Discovering and validating between-subject variations in plasma lipids in healthy subjects.

Husna Begum,Peter Little,Richie Soong,Guanghou Shui,Markus R Wenk,Yik-Ying Teo,Bowen Li,Rick Twee-Hee Ong,Amaury Cazenave-Gassiot

doi:10.1038/srep19139

Abstract

Lipid levels are commonly used in clinical settings as disease biomarkers, and the advent of mass spectrometry-based (MS) lipidomics heralds the possibility of identifying additional lipids that can inform disease predispositions. However, the degree of natural variation for many lipids remains poorly understood, thus confounding downstream investigations on whether a specific intervention is driving observed lipid fluctuations. Here, we performed targeted mass spectrometry with multiple reaction monitoring across a comprehensive spectrum of 192 plasma lipids on eight subjects across three time-points separated by six hours and two standardized meals. A validation study to confirm the initial discoveries was performed in a further set of nine subjects, subject to the identical study design. Technical variation of the MS was assessed using duplicate measurements in the validation study, while biological variation was measured for lipid species with coefficients of variation <20%. We observed that eight lipid species from the phosphatidylethanolamine and phosphatidylcholine lipid classes were discovered and validated to vary consistently across the three time-points, where the within-subject variance can be up to 1.3-fold higher than between-subject variance. These findings highlight the importance of understanding the range of biological variation in plasma lipids as a precursor to their use in clinical biochemistry.

Highlights

Clinical biochemistry has clearly demonstrated that the concentration ranges of a number of blood metabolites depend on the time of day, gender, and ethnicity[3]
Plasma lipid levels were measured in a pilot panel of eight healthy subjects across three time-points of the same day to discover the extent that blood lipids vary between fasting and post-meal intakes, and lipid species which were identified to vary significantly were evaluated in a further validation panel of nine healthy subjects
We have evaluated how plasma lipids vary across three time points in two panels of healthy subjects, and identified eight lipid species which reproducibly exhibited evidence of increasing consistently with time

Summary

Introduction

Clinical biochemistry has clearly demonstrated that the concentration ranges of a number of blood metabolites depend on the time of day (cortisol), gender (hormones, creatinine), and ethnicity (free fatty acids)[3]. The effects of age[4], gender and ethnicity[5] on plasma lipid concentrations have been determined by mass spectrometry in a healthy subject cohort representative of the US population. The majority of existing literature around mass spectrometry lipidomics has overlooked the importance of measurement reproducibility and instrument validation. This is increasingly a concern in lipidomics research as reports are surfacing about how biological findings are confounded by instrumental errors, leading to a failure to reproduce previously observed experimental findings[8,9,10]. The subjects in the validation panel were assayed twice in order to ensure that any discoveries were genuinely due to biological changes and not artefacts due to instrumental or technical variation

Objectives

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Conclusion