DOI 10.1515/cclm-2013-0525 Clin Chem Lab Med 2014; 52(4): 557–564
Jun Dong, Songlin Yu, Ruiyue Yang, Hongxia Li, Hanbang Guo, Haijian Zhao, Shu Wang and Wenxiang Chen*
A simple and precise method for direct measurement of fractional esterification rate of high density lipoprotein cholesterol by high performance liquid chromatography Abstract Background: The relationship between fractional cholesterol esterification rate in plasma or serum high-density lipoprotein (HDL) (FERHDL) and lipoprotein subfractions and other cardiovascular disease (CVD) risk factors has been demonstrated. However, the current method for measuring FERHDL requires fresh serum samples and radio active labeling of the samples, making it impractical for use in clinical laboratories. In this study, we developed a simple and precise HPLC method for the measurement of FERHDL. Correlations between FERHDL and CVD risk factors were evaluated in 119 healthy volunteers. Methods: Fasting blood samples were collected and serum was isolated within 2 h. Serum HDL was prepared by precipitation of apolipoprotein B (apoB)-containing lipoproteins with dextran sulfate and magnesium chloride. HDL fractions were divided into two aliquots and incubated at 0°C and 37°C, respectively, for 1 h. Free cholesterol in the HDL fractions was analyzed by HPLC. FERHDL was calculated as the percent decrease of free cholesterol during incubation. Results: The esterification reaction of HDL free cholesterol was not linear, but the measured FERHDL was stable when serum samples were stored at room temperature for 0.05). In contrast, dilution of HDL with normal saline significantly affected HDLFC esterification as well as FERHDL assay. FERHDL rates decreased with increased dilution ratio (Figure 2). In addition, the presence of ME in serum caused an average 15% increase of measured FERHDL. Correlation between FERHDL with and without ME was nearly linear (r = 0.998).
Biological variations of FERHDL For the study of biological variations, 20 healthy volunteers, 10 men and 10 women, were recruited. All subjects maintained a constant weight and their normal diet and lifestyle during the 2 months of study. Blood was collected four times from each individual with 2-week intervals between each sampling. Serum was isolated within 2 h post-collection, frozen, and stored in 1 mL aliquots at −80°C until analysis. The within-subject and between-subject variations (CVI and CVG) and individuality index (II) were calculated as described by Fraser [21].
Results Kinetics of HDLFC esterification Changes of HDLFC of the five individuals during incubation were shown in Figure 1. These results demonstrated
HDLFC esterification, %
100.0 80.0 60.0 40.0 20.0 0
0
2
4 Incubation time at 37°C, h
6
8
Figure 1 Line plot of time course (h) for fractional esterification rate of cholesterol (% esterification). Fasting blood samples were collected from 5 individuals into tubes containing clot-activator. Serum was isolated from blood cells within 2 h. HDL fractions were prepared by precipitating apoB-containing lipoproteins with dextran sulfate and MgCl2 and aliquoted. Aliquots of HDL fractions were placed in 37°C water bath for 0 (put in an ice water bath), 0.5, 1, 2, 4 and 8 h, respectively, and then put in ice water bath. HDLFC concentrations were measured by HPLC method.
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560 Dong et al.: Fractional esterification rate of HDL cholesterol by HPLC
A
30.0%
0.40
0.30
20.0%
HDLFC, mmol/L
FERHDL,%/h
25.0%
15.0% 10.0% 5.0% 0.0% 0%
20%
40%
60%
0.20
0.10
0.00
80%
0
2
0
2
Dilution time of HDL
B
6
8
4
6
8
35.0% 28.0%
FERHDL, %/h
Figure 2 Effect of HDL dilution on FERHDL assay. HDL fractions of three aliquots of mixed serum samples were isolated by precipitating apoB-containing lipoproteins with dextran sulfate and MgCl2. HDL was diluted with 0.9% NaCl at ratios of 0%, 20%, 40%, 60%, and 80%, respectively. HDLFC concentrations at each dilution ratio were measured by HPLC method and FERHDL were calculated. FERHDL values for y-axis were adjusted for dilution.
4 Room temperature, h
21.0% 14.0% 7.0% 0.0%
Room temperature, h
Stability of FERHDL After serum samples were incubated at room temperature and 4°C for different periods of time, concentrations of HDLFC were measured and FERHDL rates were calculated in these samples and the results are shown in Figures 3 and 4. When incubated at room temperature, HDLFC concentrations of all the five individuals decreased with increased incubation time. However, the measured FERHDL were not significantly affected when measured within 4 h of storage at room temperature. Further incubation caused slight decreases of FERHDL, especially for samples with higher FERHDL rates. Similarly, incubation of serum samples at 4°C resulted a significant reduction of HDLFC concentrations, but FERHDL stayed unchanged within 24 h. Prolonged incubation at 4°C ( > 24 h) caused slight increase of FERHDL for samples with higher FERHDL rates. These results suggested that, for measurement of initial esterification rate, fresh serum samples are not indispensable so long as the isolated serum samples are stored at room temperature for
