The contributions of temporal and spatial environmental variation to physiological variation remain poorly resolved. Rocky intertidal zone populations are subjected to thermal variation over the tidal cycle, superimposed with micro-scale variation in individuals' body temperatures. Using the sea mussel (Mytilus californianus), we assessed the consequences of this microscale environmental variation for physiological variation among individuals, first by examining the latter in field-acclimatized animals, second by abolishing micro-scale environmental variation via common garden acclimation, and third by restoring this variation using a reciprocal outplant approach. Common garden acclimation reduced the magnitude of variation in tissuelevel antioxidant capacities by approximately 30% among mussels from a wave-protected (warm) site, but it had no effect on antioxidant variation among mussels from a wave-exposed (cool) site. The field-acclimatized level of antioxidant variation was restored only when protected-site mussels were outplanted to a high, thermally stressful site. Variation in organismal oxygen consumption rates reflected antioxidant patterns, decreasing dramatically among protected-site mussels after common gardening. These results suggest a highly plastic relationship between individuals' genotypes and their physiological phenotypes that depends on recent environmental experience. Corresponding context-dependent changes in the physiological meanvariance relationships within populations complicate prediction of responses to shifts in environmental variability that are anticipated with global change.
Rationale Consumption of whole grains is negatively associated with cardiovascular disease (CVD) risk but quantification of whole‐grain intake is challenging. Alkylresorcinols (ARs) are biomarkers of whole‐grain intake. Current methods for AR quantification involve a time‐consuming multi‐step separation process that hampers applicability in large‐scale studies. Methods We developed a streamlined method to quantify ARs in human plasma based on protein precipitation and direct injection into an ultra‐high‐performance liquid chromatograph coupled to a quadrupole time‐of‐flight mass spectrometer operating in atmospheric pressure chemical ionization negative ion mode. Results Separation of five major ARs was achieved, with linearity in the 5 to 550 nmol/L range and a lower limit of detection (LOD) of 0.5 nmol/L and quantification (LOQ) of 5 nmol/L. The within‐run and between‐run precision and accuracy were below 15%, and recoveries above 90%. Once validated, the method was applied to measure concentrations of plasma ARs in subjects who participated in a randomized, crossover trial evaluating the effect of carbohydrate type on CVD risk factors. The unrefined carbohydrate diet with the highest fiber content resulted in the highest plasma AR concentration (93 ± 78 nmol/L), and was significantly different (p <0.01) from lower fiber diets (18 ± 26 nmol/L and 19 ± 26 nmol/L, simple and unrefined carbohydrate, respectively). Conclusions This method offers a simplified approach to measure concentrations of plasma ARs as an objective biomarker of whole‐grain intake that can be applied to large‐scale cohort studies.
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 4 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 2 , 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.
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