| Hyperhomocysteinemia, or elevated concentrations of homocysteine in plasma, is associated with a variety of disorders, especially vascular disease. The mechanism by which homocysteine causes damage is unknown. One of the possible mechanisms for tissue damage in hyperhomocysteinemia is impaired methylation. S-Adenosyl-homocysteine is the immediate precursor of homocysteine in the methionine cycle. Several recent studies of DNA hypomethylation have shown that impaired methylation correlates with plasma S-adenosyl-homocysteine concentration. In addition, it has been suggested the plasma S-adenosyl-homocysteine concentration may be a superior marker for disease than plasma homocysteine. Measuring S-adenosyl-homocysteine in plasma is difficult for a variety of reasons. Methods that measure underivatized S-adenosyl-homocysteine require expensive equipment and derivatization reactions can require long incubations. There are also many compounds in plasma that can potentially interfere with the analysis. The purpose of this thesis was to develop a method for plasma S-adenosyl-homocysteine measurement that had a shorter derivatization reaction time and gave a superior separation from interfering compounds without requiring specialized, expensive equipment. To that end, a shorter derivatization reaction scheme was developed, employing EDTA to prevent degradation of S-adenosyl-homocysteine. Two-dimensional liquid chromatography was performed to effect a better separation of the etheno-S-adenosyl-homocysteine derivative from other compounds and to allow the use of an internal standard, S-adenosyl-cysteine. |
