Measurement of Blood Levels

A variety of assays have been developed to quantify blood homocysteine levels, with those employing high-pressure liquid chromatography perhaps being the most common. These assays have proven to be relatively accurate and precise (coefficients of variation less than 10%), and are relatively simple and quick to perform. The development of such assays in the 1980s was the technological breakthrough that spurred the exponential increase in homocysteine-related research over the last 15-20 years and the establishment of hyperhomocysteinemia as an independent risk factor for vascular disease.

As described above, homocysteine comes in several forms in the blood, including protein-bound, mixed disulfides, homocystine, and free-reduced. Assays for homocysteine usually measure the sum total of all these forms, i.e., total homocysteine. To accomplish this, the first procedure in homocysteine assays is a reduction step to break all disulfide bonds, thus converting all homocysteine to the free-reduced form. The free-reduced form is then quantified by one of various methods.

Blood sample collection and processing are critical factors in the determination of homocysteine concentrations. Typically, blood samples for homocys-teine analysis are collected in tubes containing an anticoagulant (e.g., EDTA, heparin). Prompt separation of plasma from the blood cells after centrifuga-tion is required to avoid excess release of intracellular homocysteine into the plasma or removal of homocysteine from the plasma by meta-bolically active leucocytes after blood draw. Keeping the blood sample cold until centrifugation and separation (ideally within 4 h of blood draw) minimizes this problem. Serum homocysteine concentrations typically exceed plasma concentrations by 20%. This is likely due to the fact that blood collected to isolate serum (i.e., without an anticoagulant) must clot at room temperature for 30-60 min before centrifugation and separation. Therefore, plasma is preferred for measurement of homocys-teine. Once separated from the blood cells, the concentration of homocysteine in plasma or serum remains stable for years when stored frozen.

Another important issue in the measurement of homocysteine is the prandial state of the individual. For individuals with adequate B vitamin status, no genetic abnormalities, and no pathophysiological conditions that affect homocysteine metabolism, plasma homocysteine levels after an overnight fast are similar to levels after a meal (even high-protein meals containing methionine). However, for individuals with low vitamin B6 status or heterozygous genetic defects in cystathionine ^-synthase, postprandial homocysteine levels can be significantly higher than fasting levels. Because of the nutritional or genetic block in the conversion of homocysteine to cystathionine, there is decreased capacity to metabolize the influx of homocysteine synthesized from dietary methionine. This, in fact, is the basis for the methionine load test for detection of impaired cystathionine ^-synthase activity. In this test, baseline blood is drawn after an overnight fast, and then again 4h after consumption of a large dose of methionine dissolved in orange juice (100 mg methionine per kilogram body weight). Plasma homocysteine increases to a greater extent in individuals with low vitamin B6 status or heterozygous genetic defects in cystathionine ^-synthase than in individuals without these problems. Importantly, individuals with elevated fasting homocysteine and those with normal fasting levels, but elevated post-methionine load levels, are both at increased risk of vascular disease.

See also: Amino Acids: Chemistry and Classification; Metabolism; Specific Functions. Cobalamins. Folic Acid. Riboflavin. Vitamin B6.

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