Enzymatic assessment of cholesterol on electrophoresis gels for estimating HDL size distribution and plasma concentrations of HDL subclasses

Toledo-Ibelles, Paola; García-Sánchez, Cynthia; Avila-Vazzini, Nydia; Carreón‐Torres, Elizabeth; Posadas‐Romero, Carlos; Vargas‐Alarcón, Gilberto; Pérez-Méndez, Óscar

doi:10.1194/jlr.d004358

Cited by 24 publications

(36 citation statements)

References 29 publications

(27 reference statements)

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“…where n represents the HDL subclass: %HDLn is the relative percentage of each subclass determined by optical densitometry, and L is the plasma concentration of HDL-lipids (total cholesterol, free triglycerides, or phospholipids) determined by the commercial kits described above [13,33]. Plasma concentrations of cholesteryl esters corresponding to HDL subclasses were estimated using the difference between HDL-total cholesterol and HDL-free cholesterol, multiplied by 1.68 and expressed in milligrams per deciliter (mg/dL).…”

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

“…Gels were stained for total cholesterol, non-esterified (free) cholesterol, phospholipids and triglycerides using enzymatic mixtures previously described by us [12,13,33]. Incubation times ranged between 60 and 75 min at 37 C, in the dark, for any lipid stained on electrophoresis gels [13].…”

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

“…The relative proportions of each HDL subclass were expressed as percentages of the area under the curve of total HDL, integrated from 7.94 to 13.59 nm [33]. For the classification of the HDL subclasses, we considered the following size intervals: HDL3c, 7.94e8.45 nm; HDL3b, 8.45e8.98 nm; HDL3a, 8.98e9.94 nm; HDL2a, 9.94e10.58 nm; and HDL2b, 10.58e13.59 nm [33].…”

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

“…The electrophoresis gels were then scanned in a GS-670 BioRad densitometer (scan 1), destained and stained again for proteins with Coomassie R-250, before being scanned once more (scan 2). The relative proportions of each HDL subclass, which were determined by protein concentrations, were estimated via optical densitometry analysis (scan 2), using the following proteins as references: thyroglobulin, 17 nm; ferritin, 12.2 nm; catalase, 10.4 nm; lactate dehydrogenase, 8.2 nm; and albumin, 7.1 nm (high-molecular weight calibration kit, Amersham Pharmacia Biotech, Buckimghamshire, UK) [31,33]. The relative proportions of each HDL subclass were expressed as percentages of the area under the curve of total HDL, integrated from 7.94 to 13.59 nm [33].…”

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

“…The relative proportions of each HDL subclass, which were determined by protein concentrations, were estimated via optical densitometry analysis (scan 2), using the following proteins as references: thyroglobulin, 17 nm; ferritin, 12.2 nm; catalase, 10.4 nm; lactate dehydrogenase, 8.2 nm; and albumin, 7.1 nm (high-molecular weight calibration kit, Amersham Pharmacia Biotech, Buckimghamshire, UK) [31,33]. The relative proportions of each HDL subclass were expressed as percentages of the area under the curve of total HDL, integrated from 7.94 to 13.59 nm [33]. For the classification of the HDL subclasses, we considered the following size intervals: HDL3c, 7.94e8.45 nm; HDL3b, 8.45e8.98 nm; HDL3a, 8.98e9.94 nm; HDL2a, 9.94e10.58 nm; and HDL2b, 10.58e13.59 nm [33].…”

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

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Low concentrations of phospholipids and plasma HDL cholesterol subclasses in asymptomatic subjects with high coronary calcium scores

et al. 2015

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Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

Section: Characterization Of Hdl Subclassesmentioning

confidence: 99%

See 3 more Smart Citations

Low concentrations of phospholipids and plasma HDL cholesterol subclasses in asymptomatic subjects with high coronary calcium scores

et al. 2015

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Heterogeneity

2011

High‐Density Lipoproteins

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A comparative analysis of human plasma and serum proteins by combining native PAGE, whole‐gel slicing and quantitative LC‐MS/MS: Utilizing native MS‐electropherograms in proteomic analysis for discovering structure and interaction‐correlated differences

et al. 2017

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MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions.

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Enzymatic assessment of cholesterol on electrophoresis gels for estimating HDL size distribution and plasma concentrations of HDL subclasses

Cited by 24 publications

References 29 publications

Low concentrations of phospholipids and plasma HDL cholesterol subclasses in asymptomatic subjects with high coronary calcium scores

Low concentrations of phospholipids and plasma HDL cholesterol subclasses in asymptomatic subjects with high coronary calcium scores

Heterogeneity

A comparative analysis of human plasma and serum proteins by combining native PAGE, whole‐gel slicing and quantitative LC‐MS/MS: Utilizing native MS‐electropherograms in proteomic analysis for discovering structure and interaction‐correlated differences

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