Diurnal rhythms of serum and plasma cytokine profiles in healthy elderly individuals assessed using membrane based multiplexed immunoassay

Abstract

Background: Recent clinical studies suggest that inflammatory mediators have huge potential in individualized therapy and in efficacy screening and can be utilized as biomarkers for a plethora of pathological conditions. The standard approach for detecting and measuring these inflammatory mediators is via blood samples. Nevertheless, there is no scientific report providing solid evidence on the most suitable blood compartment that will give the optimal inflammatory mediator measurement, or regarding the diurnal variation of circulating mediators. In this study, we present the biological variability of circulating cytokines and chemokines from healthy individuals (mean age 59 years) assessed by a novel membrane-based assay.

Methods: Fifteen males and an equal number of females (all above 50 years) with no known inflammatory condition were selected. Through a planar method, named Proteome Profiler™, improved with fluorescence readout into a semi-quantitative multiplex assay, a screening of 36 inflammatory mediators was performed in serum and plasma of morning and afternoon blood withdrawals.

Results: The multiplex analysis revealed that the physiological variability of several circulating inflammatory mediators was relatively small within a cohort of 30 healthy aging subjects. There was no substantial gender effect in the inflammatory mediator profile. On the contrary, most of the cytokine/chemokine values measured in the afternoon collection were found to be higher compared to the morning ones, particularly in plasma.

Conclusions: In this study we provide evidence that circulating cytokine and chemokine levels of healthy individuals are elevated when blood is sampled in the afternoon compared to the morning, as influenced by the circulating cortisol levels. Furthermore, we report significant differences between cytokine/chemokine levels measured in serum and plasma. Our results provide essential information for future studies that will focus on examining circulating inflammatory mediator differences between healthy and diseased individuals.

Figure 1

Two-dimensional cluster analysis of blood samples. (a) The similarity between individual profiles in different conditions clustered according to the dendrogram on the top, whereas, the relationship between inflammatory molecules by the dendrogram on the left. Sample times, genders, sample types distribution has been color coded and placed below the relative individual profiles cluster analysis (dendrogram on the top). The color codes are: yellow/morning, orange/afternoon (b); blue/male, pink/female (c); brown/serum, red/plasma (d). The color-coded florescence intensity of all the values measured in the study are illustrated by the heatmap. White spots are missing values.

Figure 2

Relative expression of the four cytokines with the highest circulating levels. Four cytokines out of 36 assessed, were shown to have MFI above the threshold in every condition measured. Plasma PM has generally higher values compared to the ones measured in the other conditions (p < 0.001). ***p < 0.001 and corresponds to comparison to plasma AM. MFI, Median Fluorescence Intensity.

Figure 3

Relative expression levels of the analytes with circulating levels above threshold in (at least) two of the four conditions tested. The graph represents the selection of the analytes that showed low signals but were still above the threshold for at least two conditions. Note that all plasma PM values are significantly (p < 0.001) increased compared to Plasma AM. MFI, Median Fluorescence Intensity.