Lipoprotein fractionation by precipitation (a comparison of two methods).
Analysis of lipoproteins has become an essential routine investigation because of their close association with atherosclerotic vascular disease.,, The most popular methods adopted for lipoprotein separation are ultracentrifugation and electrophoresis. The method of ultracentrifugation is based on the separation of lipoproteins according to hydrated densities and floatation rates. The procedure is most accurate and provides separation of uncontaminated lipoprotein fractions. However, it is time consuming and requires 72 hours for the separation of all lipoproteins. The electrophoretic procedure is simple and less time consuming and provides substantial information necessary for phenotyping hyperlipoproteinemias (HLP). However, this method is based on lipid staining and densitometric quantitation of lipoproteins, which fails to provide information about absolute cholesterol and triglyceride content of individual lipoprotein classes. Hence it becomes necessary to develop a simple, inexpensive and less time consuming procedure for lipoprotein separation. Some alternatives are gel chromatography, and precipitation procedures.,,, Out of these, the precipitation procedures are becoming more popular.
In the present study, we have developed a dual precipitation procedure based on the procedure of Garcia Parra et al and compared it with the procedure of Onongbu and Lewis which we have been using in our laboratory for the last three years.
The blood samples were collected in EDTA (1.5 mg/ml). The plasma was separated by centrifugation at 3000 r.p.m. The lipoprotein fractionation was completed within 24 hours of collection. Samples were simultaneously analysed for lipoproteins by two different methods: - Method A: Onongbu and Lewis; and Method B: Garcia Parra et al.
This involves two steps:
Step I-from 1 ml of plasma, VLDL particles were separated using 10;C (w/v) Sodium Dodecyl Sulphate (SDS). The precipitate was separated as a floating pellet by centrifugation at 10,000 g for 30 minutes, and was dissolved in 1% SDS solution. This was used for VLDL cholesterol estimation.
Step II-involved precipitation of LDL and VLDL from another 1 ml aliquot of plasma using dextran sulphate and CaCl2. HDL cholesterol was estimated in the supernatant after the removal of LDL and VLDL precipitate by centrifugation.
Finally, LDL cholesterol was calculated as total cholesterol- (VLDL cholesterol + HDL cholesterol).
The original method of Garcia Parra et al was used for bulk separation of lipoproteins from normolipemic sera. We have modified this method to fractionate lipoproteins from smaller aliquots of plasma from normolipemic as well as hyerlipemic sera.
1) Ammonium sulphate A.R., saturated solution in distilled water.
2) Sodium phosphotungstate 4% (w/v) .
3) Magnesium chloride (MgCI2,.6H2O) 2 M, in distilled water.
4) Sodium chloride 0.15 M in distilled water.
5) (5)Sodium carbonate 10 per cent (w/v) .
6) Tris-HCl buffer pH 7.0 (0.05 M) in 0.15 M NaCl.
Step I-3 ml of plasma was diluted to 6 ml with tris buffer. The sample was centrifuged for 1 hr at 20,000 g. The supernatant 1 ml was removed for chylomicrons. Subnatant 4 ml were taken for separation of VLDL, Lp (a), LDL and HDL.
Step II-Concentration of 4 ml of subnatant obtained from Step I was raised to 20% saturation of ammonium sulphate by adding 1 ml of saturated ammonium sulphate. The precipitate was separated by centrifugation at 10,000 g for 30 min. The saturation of 4 ml of supernatant was adjusted to 351 by addition of 0.92 ml of ammonium sulphate. The precipitate of Lp (a) was separated by centrifugation and dissolved in 2 ml of tris buffer.
Step III-The 4 ml supernatant from Step II was adjusted to 50% saturation by adding 1.2 ml of saturated ammonium sulphate. The precipitate of crude VLDL was separated by centrifugation and dissolved in 2 ml of tris buffer.
Step IV-The 4 ml supernatant from Step III was adjusted to 60% saturation by adding 1 ml of ammonium sulphate solution and crude LDL fraction was obtained.
Step V-Crude HDL fraction was obtained by raising the saturation to 70'% of 4 ml of supernatant from Step III by adding 1.32 ml of saturated ammonium sulphate. All the lipoprotein fractions were checked by agarose gel electrophoresis to confirm that there is no contamination with albumin.
From the crude Lp (a), VLDL and LDL fractions, the purified lipoproteins were obtained by precipitation using 0.2 ml of sodium phosphotungstate and 0.05 ml of magnesium chloride solution. The crude HDL fraction was treated with 2 ml of sodium phosphotungstate and 0.4 ml of magnesium chloride solution to obtain the precipitate of pure HDL. All the precipitates were dissolved in 0.15 M sodium chloride solution, made alkaline with sodium carbonate (10% w/v). All the lipoprotein fractions were confirmed for their purity by electrophoresis on 3.8% polyacrylamide gel and then subjected to cholesterol analysis by Abells method.
The results of above methods were compared by using paired 't' test. The percentage recovery was calculated for method B as follows: The cholesterol values of HDL, LDL Lp (a) and VLDL were summed up and the sum was expressed as the percentage of total cholesterol measured of the same sample. Such calculation could not be done in method A as LDL fraction was not estimated but calculated as a difference between total cholesterol on one hand and the sum of HDL and VLDL cholesterol on the other hand.
For the convenience of comparison, Lp (a) and LDL in method B were pooled together to represent single LDL fraction.
There was no difference between HDL and VLDL cholesterol results obtained by methods A and B [Table - 1]. Estimation of LDL cholesterol by method A showed significantly high values than method B [Table - 1]. The coefficient of correlation between LDL cholesterol values estimated by method A and B was: r = 0.991 (p < 0.001). The percentage recovery of method B was 98.61 ± 2.69% (mean ± SD). Out of 26 samples analysed only 3 samples showed recovery below 95%. Out of 26 samples 4 samples were hyperlipemic (cholesterol above 250 mg/dl) and triglycerides above 150 mg/dl). The recovery of hyperlipemic sera was 98.2 ± 3.01 per cent (mean ± SD).
The two methods adopted for separation of lipoproteins in the present study, were compared individually with ultracentrifugation in their original articles., The objective of the present study was to modify the procedure of Garcia Parra et al in order to make it suitable for routine purpose; and to compare it with one of the widely accepted precipitation procedures. The original procedure of Garcia Parra et al was used for bulk lipoprotein separation from normo-lipemic sera. With minor modifications we have used it for estimations of lipoproteins from normolipemic as well as hyperlipemic sera. The procedure of Onongbu and Lewis was previously adopted for some of our earlier studies, and was suitable for routine purposes; hence it was taken for comparison.
The values of HDL and VLDL cholesterol showed no significant difference in two procedures and showed excellent coefficient of correlation. However, there was a difference in LDL cholesterol estimated by two methods. This may be because of the fact that in procedure A, LDL cholesterol is calculated as a difference between total cholesterol and (VLDL + HDL cholesterol) whereas in procedure B cholesterol content of LDL is directly measured. The amount of recovery obtained after summing up cholesterol content of all lipoproteins was nearly 97% in method B. This is as good as ultracentrifugation procedure.
Recent reports have shown difference between HDL cholesterol estimated by dextran sulphate/Ca+2 and sodium phosphotungstate/Mg+2 method with latter method giving more accurate results. However, in the present study no such difference is observed.
Out of 1000 samples analysed routinely by the method of Onongbu and Lewis, two samples failed to precipitate VLDL by sodium dodecyl sulphate (SDS). These samples were successfully fractionated by method B. The failure of SDS to precipitate VLDL may be due to the absence of beta-l-glycoprotein-1 factor which seems to be necessary for VLDL aggregation by SDS. In this respect the procedure of ammonium sulphate and sodium phosphotungstate seems to be preferable to SDS precipitation.
Another advantage of procedure-B is that it allows the separation of Lp (a) an isoform, of LDL. This fraction cannot be separated by any precipitation procedure and also reported to give interference with ultracentrifugation procedure. Hence we recommend that the procedure of Gracia Parra which depends upon the dual precipitation technique is the best precipitation procedure for lipoprotein fractionation for routine purposes.
We are thankful to the Dean, Seth G.S. Medical College and K.E.M. Hospital, Bombay, for providing the necessary facilities. We are also thankful to Dr. S. D. Bhandarkar, Hon. Physician, Endocrinology Department and Dr. (Smt.) S. G. Kinare, Prof. and Head, Department of Pathology, Seth G.S.M. College, Bombay for their valuable help.