Abstract P285: Development of a Simplified Method for the Measurement of Plasma Alkylresorcinols as a Biomarker of Whole Grain Intake and Application to a Human Clinical Trial Evaluating the Effect of Carbohydrate Quality on Cardiometabolic Risk Factors
Authors: Rodríguez-Morató Jose , Jayawardene Sarah , Dolnikowski Gregory , Galluccio Jean , Lichtenstein Alice H , Matthan Nirupa R 
Published: March 2019

Introduction:
Consumption of whole grains is associated with improvements in cardiometabolic risk factors and decreased CVD risk. Quantification of whole grain intake is challenging due to the limitations of self-reported intake; diversity among and differences in the interpretation of the term “whole grain”. Alkylresorcinols (ARs) are phenolic lipids present in the outer layer of wheat and rye grains that are considered objective biomarkers of whole grain intake. Current methods for ARs quantification involve a multi-step separation, extraction and purification processes that hampers applicability in large-scale studies.

Hypothesis:
Our aim was to develop a single-step method to measure 5 predominant ARs (C17:0, C19:0, C21:0, C23:0 and C25:0) in human plasma, and to validate this method by measuring plasma ARs levels in subjects who participated in a randomized, cross-over trial evaluating the effect of carbohydrate quality on CVD risk factors. We hypothesized that the direct method would distinguish between low- and high-whole grain intake and be more rapid and cost effective than prior methods.

Methods:
A dilute-and-shoot strategy based on plasma (20 μL) dilution with methanol for protein precipitation, followed by centrifugation and direct injection into an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometer (UHPLC/Q-TOF-MS; Agilent Technologies), using negative electrospray ionization. C19:0-D
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was used as internal standard. Separation was performed using a C18 column. Run time was 11 minutes/sample. Method validation was based on the following criteria: linearity, working range, lowest limit of quantification (LOQ), accuracy and precision. This method was then used to quantify ARs in fasting plasma samples from postmenopausal women and men (N=11, 65±8 years, BMI 29.8±3.2 kg/m
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, LDL-C ≥2.6 mmol/L) who consumed each of 3 isocaloric diets (60%E carbohydrate, 15%E protein, 25%E fat) enriched in either simple, refined, or unrefined carbohydrate-containing foods for 4.5 weeks in a randomized crossover design, with 2-week washout periods.

Results:
(1) Analytical: The method showed linearity (>0.999) in the range 2 to 100 ng/mL, and had acceptable values for accuracy and precision with a LOQ of 2 ng/mL. (2) Applicability: Total fiber content of the simple, refined and unrefined carbohydrate diets was 8.6, 9.6 and 19.5g/1000kcal, respectively. This was reflected in plasma AR levels, being significantly higher (p<0.05) after subjects consumed the unrefined (124.8±55.8 pmol/mL) compared to the simple (29.5±10.3 pmol/mL) and refined (26.8±9.0 pmol/mL) diets. C21:0 and C19:0 were the major ARs present in plasma.

Conclusion:
This simplified method offers a direct and rapid strategy to accurately measure AR in human plasma that can be scaled up for large studies, and provides an objective assessment of whole grain intake.