588 Thurs&ty, June 22, 2006: Poster Session P17 Novel technologies for risk determination separation method used to separate lipoproteins (LP) in human serum with flu- orescence imaging. This allows us to determine the densities of LPs as well as the concentrations of cholesterol in LDL and triglycerides in triglyceride-rich lipoproteins (TRL). Metbods: We combine NBD C6-ceramide fluorescence imaging with DGU to obtain a LP density profile. This work demonstrates the feasibility of measuring lipoprotein densities while simultaneously obtaining the TG content of TRL as well as cholesterol content in LDL by using fluorophore labeling of the lipoproteins. Results: The major outcome of this research is a novel analytical method that couples density gradient ultracentrifugation and fluorescence imaging to separate LP and quantify TG and cholesterol simultaneously. Conclusions: We demonstrate the viable application of fluorescence imag- ing to determine TG content of TRL and cholesterol content LDL, while simultaneously separating lipoproteins by their respective densities. Funding: This work was supported by the NIH Heart, Lung, & Blood Institute (HL 068794). Th-P 17:4291 A R A P I D M I C R O F L U I D I C ASSAY F O R HDL-C AND l J HDL SUBCLASS D I S T R I B U T I O N C. Li I , D. Deng I , D. Jing I , W. Love I , M. Kratzmeier 2 , F. Bek 2, X. Tian 2, R. Romeo I , O. Mueller I , R. Superko 3 . 1Agilent Technologies, Santa Clara, USA: "~Agilent Technologies, Waldbrotm, Germany: 3 Fuqua Heart Center; Piedmont Hospital, Atlanta,USA Objective: HDL-C and HDL subclass distribution are clinically useful but current laboratory methods to determinesubclass distribution are cumber- some, expensive, and time-consuming. We investigated a rapid microfluidic method based on the Agilent 2100 Bioanalyzer for determining HDL-C and HDL subclass distribution. Methods: The Bioanalyzer, a benchtop instrument, separates lipoproteins on an electrophoresischip. Each test requires less than one microliter of serum. The Bioanalyzer can run 12 samples plus a control on each chip, and produces a result in less than three minutes per sample. Serum samples from 20 subjects were analyzed for HDL subclass distribution using the Bioanalyzer; the Lipoprint(R) system (Quantimetrix); and gradient gel elec- trophoresis (GGE). Total HDL-C was determined by the Bioanalyzer and also by standard enzymatic methods. Results: Bioanalyzer electropherogramsshowed several distinct HDL particle subfractions with increasing size. Quantitative measure of these sub- classes was reproducible (CV = 5% to 10%). The third subclass correlated well with other measures of large HDL, namely, HDL-L obtained on the Lipoprint system (r=0.89) and HDL 2b obtained by GGE (r=0.90). HDL-C measured on LipidChip was reproducible (CV=4%) and correlated to enzymatically determined HDL-C (r=0.90). Conclusions: The Bioanalyzer microfluidic chip-based assay provides HDL-C and HDL subclass distribution results that correlate well with exist- ing methods. This assay speed, simplicity, and accuracy provide clinically important advantages over current assays. Funding: Funding was provided by Agilent Technologies. Th-P 17:4301 CORONARY RISK ASSESSMENT USING A RAPID l J M I C R O F L U I D I C L I P O P R O T E I N ASSAY C. Li I , D. Deng 1 , D. Jing 1 , R. Romeo 1 , M. Kratzmeier 2 , F. Bek 2, X. Tian 2, O. Mueller 1 , D. Schreiber 3 , H. Sussman 3. 1Agilent Technologies, Santa Clara, USA: 2 A gil ent Technologies, Waldbrotm, Germany: 3Stanford Universi~ Medical Centre; Palo Alto, USA Objective: A rapid, simple assay of total HDL-C, LDL-C, and HDL subclass distribution was developed on the Agilent 2100 Bioanalyzer. This assay was used to test serum from normal subjects and from patients with suspected acute coronary syndrome (ACS) to determine a possible role for lipoprotein parameters in assessing ACS risk. Methods: Serum samples from 24 patients presenting with non-traumatic chest pain at the Stanford University Medical Center Cardiac Emergency Department (ED) were measured for total HDL-C, LDL-C, and HDL subclass distribution on LipidChip using the Bioanalyzer. Serum samples from 20 subjects without chest pain were also analyzed. Results: The average total HDL-C was significantly lower (Students t-test, 2-tailed, p<0.03)and total LDL-C significantly higher (p<0.01) in patients with chest pain compared to those without chest pain. As well, the ratio of HDL-C to LDL-C was significantly lower in the group of subjects with chest pain (p<0.01). Furthermore, the HDL subfraction distribution was markedly different between the two groups. The ratio of large HDL particles to small HDL particles was significantly lower in subjects with chest pain compared to the control group (p< 0.03). Conclusions: HDL-C, LDL-C, and HDL subclass distribution can distin- guish between patients with symptoms of ACS and normal individuals. Thus, the Bioanalyzer could be used to rapidly assign a patient to a group with a lipid profile that has a higher frequency of cardiovascular disease. Funding: Funding for this study was provided by Agilent Technologies, Inc. I Th-P17:4311 ANALYSIS OF F R E E FATTY ACIDS IN I A T H E R O S C L E R O T I C PLAQUES C. Pettinella 1 , S.H. Lee 2, F. Cipollone 1 , C. Patrono 3, I.A. Blair 2 . 1Center of Excellence On Aging, Universi~ G. d'Anmmzio, Chieti, Italy: 2 Universi~ of Pennsyh,ania, Philadelphia, USA; 3 Universi~ of Ronw la Sapienza, Ronw, Italy Objectives: Prognostic evaluation of atherosclerotic plaques, initially based upon a quantitative approach linked to the severity of arterial stenosis, has pro- gressed to a qualitative approach of plaque composition. Thus, we developed a targeted lipidomic analysis to qualify and quantify the principal fatty acids present in human plaques. Methods: Fatty acids extracted from 10 carotid plaques were ana- lyzed using stable isotope dilution liquid chromatography/electrospray ion- ization/tandem mass spectrometry. The analysis was performed on esters derivatives with the ESI source in the positive ionization mode. Results: We performed the analysis of the 10 major fatty acids (myris- tic, palmitic, steaxic, oleic, linoleic, alpha and gamma-linolenic, arachi- donic, eicosapentenoic, docosahexenoic) previously converted to trimethy- laminoethyl (TMAE) esters. We found a quantitation limit in the order of pg on column for all the acids. We also verified the linearity of response to increasing amount of fatty acids (both on standard and tissue) and we found r2 values close to 0.99, the precision was checked in inter and intra-day assays. Conclusions: We validated a specific and selective method to analyze 10 or more acids. Our data illustrate that formation of TMAE iodide derivatives coupled with analysis by stable isotope dilution LC/MRMIMS provides an alternative to GC/MS for the sensitive and specific analysis of saturated and unsaturated fatty acids that compares favorably with other traditional GC and HPLC methods. Funding: NIH grant P50 HL70128 I Th-P17:4321 H I G H D E N S I T Y L I P O P R O T E I N QUANTITATION AND SUB-FRACTIONATION ANALYSIS BY MICROFLUIDICS T E C H N O L O G Y O. Mueller I , D. Deng I , W. Love I , D. Jing I , M. Kratzmeier 2 , X. Tian 2, F. Bek 2, C. Li 1 , R. Romeo 1 , M. McNulty 1 . 1Agilent Technologies, Santa Clara, USA: "~Agilent Technologies, Waldbronn, Gerntany Objective: Development of a low-cost, highly precise, and easy-to-use method for the analysis of total HDL-Cholesterol (HDL-C) and HDL sub-fractionation. Methods: A new microfluidics-based method for HDL-C quantitation and sub-fractionation is introduced. Results: To date, several techniques are available for the determination of HDL-C levels as well as for sub-pattern analysis of HDL samples. Most of these techniques suffer severe disadvantages such as complicated and lengthy procedures, imprecision of results, and high cost per analysis that prevent them from routine use in the clinical laboratory. Here, we present a simple, inexpensive, and highly precise procedure for the single-step analysis of HDL-C and HDL sub-fractionation. The approach uses microfluidics technology for the electrophoretic separation of serum samples in microfabricated chips. The solution consists of an instrument, which carries out sample loading, injection, separation and detection, as well as software that automatically analyses the results based on internal standards and controls. Sample molecules axe detected by means of LIF as they pass the detection window. Electrophoretic traces axe displayed on the computer screen and analyzed to result the total amount of HDL in each sample, as well as relative amounts of HDL subspecies. This simple procedure, where each sample is analyzed in less than 2 minutes, is easy to use and can potentially be caxried out by laboratory technicians. Conclusions: Microfluidics analysis provides an inexpensive, easy-to-use, and highly accurate approach to HDL-C quantitation and sub-fractionation. XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006