α-1 acid glycoprotein (AGP) presents several isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). Changes in the isoforms of AGP have been related to cancer and liver or cardiovascular diseases (CVD). However, to our knowledge, the role of changes in the isoforms of AGP as biomarker of CVD has not been addressed. As differences in its glycosylation or in its amino acid composition can lead to differences in the total charge and/or mass, CZE is a suitable technique for the analysis of the isoforms of the intact AGP. Unfortunately, although its CZE-UV analysis only takes some minutes, currently used methods for AGP purification can take up to 4 days, with high solvent and materials consumption. In this work, miniaturized methods developed in our laboratory for AGP purification are presented. These methods were based on immunoaffinity chromatography (IAC), and ultracentrifuge filter devices. After purification, AGP isoforms profile was analyzed by CZE-UV. The optimum purification process was based on double IAC purification with columns of 500 ¿L volume, using 100 ¿L of sample which was concentrated to 5 ¿ 20 ¿L. The whole analysis method took about 4.5 h. This methodology was successfully applied to serum, plasma, and supernatants from blood vessels. Statistical techniques were applied to analyze plasmas from healthy donors, abdominal aortic aneurysm (AAA) and carotid atherosclerosis (CTA) patients. Linear discriminant analysis (LDA) provided very good classification of the samples and fairly good predictive power for the groups healthy vs patients, healthy vs AAA, and healthy vs CTA, pointing out the potential role of AGP isoforms profile as biomarker of CVD.Currently, we are working to couple online the IAC step with the CE-UV analysis by preparing icroconcentrators (based in magnetic particles or chromatographic supports) in the silica capillaries used for the CE-UV analysis. This will lead to higher automation and miniaturization of the process, diminishing the solvent consumption, shortening the analysis time and facilitating its implementation in microchip format.

Financial support from Comunidad de Madrid (Project S-GEN/0247/2006) and the Spanish Ministry of Science and Innovation (projects CTQ2006-05214, PCI2006-A7-0646, CTQ2009-09399, HH2006-0013, and PSE-010000-2008-6) is acknowledged. Ángel de la Puerta acknowledges the Spanish National Research Council (C.S.I.C.) for a contract in the JAEdoc program. Sara Ongay acknowledges C.S.I.C. for a contract to perform Ph.D. Thesis.

Trabajo presentado al II International Workshop on Analytical Miniaturizatiton: "lab-on-a-chip" celebrado en Oviedo (España) en junio de 2010.-- et al.

Peer reviewed