Dear Rian Ka Praja

Not exactly sure,but from my past knowledge, HDL can be isolated by:

1-Ultracentifugation over night

2- Chemical precipitation methods

3-Techniques to directly measure HDL

4-NMR spectroscopy can,i guess the sub-components of HDL including nascent HDL particles, HLD-1 and HDL-2

Yes there could be other methods and sharing my informationonly

regards

Hello!

In our laboratory we isolate lipoproteins with density gradients of KBr. Firstly, we prepare a NaCl stock solution of 1,006g/ml (in literature you can find what you need to prepare it). You also need a high denstity solution of KBr (1,340g/ml).

Density range of HDL lipoproteins is 1.063–1.210 g/mL. You have to do a initial step in order to remove the fraction that you do not need (vLDLs, Chylomicrons and LDLs in case you do not want them).

The initial density of physiological serum is around the same density of the stock solution. So, you need to apply the following formula:

gr of KBr = [volume ∗ (  final density- intial denisty )]/[1 − (0.312 ∗ final density)]

Volume=the final volume that you want in your ultrcentrifugue tube.

The initial density is 1,006g/ml and you want to take your density to 1,063g/ml (final density). Apply the formula, the result is in g, we know that to obtain a solution of KBr of 1,340g/ml we need 57,40g of KBr in 117,9ul stock solution. For a rule of three you will know how ul of 1,340g/ml you need to add to take the density to 1,063. So in your tube you will have your X ml of sample, X ml of solution of 1,006g/ml and X of 1,340g/ml depending on the ml you want as a final volume.

Utracentrifugate the samples for 18 or 20 hours at 36000rpm at 4ºC. After that, remove the surface layer and start the process again taking the density of the sample to 1,210g/ml. Now rememer that after the first step your initial density is 1,063g/ml and that you need a solution with that density!!. Ultracentrifugate the samples at 36000 rpm, for 22 or 24 hours at 4ºC.

Now, in your uplayer fraction are contained your HDLs. You can prepare an agarose gel in order to check the purity, Very important!! Check always the density of your solutions to not make mistakes!!

Best regards,

Natàlia

This is shared from an article on NMR spectroscopy

" Several analytical approaches can be used for accurately measuring blood LPs, such as gel electrophoresis and Gel-Permeation High Performance Liquid Chromatography(GP-HPLC), but density gradient Ultra-Centrifugation (UC) represents the “gold standard method” for lipoproteins isolation and quantification [6]. Nevertheless, LP analysis by UC is time consuming and labor intensive as it requires numerous sample handlings and specific enzymatic assays are needed to further estimate their composition (usually Cho or TGs content) [7]. High-field 1H Nuclear Magnetic Resonance (1H-NMR)-based lipoprotein profiling has proven to be a valuable alternative to the standard quantification methods of total lipoproteins. 1H-NMR, which is normally used for structure elucidation and chemical mixture quantifications, has one more advantage, namely that it is sensitive to the size (translational and rotational diffusion) and density of macromolecules and supramolecular aggregates [8]. This makes NMR a unique platform for investigating Lipoprotein Particle Distributions (LPDs) primarily because different LP fractions and subfractions have different magnetic susceptibilities which will broadcast different signals whose amplitude reflects the particles concentration [9]. Moreover, the minimum sample pre-treatment and the possibility of gaining relevant biochemical information with a single rapid experiment make 1H-NMR spectroscopy a preferable/valuable screening tool for diagnostics as well as for large scale epidemiological investigations [10]. When combined with multivariate regression, NMR spectroscopy can be used to efficiently and accurately determine LP concentrations as well as TG and Cho content in specific lipoprotein fractions. However, the NMR prediction methods still depend on calibration with reference methods such as UC, gel electrophoresis or GP-HPLC. "

" Several analytical approaches can be used for accurately measuring blood LPs, such as gel electrophoresis and Gel-Permeation High Performance Liquid Chromatography(GP-HPLC), but density gradient Ultra-Centrifugation (UC) represents the “gold standard method” for lipoproteins isolation and quantification [6]. Nevertheless, LP analysis by UC is time consuming and labor intensive as it requires numerous sample handlings and specific enzymatic assays are needed to further estimate their composition (usually Cho or TGs content) [7]. High-field 1H Nuclear Magnetic Resonance (1H-NMR)-based lipoprotein profiling has proven to be a valuable alternative to the standard quantification methods of total lipoproteins. 1H-NMR, which is normally used for structure elucidation and chemical mixture quantifications, has one more advantage, namely that it is sensitive to the size (translational and rotational diffusion) and density of macromolecules and supramolecular aggregates [8]. This makes NMR a unique platform for investigating Lipoprotein Particle Distributions (LPDs) primarily because different LP fractions and subfractions have different magnetic susceptibilities which will broadcast different signals whose amplitude reflects the particles concentration [9]. Moreover, the minimum sample pre-treatment and the possibility of gaining relevant biochemical information with a single rapid experiment make 1H-NMR spectroscopy a preferable/valuable screening tool for diagnostics as well as for large scale epidemiological investigations [10]. When combined with multivariate regression, NMR spectroscopy can be used to efficiently and accuratelcy determine LP concentrations as well as TG and Cho content in specific lipoprotein fractions. However, the NMR prediction methods still depend on calibration with reference methods such as UC, gel electrophoresis or GP-HPLC. "

Reference- Article Quantification of Lipoprotein Profiles by Nuclear Magnetic R...

please see this PDF file

I think it is helpful

Serum LDL- and HDL-cholesterol determined by ultracentrifugation and HPLC

There are several commercial kits for isolation and measurement of HDL-cholesterol. Chemical precipitants are added to serum samples& ultracentrifugarion is done at low temperature (cold centrifuge). Supernatant is finally used to estimate HDL_cholesterol with the same working solution for total cholesterol measurement.

Best wishes

Please find the knowledge you look for in the attached chapter. We have isolated the three classes of lipoproteins, HDL. LDL and VLDL. the method is denefiely accurate but needs to practice.