Quantitative determination of phosphatidylcholine hydroperoxides during copper oxidation of LDL and HDL by liquid chromatography/mass spectrometry.

1-Palmitoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 16:0/18:2-OOH) and 1-stearoyl-2-linoleoylphosphatidylcholine monohydroperoxide (PC 18:0/18:2-OOH) were measured by liquid chromatography/mass spectrometry (LC/MS) using nonendogenous 1-palmitoyl-2-heptadecenoylphosphatidylcholine monohydroperoxide as an internal standard. The calibration curves for synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, which were obtained by direct injection of the internal standard into the LC/MS system, were linear throughout the calibration range (0.8-12.8 pmol). Within-day and between-day coefficients of variation were less than 10%, and the recoveries were between 86% and 105%. The limit of detection (LOD) and the limit of quantification (LOQ) were determined using synthetic standards. The LOD (signal-to-noise ratio 3:1) was 0.01 pmol, and the LOQ (signal-to-noise ratio 6:1) was 0.08 pmol for both PC 16:0/18:2-OOH and PC 18:0/18:2-OOH. With use of this method, the concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH in the lipoprotein fractions during copper-mediated oxidation were determined. We prepared oxLDL and oxHDL by incubating native LDL and native HDL from human plasma (n = 10) with CuSO(4) for up to 4 h. The time course of the PC 16:0/18:2-OOH and PC 18:0/18:2-OOH levels during oxidation consisted of three phases. For oxidized LDL, both compounds exhibited a slow lag phase and a subsequent rapidly increasing propagation phase, followed by a gradually decreasing degradation phase. In contrast, for oxidized HDL, both compounds initially exhibited a prompt propagation phase with a subsequent plateau phase, followed by a rapid degradation phase. The analytical LC/MS method for phosphatidylcholine hydroperoxides might be useful for the analysis of biological samples.

Peripheral leukocyte anomaly detected with routine automated hematology analyzer sensitive to adipose triglyceride lipase deficiency manifesting neutral lipid storage disease with myopathy/triglyceride deposit cardiomyovasculopathy.

Adipose triglyceride lipase (ATGL) deficiency manifesting neutral lipid storage disease with myopathy/triglyceride deposit cardiomyovasculopathy presents distinct fat-containing vacuoles known as Jordans' anomaly in peripheral leucocytes. To develop an automatic notification system for Jordans' anomaly in ATGL-deficient patients, we analyzed circulatory leukocyte scattergrams on automated hematology analyzer XE-5000. The BASO-WX and BASO-WY values were found to be significantly higher in patients than those in non-affected subjects. The two parameters measured by automated hematology analyzer may be expected to provide an important diagnostic clue for homozygous ATGL deficiency.