Background: Magnetic bead purification for the analysis of low-abundance proteins in body fluids facilitates the identification of potential new biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The aims of our study were to establish a proteome fractionation technique and to validate a standardized blood sampling, processing, and storage procedure for proteomic pattern analysis. Methods: We used magnetic bead separation for proteome profiling of human blood by MALDI-TOF MS (mass range, 1000 -10 000 Da) and studied the effects on the quality and reproducibility of the proteome analysis of anticoagulants, blood clotting, time and temperature of sample storage, and the number of freeze-thaw cycles of samples. Results: The proteome pattern of human serum was characterized by ϳ350 signals in the mass range of 1000 -10 000 Da. The proteome profile showed timedependent dynamic changes before and after centrifugation of the blood samples. Serum mass patterns differed between native samples and samples frozen once. The best reproducibility of proteomic patterns was with a single thawing of frozen serum samples. Conclusion: Application of the standardized preanalytical blood sampling and storage procedure in combina-

A novel analytical platform based on liquid chromatography and tandem mass spectrometry using atmospheric pressure photoionization was applied for the simultaneous quantification of free and esterified ␤ -sitosterol, campesterol, brassicasterol, and stigmasterol. The total time for sample pretreatment and analysis could be reduced from ‫ف‬ 3 h [gas chromatography-mass spectrometry (GC-MS)] to 15 min. The detection limits of the different phytosterols ranged between 0.25 and 0.68 g/l. Linear ranges were between 1 and 1,000 g/l. The within-run and between-run variabilities ranged between 1.4% and 9.9%. The analytical sensitivity was at least 150-fold higher compared with GC-MS. Our new method allows a rapid and simultaneous determination of free and esterified phytosterols in serum. Phytosterols are common components of plant foods, especially vegetable oils, seeds, nuts, and cereals (1). They are structurally similar to cholesterol, differing only in the number of carbons or double bonds in the side chain. An average Western diet contains ‫ف‬ 200-400 mg of phytosterols per day (e.g., ␤ -sitosterol, campesterol, stigmasterol, and brassicasterol). All serum and tissue phytosterols are derived exclusively by intestinal absorption. Therefore, serum levels of phytosterols reflect dietary plant sterol intake and intestinal absorption (2). The individual differences in plasma phytosterol concentrations are highly heritable (3). Compared with dietary cholesterol, the intestinal absorption rate of dietary phytosterols is markedly lower, because the bulk of absorbed phytosterols is immediately secreted into the intestine by the ATP-binding cassette half-transporters ABCG5 and ABCG8 of the enterocytes (4). Mutations in either of the transporter genes have been identified as the cause of sitosterolemia, a rare autosomal recessive lipid disorder that is characterized by markedly increased serum phytosterol concentrations (e.g., ␤ -sitosterol Ͼ 50 mg/l) as a consequence of hyperabsorption and impaired biliary secretion of neutral sterols (5-7). These patients develop premature coronary heart disease (8, 9). A recent study in patients admitted for elective coronary artery bypass graft surgery supports the hypothesis that even slightly increased campesterol (3.8 Ϯ 1.6 mg/l) and ␤ -sitosterol (3.1 Ϯ 1.1 mg/l) concentrations in serum may contribute to the risk of coronary heart disease (10).Sensitive analytical methods are necessary to detect physiological phytosterol concentrations and even slightly increased concentrations in human serum. Currently available methods for the measurement of phytosterols in serum are based on GC-MS. However, this analytical platform is time-consuming and requires a laborious pretreatment procedure (hydrolysis, liquid-liquid extraction, and derivatization) and a large sample volume (11,12). Recently described liquid chromatography-mass spectrometry (LC-MS) methods for the determination of cholesterol and oxidized metabolites in human plasma also include time-consuming extraction and hydroly...

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We previously identified two inbred mouse strains, C57BL/6J and CASA/Rk, with different plasma plant sterol levels. An intercross between these strains revealed a broad plasma plant sterol locus on chromosome 14, which peaked at 17 centimorgan (cM) with a maximum logarithm of the odds score of 9.9. Studies in a chromosome 14 congenic strain, 14KK, with a 4-60 cM CASA/Rk interval on the C57BL/6J background revealed that males, but not females, had decreased plasma plant sterol levels and intestinal cholesterol absorption. In two subcongenic strains, 14PKK and 14DKK, with 4-19.5 and 19.5-60 cM CASA/Rk intervals, respectively, both males and females had decreased plasma plant sterol levels and decreased intestinal cholesterol absorption. Compatible with the decreased plasma plant sterol phenotype, 14PKK mice had increased biliary plant sterol excretion, whereas 14DKK mice did not. Therefore, gender-dependent interactions of genes at the 14PKK and 14DKK intervals are likely to underlie the 14KK interval effect on plasma plant sterol levels and sterol absorption from the intestine. These studies confirm the plasma plant sterol locus on mouse chromosome 14 and provide evidence that there are at least two sets of genes operating: one set affecting intestinal sterol absorption and biliary excretion, and the other set mainly affecting intestinal sterol