A comparison of anion exchange and steric exclusion HPLC assays of mouse plasma lipoproteins

Jonathan Neyer, Christain Espinoza, Terence M. Doherty, Luppe Luppen, Subhash C. Tripathi, Hiroyasu Uzui, Pinky V. Tripathi, Gerald Lee, Prediman K. Shah, and Prediman B. Rajavashisth

Medicine Dept., Cedars-Sinai Medical Center, Los Angeles, CA 90048, CA

Corresponding Author: rajavashisth{at}cshs.org

We compared the precision and accuracy of two HPLC methods (anion exchange [AE] and steric exclusion [SE]) for analysis of mouse lipoprotein profiles by determining coefficients of variability (CVs) under varying conditions. CVs for AE and SE were comparable on fresh samples (average CVs 7.7 ± 6.2% and 5.8 ± 2.7% for AE and SE respectively). There was an inverse linear relationship between subfraction curve area and CV (r = -0.65 [AE] and –0.50 [SE]), consistent with interpretation that as curve area decreased, error variance increased and signal-to-noise ratio decreased. Sample storage did not affect SE. In contrast, with AE, alterations in measured lipoproteins were apparent after 7 days storage, including a decrease in the HDL subfraction (66.8% [baseline] vs. 15.9% [1 week]; p < 0.01) and an increase in areas under LDL and VLDL peaks (LDL: 23.4% vs. 42.1% , VLDL: 9.9% vs. 41.9% [1 week vs. baseline for both comparisons; p < 0.02 and p < 0.03 respectively]). Concomitant with decreasing HDL area, reproducibility deteriorated with duration of storage. Analysis of the effects of decreasing sample injectate volume below 25 mL on SE lipoprotein subfractions revealed that area under LDL and VLDL peaks decreased (26.8% and 25.4% respectively; p < 0.001) and persisted as volume was decreased further. Areas under all lipoprotein subfractions measured with either AE or SE were linearly correlated with the amount of cholesterol measured by a colorimetric assay (r = 0.69 [AE] and 0.87 [SE]). Both AE and SE yield reproducible, accurate, and rapid measurements of lipoproteins from small amounts of serum. AE yields more sensitive, high amplitude, well-defined peaks that can be easily distinguished, and necessitates the use of smaller sample volumes compared to SE, but sample storage causes alterations in the chromatogram. SE appears better suited to serial analyses involving stored samples.

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