure has the advantage of being relatively specific (i.e., free from most forms of interference) and highly sensitive, but it is more time-consuming than the simpler nonchromatographic methods. Different methods may differ with respect to their specificity and their sensitivity to problems of interference as well as in the precautions that are needed to avoid oxidative destruction of the vitamin during the assay. Careful validation and robust quality-control procedures are essential.

Plasma or serum levels below 11 p.moll—1 (<0.2mg per 100ml) are considered to be evidence of biochemical deficiency, and if this is severe and prolonged, the risk of clinical deficiency, i.e., scorbutic signs and symptoms, gradually increases. Intakes below around 20mgday—1 are likely to result in plasma levels in this range. Studies of human volunteers in the middle of the twentieth century showed clearly that an intake of 10 mg vitamin C per day in a healthy adult is sufficient to prevent clinical scurvy, and this small amount is also sufficient to cure scorbutic signs and symptoms (Table 1).

Assay methods based on urinary excretion of vitamin C have been used to study status, but they are too cumbersome and difficult to interpret to be useful in population studies. There are no well-established functional assays available to define vitamin C status and requirements at present. An older method known as the 'Hess test,' which measures relative capillary fragility under pressure or suction (Table 2), is useful only if subclinical scurvy is present and is rarely attempted today. Studies of collagen crosslinks or oxidative damage to macro-molecules such as DNA or lipids may yield evidence about functional status in the future, but this remains a research challenge and is not yet an available option for routine studies or surveys.

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