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Comparison of Lipoprotein Separation and Lipid Analysis Methodologies for Human and Cynomolgus Monkey Plasma Samples

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Abstract

To assess cardiovascular risk in both clinical and basic research settings, it is imperative to be able to accurately measure plasma lipid levels. Here, methods commonly used to measure lipoproteins and lipids: ultracentrifugation (UC), fast protein liquid chromatography (FPLC), Roche auto-analyzer, and enzymatic assays were tested and compared. Plasma samples from 20 healthy humans and 22 cynomolgus monkeys were analyzed for their total cholesterol (TC), cholesterol in low density lipoproteins (LDL) and high density lipoproteins (HDL), and triglycerides (TG). Major lipid classes from UC and FPLC separated lipoprotein fractions from human plasma were further characterized by liquid chromatography-mass spectrometry analysis. All the tested methods showed acceptable performance with Roche analyzer among the best in approximate dilution linearity and recovery for most lipids as well as in repeatability between measurements of the same samples. TC, LDL, HDL, and TG values measured in human vs. monkey were—183.9 ± 35.5 (mean ± SD) vs. 105.6 ± 24.6 mg/dl, 106.0 ± 30.1 vs. 42.8 ± 13.0 mg/dl, 50.0 ± 11.4 vs. 53.4 ± 14.8 mg/dl, and 107.6 ± 50.7 vs. 58.0 ± 52.3 mg/dl. While no single method was uniformly the best, we recommend the Roche analyzer for routine measurements. UC or FPLC separation is needed for further functional characterization for specific lipid fraction. We have shown athero-protective profile in cynomolgus monkey compared with humans.

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Acknowledgments

We would like to thank Christine Brzostowski for providing cynomolgus monkey plasma, Doree Gorden for collecting human plasma samples, and Dan Xi and Vinit Shah for LC/MS sample process and analysis. For disclosing conflict of interests, all authors are employees and may hold stocks or stock options of Merck & Co.

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Authors and Affiliations

  1. Division of Cardiovascular Disease, Merck Research Laboratories, Rahway, NJ, 07065, USA

    Seongah Han, David McLaren, Vivienne Mendoza, Ray Rosa, Neil Geoghagen, Jose M. Castro-Perez, Thomas P. Roddy, Douglas Johns, Brian K. Hubbard & Jing Li

  2. Division of Bioanalytics and Pathology, Merck Research Laboratories, Rahway, NJ, 07065, USA

    Amy M. Flattery, Sang Ho Lee & Gail Forrest

  3. Division of Early Development Statistics, Merck Research Laboratories, Rahway, NJ, 07065, USA

    Richard Raubertas

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  1. Seongah Han
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Correspondence to Seongah Han.

Additional information

Clinical relevance: Clinical trial has demonstrated that cardiovascular disease risk can be reduced through lipid-lowering therapy. Reliable laboratory measurements of plasma lipoproteins and lipid measurement are important in research investigations and clinical practice for lipid-lowering therapy and for assessing cardiovascular risks. This study provide comparability of measurement results using four common methodologies (ultracentrifugation, fast protein liquid chromatography, Roche auto-analyzer, and enzymatic assays) in healthy human and cynomolgus monkey plasma samples. We recommend the Roche analyzer for routine measurements while UC or FPLC separation is needed for further functional characterization for specific lipid fraction.

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Supplementary Table 1

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Supplementary Table 2

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Supplementary Table 3

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Supplementary Figure 1

Comparison of methods by correlation and by Bland–Altman plot for human plasma. Linear regression lines and Bland–Altman plots for a total cholesterol (TC), b LDL cholesterol (LDL-C), c HDL cholesterol (HDL-C), and d TG in human plasma, comparing ultracentrifugation to each of the other three methods (PPT 302 kb)

Supplementary Figure 2

Comparison of methods by correlation and by Bland–Altman plots for cynomolgus plasma. Linear regression lines and Bland–Altman plots for a total cholesterol (TC), b LDL cholesterol (LDL-C), c HDL cholesterol (HDL-C), and d TG in cynomolgus plasma, comparing ultracentrifugation to each of the other three methods (PPT 311 kb)

Supplementary Figure 3

Relation of cholesterol in LDL or HDL and apolipoproteins. LDL cholesterol, HDL cholesterol, apoB, and apoA1 in cynomolgus plasma samples were determined using Roche analyzer. a Plot of plasma LDL cholesterol concentration against apoB level. b Plot of plasma HDL cholesterol concentration against apoA1 level (PPT 83 kb)

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Han, S., Flattery, A.M., McLaren, D. et al. Comparison of Lipoprotein Separation and Lipid Analysis Methodologies for Human and Cynomolgus Monkey Plasma Samples. J. of Cardiovasc. Trans. Res. 5, 75–83 (2012). https://doi.org/10.1007/s12265-011-9340-9

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  • DOI: https://doi.org/10.1007/s12265-011-9340-9

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