A rapid method for separation of plasma low and high density lipoproteins for tocopherol and carotenoid analyses

Ultracentrifugation (UC) is the method most often employed for separation and quantification of lipoproteins. Because this procedure requires expensive laboratory equipment, a large volume of fresh sample and an inordinate amount of time, it may not be ideal for routine clinical/experimental use. The aim of the current study was to evaluate a method which combines selective precipitation (HDL-P) and immunoseparation (LDL-I) for the rapid and reliable isolation of high density lipoproteins (HDL) and low density lipoproteins (LDL) specifically for vitamin E and carotenoid determination within these fractions. Cholesterol and triacylgylcerol concentrations within the HDL and LDL were also determined to enable expression of vitamin E and carotenoid concentrations per gram of lipid. Isolation of lipoproteins by UC was used as the reference method (HDL-UC/LDL-UC). There were no significant differences between methods for alpha- and gamma-tocopherol in LDL and HDL. Carotenoids measured in HDL and LDL were comparable between the methods. The exception was higher lutein/zeaxanthin concentration in HDL-P and LDL-I compared to HDL-UC and LDL-UC, respectively. Additionally, lycopene concentration was significantly lower in LDL-I compared to LDL-UC. In comparing vitamin E and carotenoid values in lipoproteins separated from fresh and frozen plasma by the direct method, there was no difference in alpha-tocopherol or the majority of carotenoids measured. In conclusion, a combination of selective precipitation and immunoseparation of fresh or frozen plasma for subsequent alpha- and gamma-tocopherol analyses provides an accurate and reliable alternative to lipoprotein separation by UC. Additionally, carotenoid concentrations in HDL separated by selective precipitation and analyses of alpha- and beta-carotenes and beta-cryptoxanthin in LDL separated by immunoseparation are also reliable, while lycopene and lutein/zeaxanthin concentrations in LDL-I are not readily comparable to LDL-UC.