New analytical methods for the determination of homocysteine in human plasma.

摘要

Homocysteine (Hcy) has been recently identified as an independent risk factor for CVD. Hcy, a sulfur-containing amino acid, is involved in the synthesis and metabolism of the essential amino acid methionine. In plasma, Hcy exists as 70--80% protein-bound and 20--30% disulfides. Reduced Hcy is present only in trace amounts. Hcy levels in plasma represent total Hcy (tHcy), which encompasses all forms of Hcy in vivo. Normal levels of tHcy are in the range of 5--15 micromolar and increase with an increased risk of cardiovascular disease to greater than 100 micromolar.; The primary objective of this thesis project was to develop selective and sensitive methodologies for the determination of Hcy in biological samples. First, methods employing both conventional CEEC (Chapter 2) and LC/MS/MS (Chapter 3) were developed to determine the concentration and calculate the mass balance of Hcy in human plasma. In the future, these methods will be available for application in a clinical trial to determine the mass balance of all forms of Hcy in vivo in healthy subjects and its alteration during hyperhomocysteinemia. Next, the optimized conventional CEEC method was transferred to a microchip CE system (Chapters 4 and 5). The microchip CEEC method demonstrated the feasibility of a miniaturized clinical analyzer for Hcy in human plasma.