High-performance aqueous gel permeation chromatography of serum lipoproteins: Selective detection of cholesterol by enzymatic reaction

doi:10.1016/S0378-4347(00)80100-0

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 223, Issue 2, 8 May 1981, Pages 285-293

https://doi.org/10.1016/S0378-4347(00)80100-0 Get rights and content

Abstract

A rapid method for the quantitation of cholesterol in each lipoprotein fraction has been developed which utilizes high-performance aqueous gel permeation chromatography followed by enzymatic reaction using reaction-type high-performance chromatography.

Cholesterol in serum lipoproteins eluted from the column could be sensitively and selectively detected by the absorbance at 550 nm following the enzymatic reaction. The sensitivity of the detection for cholesterol measured by A₅₅₀ was compared with that for protein measured by A₂₅₀ using the standard lipoprotein fractions: low-density lipoprotein (LDL) and high-density lipoproteins (HDL₂ and HDL₃). The effects of changing the flow-rate and lengthening the column on the resolution of LDL and HDL were examined. Analyses of serum protein and cholesterol were performed with this method for human and animal subjects.

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Enzymatic determination of cholesterol and triglycerides in serum lipoprotein profiles by asymmetrical flow field-flow fractionation with on-line, dual detection
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Electrophoretic and chromatographic techniques have been used to separate LPs and to obtain LP profiles that can be easily interpreted for diagnostic purposes [6,7]. The use of specific enzymatic reagents for selective detection and quantification of the LP-associated CHOL has been proposed for both electrophoretic [8–10] and chromatographic methods [11–13]. Size exclusion chromatography (SEC) with enzymatic reaction detection has been the most-applied method.
Alterations of lipoproteins (LPs) and related lipid levels in blood serum are correlated to the risk of coronary artery disease (CAD). Fast, possibly automated methods to obtain complete, multi-parametric LP profiles are therefore welcome to be developed for routine, clinical analysis practice. In this work, asymmetrical flow field-flow fractionation (AF4) with on-line, dual post-fractionation reaction detection (PFRD) is applied to develop a method for single-run, simultaneous quantification of cholesterol (CHOL) and triglycerides (TGs) in each fractionated LP class. The enzymatic reagents used for the post-fractionation reaction are available as commercial kits for certified, standard clinical protocols for the analysis of CHOL and TGs in serum. Using CHOL and glycerol as reference standards, a new procedure is applied to optimize the experimental conditions for PFRD-based, quantitative analysis. Upon optimized PFRD and AF4 conditions, results obtained for the determination of total CHOL (TC), TGs, HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) in a set of serum samples from healthy donors are found in agreement with the values provided by a clinical laboratory. The intra-day and inter-day precisions of the method were found always lower than 10% (CV). When the method was applied to serum samples from patients affected by sepsis, differences in CHOL and TG profiles between patients and healthy donors were observed.
Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides
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A method for the fractionation of lipoproteins based on size-exclusion chromatography (SEC) or gel permeation chromatography was developed that could separate HDL, LDL and VLDL in less than 60 min [16]. The cholesterol and triglyceride distribution over the lipoprotein fractions could be determined with enzymatic methods applied on collected fractions, but on-line quantification of one of the analytes associated to the different (sub)fractions of lipoproteins after their separation by SEC is also possible with a post-column enzymatic reaction setup [17,18]. Finally, Usui et al. showed that both the cholesterol and the triglyceride content of the separated fractions can be determined on line, by splitting of the column effluent and using two post-column reaction systems simultaneously [19].
A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not possible with the size-exclusion chromatography methods applied in routine analysis. Different channel geometries and flow programs were tested and compared. The use of a short fractionation channel was shown to give less sample dilution at the same fractionation power compared to a conventional, long channel. Different size selectivities were obtained with an exponential decay and a linear cross flow program. The ratio of the UV absorption signal to the light scattering signal was used to validate the relation between retention time and size of the fractionated particles.
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Complementary use of counter-current chromatography and hydroxyapatite chromatography for the separation of three main classes of lipoproteins from human serum
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High-density, low-density and very-low-density lipoproteins (HDLs, LDLs and VLDLs) were purified from human serum by the combined use of counter-current chromatography (CCC) and hydroxyapatite chromatography. Polymer-phase CCC of human serum using the cross-axis coil planet centrifuge yielded two lipoprotein fractions, one containing HDLs and LDLs and the other VLDLs and serum proteins. Each fraction was concentrated and subjected to hydroxyapatite chromatography to obtain three lipoprotein fractions, all free from serum proteins. Each lipoprotein was confirmed by agarose gel electrophoresis.
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Lipoproteins were separated by counter-current chromatography using the type-XLL coil planet centrifuge. The separation was performed with a polymer phase system composed of 16% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow rate of 0.5 ml/min. About 5 ml of the sample solution containing approximately 150 mg of a lipoprotein mixture were loaded. High- and low-density lipoproteins were resolved within 12 h. Each component was detected by gel electrophoresis with oil red staining.

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¹: Present address: Sanyo Gakuen Junior College, Okayama, Okayama Prefecture, Japan 703.

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High-performance aqueous gel permeation chromatography of serum lipoproteins: Selective detection of cholesterol by enzymatic reaction

Abstract

J. Chromatogr.

J. Amer. Oil Chem. Soc.

J. Biochem.

J. Biochem.

J. Biochem.