Effect of labeling of plasma lipoproteins with [3H]cholesterol on values of esterification rate of cholesterol in apolipoprotein B-depleted plasma

The fractional esterification rate of cholesterol in apolipoprotein B (apoB)-depleted plasma (FERHDL) is a good indicator of particle size distribution in high density lipoprotein (HDL) and low density lipoprotein (LDL). However, there has been a discrepancy in the absolute values of FERHDL published by different laboratories. Because the main difference between the methods was in the labeling of lipoproteins with [3H]cholesterol we investigated the effect of using Corning immunoplates and paper discs as carriers of the labeled unesterified cholesterol. We found that Corning plates trap some 3H-labeled free cholesterol, which is released during incubation at 37°C. This means that this additional 3H-labeled free cholesterol is exposed to lecithin: cholesterol acyltransferase (LCAT) for a shorter time and artificially decreases FERHDL. Using paper discs discarded before incubation as carriers of the 3H-labeled free cholesterol results in complete labeling of HDL and thus yields higher values of FERHDL. —Dobiášová, M., L. Adler, T. Ohta, and J. Frohlich. Effect of labeling of plasma lipoproteins with [3H]cholesterol on values of esterification rate of cholesterol in apolipoprotein B-depleted plasma. J. Lipid Res. 2000. 41: 1356–1357. The fractional esterification rate of cholesterol in apolipoprotein B (apoB)-depleted plasma (FERHDL) is a good indicator of particle size distribution in high density lipoprotein (HDL) and low density lipoprotein (LDL). However, there has been a discrepancy in the absolute values of FERHDL published by different laboratories. Because the main difference between the methods was in the labeling of lipoproteins with [3H]cholesterol we investigated the effect of using Corning immunoplates and paper discs as carriers of the labeled unesterified cholesterol. We found that Corning plates trap some 3H-labeled free cholesterol, which is released during incubation at 37°C. This means that this additional 3H-labeled free cholesterol is exposed to lecithin: cholesterol acyltransferase (LCAT) for a shorter time and artificially decreases FERHDL. Using paper discs discarded before incubation as carriers of the 3H-labeled free cholesterol results in complete labeling of HDL and thus yields higher values of FERHDL. —Dobiášová, M., L. Adler, T. Ohta, and J. Frohlich. Effect of labeling of plasma lipoproteins with [3H]cholesterol on values of esterification rate of cholesterol in apolipoprotein B-depleted plasma. J. Lipid Res. 2000. 41: 1356–1357. We have previously demonstrated that the fractional esterification rate of cholesterol in apolipoprotein B (apoB)-depleted plasma (FERHDL) reflects the particle sizes of plasma high density lipoprotein (HDL) and low density lipoprotein (LDL) (1Dobiášová M. Stříbrná J. Sparks D.L. Pritchard P.H. Frohlich J.J. Cholesterol esterification rates in very low density lipoprotein and low density lipoprotein-depleted plasma: relation to high density lipoprotein subspecies, sex, hyperlipidemia, and coronary artery disease.Arterioscler. Thromb. 1991; 11: 64-70Google Scholar, 2Dobiášová M. Stříbrná J. Pritchard P. Frohlich J. Cholesterol esterification rate in plasma depleted of very low and low density lipoprotein is controlled by the proportion of HDL2 and HDL3 subclasses: study in hypertensive and normal middle aged and septuagenarian men.J. Lipid Res. 1992; 33: 1411-1418Google Scholar, 3Dobiášová M. Frohlich J.J. Understanding the mechanism of LCAT reaction may help to explain the high predictive value of LDL-HDL cholesterol ratio.Physiol. Res. 1998; 47: 387-397Google Scholar, 4Tan M.H. Loh C. Dobiasova M. Frohlich J. Fractional esterification rate of HDL particles in patients with type 2 diabetes.Diabetes Care. 1998; 21: 139-142Google Scholar, 5Ohta T. Saku K. Takata K. Nagata N. Maung K.K. Matsuda I. Fractional esterification rate of cholesterol in high density lipoprotein (HDL) can predict the particle size of low density lipoprotein and HDL in patients with coronary heart disease.Atherosclerosis. 1997; 135: 205-212Google Scholar). This results from the fact that the smaller lipoprotein particles are better substrates for plasma lecithin:cholesterol acyltransferase (LCAT) than the larger particles. However, there has been a discrepancy in the absolute values for this parameter between our laboratories. For example, in children and in patients with coronary artery disease (CAD), the FERHDL values reported differed by more than 100% (5Ohta T. Saku K. Takata K. Nagata N. Maung K.K. Matsuda I. Fractional esterification rate of cholesterol in high density lipoprotein (HDL) can predict the particle size of low density lipoprotein and HDL in patients with coronary heart disease.Atherosclerosis. 1997; 135: 205-212Google Scholar, 6Ohta T. Kakiuti Y. Kurahara K. Saku K. Nagata N. Matsuda I. Fractional esterification rate of cholesterol in high density lipoprotein is correlated with low density lipoprotein particle size in children.J. Lipid Res. 1997; 38: 139-146Google Scholar, 7Dobiášová M. Urbanová Z. Rauchová H. Šamánek M. Frohlich J.J. High-density lipoprotein subclasses and esterification rate of cholesterol in children—effect of gender and age.Acta Paediatr. 1998; 87: 918-923Google Scholar, 8Frohlich J. Rae J. Spinelli L. Adler L. Dobiasova M. Gender differences in prediction of coronary artery disease (CAD) by plasma lipoproteins, LCAT measurements. 11th International Symposium on Atherosclerosis, Paris, October, 1997.Atherosclerosis. 1997; 134: 157Google Scholar). The measurement of FERHDL is based on estimation of the radioactivity of free and esterified cholesterol in plasma depleted of apoB lipoproteins (HDL-plasma), labeled with 3H-labeled free cholesterol at 4°C, and then incubated for 30 min at 37°C. Under these conditions only the free cholesterol on HDL is a substrate for lecithin:cholesterol acyltransferase (9Dobiášová M. Frohlich J. Assays of lecithin cholesterol acyltransferase (LCAT).in: Ordovas J.M. Methods in Molecular Biology: Lipoprotein Protocols. Humana Press, Totowa, NJ1998: 217-230Google Scholar). We compared different labeling methods, using EDTA plasma depleted of apoB lipoproteins by precipitation with phosphotungstate and MgCl2 (10Burstein M. Scholnick H.R. Morfin R. Rapid method for isolation of lipoproteins from human serum precipitation with polyanions.J. Lipid Res. 1970; 11: 583-595Google Scholar); HDL-plasma from 21 subjects was used in three experiments. In the first experiment (DISCS) we used our standard procedure (9Dobiášová M. Frohlich J. Assays of lecithin cholesterol acyltransferase (LCAT).in: Ordovas J.M. Methods in Molecular Biology: Lipoprotein Protocols. Humana Press, Totowa, NJ1998: 217-230Google Scholar). Paper discs containing homogeneously dispersed [3H]cholesterol (0.075 μCi) were immersed in diluted HDL-plasma (100 μL of Tris-saline buffer and 50 μL of HDL-plasma in glass tubes on ice). By this procedure HDL is homogeneously labeled with [3H]cholesterol, transferred from discs after 18 h of incubation at 4°C. The discs are then removed and the tubes placed in a shaking water bath for 30 min at 37°C. In the second experiment (CORNING 1) tissue culture plates were used for labeling as described by Ohta et al. (11Ohta T. Saku K. Takata K. Nakamura R. Ikeda Y. Matsuda I. Different effects of subclasses of HDL containing apoA-I but not apoA-II (LpA-I) on cholesterol esterification in plasma and net cholesterol efflux from foam cells.Arterioscler. Thromb. 1995; 15: 956-962Google Scholar): here the [3H]cholesterol was incorporated onto polystyrene tissue culture wells (Corning, Acton, MA). Absolute ethanol (100 μL) containing 0.2 μCi of [3H]cholesterol was placed in the wells and then dried off by flushing with nitrogen. One hundred μL of plasma depleted of apoB lipoproteins in 400 μL of phosphate-buffered saline was added to each well and [3H]cholesterol was equilibrated with the unesterified cholesterol of each sample by incubation at 4°C for 16 h. The Corning plates were thentransferred into a shaking water bath and incubated for 30 min at 37°C. A third experiment was carried out to assess the effect of the Corning polystyrene tissue culture wells (CORNING 2): The procedure was the same as in CORNING 1 but before the incubation at 37°C the labeled samples of HDL-plasma were transferred from the Corning plates to new glass tubes and processed as described above. In all three experiments lipid extracts were dried under nitrogen, and unesterified and esterified cholesterol was separated by thin-layer chromatography (TLC) and detected with iodine vapors. The resulting spots were cut from the plates, transferred into scintillation vials, and counted (9Dobiášová M. Frohlich J. Assays of lecithin cholesterol acyltransferase (LCAT).in: Ordovas J.M. Methods in Molecular Biology: Lipoprotein Protocols. Humana Press, Totowa, NJ1998: 217-230Google Scholar). Radioactivity was measured in samples before and after a 30-min incubation at 37°C to determine whether there was additional flux of [3H]cholesterol from the Corning wells (CORNING 1). Results shown in Table 1 confirm that the polystyrene material of the Corning plates trapped some 3H-labeled free cholesterol and that it was released during incubation at 37°C. Thus some of the 3H-labeled free cholesterol was not utilized during the entire 30 min of incubation.TABLE 1.3H radioactivity in 10-μL samples before and after 30-min incubation at 37°C and FERHDL in the three experimentsDISCSCORNING 1CORNING 2BeforeAfterBeforeAfterBeforeaSame samples as in CORNING 1.Afterdpm5645 ± 7965957 ± 507956 ± 3931869 ± 643956 ± 393988 ± 399FERHDL19.55 ± 9.8210.22 ± 5.1319.20 ± 8.47Values given as means ± SD.a Same samples as in CORNING 1. Open table in a new tab Values given as means ± SD. FERHDL values were significantly different between DISC and CORNING 1 experiments but were similar in DISCS and CORNING 2 experiments. This means that [3H]cholesterol released from the Corning wells during the incubation (and not used for the LCAT reaction) artificially decreases FERHDL (Table 1). However, the FERHDL values, from both CORNING 1 and 2 experiments are highly correlated with the DISCS values (Fig. 1). In conclusion, the trapping of labeled free cholesterol on Corning plates explains the differences in absolute values of FERHDL between the two laboratories. This work was supported by grant 306-96-K220 from the Grant Agency of the Czech Republic, and by the British Columbia Heart and Stroke Foundation.

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